Polymeric chemical microsensor with fluorogenic molecular probe, manufacturing process and use for t

专利摘要:
Polymeric chemical microsensor with fluorogenic molecular probe, manufacturing process and use for the controlled release of bioactive substances and other applications. The present invention relates to low cost polymeric chemical microsensors, as well as to their method of obtaining, which are devices sensitive to variations in oxygen concentration in samples and which comprise one or more fluorogenic molecular probes that are derivatives of metaphthalocyanine and/or metalloporphyrin anchored in an inert and stable polymer matrix of a xerogel and deposited as a homogeneous film on a microplate or microtitre plate type test plate. The microsensors are suitable for those operations of the biochemical laboratory that require the handling of multiple samples of small volume, for example, immunological techniques or cell-based assays, such as the monitoring of metabolic studies related to the cellular proliferation of eukaryotes and prokaryotes, the cytotoxicity and cellular senescence and the mitochondrial respiration chain, among other applications, and can also contain and release controlled bioactive substances in a controlled manner. (Machine-translation by Google Translate, not legally binding)
公开号:ES2554077A1
申请号:ES201430899
申请日:2014-06-12
公开日:2015-12-15
发明作者:Juan Antonio DÍAZ MARTÍN；Joaquín MATILLA FUENTES；Juan Antonio SÁNCHEZ ARIAS
申请人:Juan Antonio DÍAZ MARTÍN；Joaquín MATILLA FUENTES；Juan Antonio SÁNCHEZ ARIAS；
IPC主号:

专利说明:

5
10
fifteen
twenty
25
30
35
40
Four. Five
DESCRIPTION
Polymeric chemical microsensor with fluorogenic molecular probe, manufacturing and use process for the controlled release of bioactive substances and other applications
Technical sector of the invention
This invention is included in the field of Chemistry, and specifically in the field of biomedical technologies, and refers to the coating of microplates with an oxygen sensitive film, being able to be preloaded with a variety of bioactive substances simultaneously, or more specifically, a biofunctional and bioactive film, of a biocompatible, chemically inert and stable material, which contains substances capable of responding to signals from the physical and / or physiological environment that surrounds it, inducing biological and physical responses, such as the variation in the concentration of dissolved oxygen in said medium and in such a way that it does not require the addition of additional reagents, complex manipulations or long incubation periods as those required in other techniques of preparation of this type of devices, so that they are of application, of simple, effective, reproducible and economical way, for in vitro and ex vivo laboratory studies, of the variac ion of the concentration of oxygen produced in biological processes, both in ultra-fast and fast or slow kinetic.
Background of the invention
In the processes of aerobic respiration of both subcellular organs and living cells, tissues and whole organisms, oxygen acts as the final acceptor of electrons, so it is consumed continuously and, consequently, can provide information on its activity, metabolic status, viability and / or physiological response to stimuli such as the action of a drug, environmental stress, toxins or effectors. Therefore, the measurement of variations in the concentration of dissolved oxygen in a medium is of paramount importance in the monitoring of many chemical and biochemical processes.
There are several methods for measuring dissolved oxygen; among them we could highlight electrochemical methods, polarographic methods (Clark Cell) and chemical methods (determination of oxygen by Winckler). However, most of these procedures have serious problems for the measurement of oxygen consumption in biochemical processes, since, due to the technical complexity of many of them, their little or no capacity for miniaturization and continuous process monitoring is linked, Not to mention the obligation, on the part of the user, to carry out permanent maintenance operations (cleaning, calibration, change of membrane and electrolyte, polishing of the anode, etc.).
Therefore, each day increases the interest in the use of new optical sensors for the measurement of oxygen concentration, using substances that are capable of absorbing light in the visible region and deactivated by fluorescent emission (fluorogenic molecular probes), through the known effect of quenching or fluorescent attenuation caused by oxygen on a large number of fluorogenic molecular probes (Mills, A .; Platinum Metals Rev., 1997,41, (3), 115-126; LingLing X. et al. , Chinese Science Bulletin, 2007, 52 (2), 188-193). Therefore, these types of compounds are useful for quantitatively recognizing the presence of a certain analyte, through changes in their optical properties.
Fluorescence is limited to a relatively small number of systems that incorporate certain structural characteristics (groups with coordinating capacity
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
of cations, such as macrocycles of different sizes containing atoms such as nitrogen, oxygen and sulfur, in which the fluorescence intensity depends on the number of rings and the degree of condensation) and in chemical environments capable of supporting and favoring the process . According to various studies, the best sensitivity for the detection of molecular oxygen in fluorescent probes derived from platinum metalloporphyrins, which absorb and emit in the visible spectrum (typically at 540 and 655 nanometers, respectively), with long state times, seems to have been demonstrated. excited (> 10 psec) and, many of them, are commercially accessible.
Summarizing all of the above, the known technology consists in the use of a fluorogenic molecular probe capable of being excited with a photon towards high (and unstable) levels of electronic and vibrational energy, so that the molecule tends to return to its fundamental state of energizes, releasing the energetic excess in the form of a photon. However, during the process, part of the energy dissipates, so that the photon emitted by the fluorogenic molecular probe is of lower energy, or what is the same, longer wavelength than initially absorbed, according to the law Stokes In addition, the intensity and wavelength of the emitted light depends on both the chemical structure of the fluorogenic molecular probe and the chemical medium in which it is located, and can be deactivated by a large number of factors, always taking into account that the processes "quenching" require physical contact between fluorogenic molecular probe and "quencher" (<2 A).
Consequently, not all known fluorogenic molecular probes are ideal for the intended applications, since they do not allow intimate contact between their coordination center and the oxygen molecule that acts as a “quencher”, which was believed to be solved by use of fluorogenic molecular probes capable of dissolving in the culture medium in which the experiment is carried out. However, given this possibility, some authors have already highlighted the risks of the presence of some metal ions when directly affecting the cells subject to the test, given the known cytotoxicity of some of the metal cations present in the structure of the complexes that are commonly used as fluorogenic molecular probes (ruthenium, palladium, platinum, etc.); This aspect is very relevant for the use of these probes in long-term studies, such as lymphoproliferation and cytotoxicity studies, regardless of the demonstration of their safety at very short times (Papkovsky, DB and Fernandes, R., EP1601955B1 ). In addition, various porphyrins have been used for the treatment of tumors through the so-called photodynamic therapy, which allows the selective destruction of tumor cells with visible light, combining a photosensitizer (water-soluble porphyrin) and oxygen, through the generation of reactive oxygen species (ROS) (Ko Y.-J. et al., Bioorg. Med. Chem. Lett. 2007.17 2789-2794; Z Hu et al., Biomedicine & Pharmacotherapy 2009, 63, 155-164). Similarly, hematoporphyrin IX and its derivatives have been frequently analyzed as photo-insecticides, that is, as photoactivatable molecules for use as pesticides in insect control (Pujol -Lereis, LM et al., Revista QufmicaViva 2011, 10, 139-153), given its ability, as a consequence of photoactivation, to produce reactive oxygen intermediates (ROI).
Consequently, in view of the growing scientific bibliography on the subject in question, the extreme interest in the development of new chemically inert sensors is deduced, such as high performance miniaturized devices based on polymeric materials with a silicon-based structure, obtained with sol-gel technology (Young, SK, Material Matters 2006, 1.3, 8), which use non-destructive detection methods, on a minimum amount of sample, with measurements in situ and in real time, using simple and affordable instrumentation in any laboratory.
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
The simplest known method of fixing the fluorogenic molecular probe to the matrix takes advantage of the adsorption phenomenon (Wodnicka M. et al., J Biomol Screen 2000, 5, 141), based on intermolecular attraction by ionic, hydrophobic or Van der Waal, according to the nature of the molecules involved. Although it is a very simple method, it lacks specificity and the gradual loss of the fluorogenic molecular probe due to desorption is expected, which influences the sensitivity, specificity and reproducibility of the measurement (due to the possible loss of fluorescent emission) and its reliability (due to the possible interaction between the probe and the biological material used). This indicates that there is no known method of permanent fixation, economically profitable and without potentially toxic residues, from the fluorogenic probe to the silicon matrix, which would be highly recommended to achieve chemically inert sensors, for the measurement of variations in the concentration of oxygen present in biological media.
The safest method of fixation described so far consists in the previous formation of one (or several) strong bonds, such as covalent bonds, between a fluorogenic molecular probe, conveniently substituted and the sillcea matrix (Figueira, F. et al., J. Porphyrins Phthalocyanines 2011; 15: 517-533). As a result of this need, several works can be cited in which an amide bond is created between tetracarboxyphenylporphyrin and 3-aminopropyl silica gel (Benedito, FL et al., Applied Catalysis A: General 2003, 250, 1-11; Rahimi , R et al., ECSOC 14, 2010), the creation of a covalent bond between tetracacarboxyphenyl- and tetrapropyl metalloporphyrins of platinum and biomolecules (Sagner, G. et al., 1999, US006004530A), the formation of an amide bond between various tetracacarboxyphenyl metalloporphyrins and a functionalized alkoxy (3- aminopropyltriethoxysilane) (Cohauila, MI, Doctoral Thesis, 2011) and tetrasulfophthalocyanines (Coahuila MI, J. Sol-Gel Science and Technology, 2006, 37, 117-120) included in the sllice network.
Obviously, although with good results, from a chemical and technical point of view, the above procedures present some difficulties, such as the use of reagents, by-products and solvents that are difficult to remove from the reaction medium, the use of high functionalized silicon derivatives price, high reaction temperatures and solvents that are not compatible with the polymers with which the microplates are manufactured (polystyrene, polycarbonate, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyethylene, cyclo-olefin polymers, etc.).
One of the main difficulties faced by laboratory personnel in the field of biological tests is the increasing sophistication of experimental and screening techniques carried out in microplates. Among the possible applications of the use of fluorogenic molecular probes, for the measurement of variations in oxygen concentration, due to cellular metabolism, we can mention, for example, the proliferation of T lymphocytes (responsible for coordinating the cellular immune response and to facilitate the necessary cooperation to develop the production of antibodies by B lymphocytes and all forms of cell-mediated immunity) is one of the first events that takes place in an immune response. The lymphatic proliferation technique allows to evaluate this functional capacity and consists in the culture of peripheral blood, with and without proliferative stimulation. Cell proliferation is expressed as a Stimulation Index, or, what is the same, the ratio between the values obtained in stimulated and non-stimulated cultures. Therefore, the technique of lymphoproliferation or lymphoblastic transformation allows to detect immunodeficiencies of cellular type, by measuring the immunological impact and, consequently, the evaluation of the immunological impact on the adaptive or acquired immune system of vertebrates. Said immune system can be influenced by numerous
5
10
fifteen
twenty
25
30
35
40
Four. Five
Circumstances such as: chemical substances, drugs, food additives, environmental characteristics, handling conditions, selection and genetics, etc.
That is why research within the field of immunology and in laboratory studies of the stimulation of in vitro growth of lymphocytes, both in primates and in other animal species (cattle, horses, pigs, sheep, etc.), gives us idea of the proportion of lymphocytes present in the individual, which is directly associated with their immunological responsiveness. Depending on the state of the art, to carry it out, 2 ml of whole blood with heparin, stored in the same extraction syringe, are needed. The rapid and correct mixing of the blood with the anticoagulant must be ensured by gentle inversion of the syringe, and sterile conditions must be maintained in the extraction and preservation of the sample, without refrigeration, to keep at room temperature.
It also requires analyzing the sample as soon as possible after it is removed and no later than 12 hours, taking into account that, for the taking of said sample, the subject must be fasting. The current method, most commonly used is that in which the sensitized lymphocytes contact the antigen, so that they proliferate and divide, measuring the degree of proliferation with tritiated thymidine (previously added to the culture medium) attached to the nucleotides which are going to be used for DNA synthesis. The standard protocol consists of:
- Isolation of lymphocytes, from whole blood, prior separation in Ficoll-isopaque gradient.
- Wash several times with cell culture medium and adjust the concentration to 5 million cells / ml.
- Distribution in 96-well microtiter plates, to which the solution of the problem antigen is added to the appropriate concentration. Some wells are not added antigen and are used as negative controls.
- Incubation for 72 hours at 37 ° C and in an atmosphere of 5% CO2.
- Collection of the cells on fiberglass filters and transfer to vials, containing a scintillation liquid. Subsequently, the emitted beta radiation is determined, which will be directly proportional to the number of cells in mitosis.
- The proliferation rate is established by comparison between the radioactivity of the problem wells and the controls.
Consequently, it would be very useful a technically feasible invention that would allow the application of an alternative procedure for in vitro research, within the field of immunology and related biochemical studies, that is capable of giving information not only of endpoint, such as the current ones, but in real time and that it is also fast, simple, effective, reproducible, economical, measurable with standard devices in any laboratory, within the limits of adequate detection and quantification, without toxic or dangerous residues, and, consequently, that exceeds all inconveniences mentioned above and avoid the technical limitations of the current biochemical methods (permanent maintenance of expensive equipment and sophisticated facilities, which require authorized and highly trained personnel, prior isolation of cells, from whole blood, several intermediate processes of washing between different reactions and treatments, possible Quality of residual contamination between washes, slow processing speed, use of expensive, dangerous and difficult to destroy radioactive reagents, etc.). Furthermore, in this field, a device capable of measuring lymphoblastic transformation, induced by
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
mitogens and / or by specific antigens, directly in whole blood without the need for prior isolation of peripheral blood mononuclear cells (PBMC), which is impossible with current colorimetric methods.
In addition to its greater simplicity of processing and avoiding the loss of lymphocyte populations and non-lymphoid cells, due to the centrifugation gradient, the use of whole blood allows it to advantageously reproduce the conditions that occur in vivo (Shifrine M et al. , Am. J. Vet. Res., 1978, 39 (4), 687-690), being able to study the potential influence on lymphocyte proliferation of other blood cells present in the blood, such as erythrocytes, through mechanisms such as production of cytokines that accelerate cell proliferation.
That is why it can be said that none of the current methods, manual or automatic, in tubes or microplates, patented or not, are capable of responding to the set of technical problems that have been raised above: costly and permanent maintenance of equipment and highly sophisticated facilities, experiments carried out exclusively by specifically trained and officially authorized personnel, need for prior isolation of cells (from whole blood), slow processing speed due to the requirement in the current protocols of various intermediate processes of washings and additions of specific reagents between the different test stages, the use of a high number of pipettes and accessories, or robotic systems, with the consequent economic cost and time, very long periods of incubation and measurement (between 5 and 7 days ), exclusive obtaining of endpoint information, without intermediate tracking data of the various types bles involved in the process, high probability of residual contamination between washes, with the use of expensive, dangerous and difficult destruction radioactive reagents, etc.
In view of the problems detected in the technical field and discussed here, the present invention offers a new device for measuring variations in the concentration of dissolved oxygen in a sample and for detecting and monitoring the biological processes that produce them, which is a Microplate with low-cost optical sensor for biological assays, constituted to solve the exposed problem and appropriate for all types of chemosensitivity studies related to eukaryotic cell proliferation (for example, lymphoblastic transformation test) and prokaryotes (for example , antibiograms for the selection of antimicrobials in in vitro susceptibility studies), cytotoxicity and mitochondrial respiration chain, both in subcellular fractions, in isolated cells, in tissues and in complete biological fluids, preferably blood and urine, through the use of a plate reader capable of measuring fluorescence or fluorescence in resu time elto, both in lower and upper reading mode. The polymeric chemical microsensor described herein consists of a matrix in the form of a xerogel doped with one or several fluorogenic (fluorescent) molecular probes carrying groups with adequate reactivity for the intended purposes, whether commercial or not, in an amount suitable for that, on the one hand, they are useful in the measurement of variations in the concentration of dissolved oxygen, during the detection and monitoring of biological processes and, on the other, of permanently joining the matrix thanks to the specific chemical characteristics that the various phases of the sol-gel process with which the xerogel is prepared, avoiding possible unwanted effects, inherent in the free probe. The present invention, in one of the most preferred embodiments, can incorporate one or several bioactive substances with different specific affinity to establish a physical-chemical interaction with the surface of the solid matrix, capable of being adsobed on the surface of said matrix and, therefore, gradually be released to the reaction medium in an amount effective to produce the intended effects.
5
10
fifteen
twenty
25
30
35
40
Four. Five
Definitions
Unless the context clearly indicates otherwise, the following terms are used herein, with the meanings provided below:
- The term "adsorption" refers to the accumulation of substances on a surface or interface, for example gas-solid. The matrix on which said adsorption occurs is called adsorbent while the material that accumulates on the surface is called adsorbate.
- The term "suitable" refers to a substance, substituent, process or quantity appropriate and compatible with the compounds, products, compositions and devices useful in the field of application of the present invention, as may be determined by one skilled in the art using only routine methods of experimentation, and without the need for a covert inventive skill.
- The term "affinity" refers to the ability of an adsorbate to bind to the surface of a specific adsorbent and form a joint through ionic interactions, attraction of van der Waals or chemical-type bonds, which determines the retention capacity of the adsorbed by the adsorbent.
- The term "alkoxy" refers to any organic radical derived from an alcohol upon loss of hydroxylic hydrogen, of formula RO-, where R is an alkyl group, as defined below.
- The term "alkyl" is used to refer to radicals of hydrocarbon, linear, branched or cyclic chains, which have 1 to 20 carbon atoms and which are attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. The alkyl groups may be optionally substituted independently by 1 to 3 groups independently selected from halogen, hydroxyl, amino, amino C1-C6 alkyl, nitro, cyano, isocyanate, isothiocyanate, C1-C20 alkyl, C1-C20 perfluoroalkyl, -O-perfluoroalkyl C1-C20, -S-perfluoroalkyl C1-C20, C1-C6 alkoxy, -OCHF2, - C (O) CH3, -C (O) O C1-C3 alkyl, -C (O) NH2, -S (O) 2CH3 , C3-C6 cycloalkyl, -CH2- C3-C6 cycloalkyl, pyridinyl, -CH2-pyridinyl, thienyl, CH2-thienyl, furanyl, CH2-furanyl, oxazolyl, CH2-oxazolyl, phenyl, benzyl, phenoxy, in which the group alkyl and the rings of the cycloalkyl, pyridinyl, thienyl, furanyl, oxazolyl, phenyl, benzyl, phenethyl and phenoxy groups may be optionally substituted by 1 to 3 groups independently selected from among halogen, hydroxyl, amino, Ci-C6 aminoalkyl, nitro , cyano, isocyanate, isothiocyanate, Ci-C6 alkyl, C3-C6 cycloalkyl, Ci-C3 perfluoroalkyl, - O-perfluoroalkyl C-tC3, -S-per CtC3 fluoroalkyl, C-, C3 alkoxy, -OCHF2, -CN, -COOH, - CH2CO2H, -C (O) CH3, -C (O) Oalkyl, -C (O) NH2, -S (O) 2CH3.
- The term "alkenyl" refers to hydrocarbon chain radicals containing one or more double carbon-carbon bonds, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-butenyl, 1,3-butadienyl, etc. Alkenyl radicals may be optionally independently substituted by 1 to 3 groups independently selected from halogen, hydroxyl, amino, CtC6 aminoalkyl, nitro, cyano, isocyanate, isothiocyanate, CrC20 alkyl, C-perfluoroalkyl, CrC20 -O-perfluoroalkyl, -S-perfluoro-C1-C20 alkyl, C1-C6 alkoxy, -OCHF2, -C (O) CH3, -C (O) O C1-C3 alkyl, -C (O) NH2, -S (O) 2CH3, C3-cycloalkyl C6, -CH2-C3-C6 cycloalkyl, pyridinyl, -CH2-pyridinyl, thienyl, CH2-thienyl, furanyl, CH2-furanyl, oxazolyl, CH2-oxazolyl, phenyl, benzyl, phenoxy, in which the alkyl group and the rings of the cycloalkyl, pyridinyl, thienyl, furanyl, oxazolyl, phenyl, benzyl, phenethyl and phenoxy groups may be optionally substituted by 1 to 3 groups independently selected from among halogen, hydroxyl, amino,
5
10
fifteen
twenty
25
30
35
40
Four. Five
C1-C6 aminoalkyl, nitro, cyano, isocyanate, isothiocyanate, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, -O-perfluoroalkyl C1-C3, -S-perfluoroalkyl C1-C3, C1C3 alkoxy, - OCHF2, -CN, -COOH, -CH2CO2H, -C (O) CH3, -C (O) Oalkyl, -C (O) NH2, -S (O) 2CH3.
- The term "analyte" refers to a chemical species of inorganic, organic or biochemical nature and is determined in a sample, by means of a set of operations and techniques applied to the analysis of said sample.
- The term "antibiogram" refers to a method of phenotypic study of sensitivity to antimicrobials, which consists of facing a standardized bacterial inoculum at a single or at different concentrations of antibiotics, allowing microorganisms to be classified into several clinical categories, as sensitive, intermediate or resistant and allows to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (CMB).
- The terms "antibiotic" and "antimicrobial" refer to substances of origin
Biological or synthetic and can be categorized as bactericidal, if they kill susceptible bacteria, or bacteriostatic, if they only reversibly inhibit the growth of bacteria. Among them we can select, by way of illustration and without limiting the scope of the invention, the following: fosfomycin, vancomycin, penicillin, ampicillin, amoxicillin, amoxicillin / clavulanic acid, ampicillin / sulbactam, ticarcillin, piperacillin, piperacillin / tazobactam, ce , tazobactam, avibactam, cefazolin, cefuroxime, cefoxitin, cefpodoxime, cefditoren, cefotetan, ceftazidime, ceftazidime / clavulanic acid, cefotaxime, cefotaxime / clavulanic acid, cefepime cephepimidem, clavimenemimene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, pyramidene, peptide pivoxil, ertapenem, nitrofurantolna, polymyxin, streptomycin, kanamycin, gentamicin, tobramycin, amikacin, netilmicin, neomycin, nalidixic acid, ciprofloxacin,
levofloxacin, nemonoxacin, moxfloxacin, ozenoxacin, finafloxacin, prulifloxacin, ulifloxacin, zabtoxacin, delafloxacin, quinupristin, dalfopristin, linopristin,
flopristine, pristinamycin, colistin, oxollnic acid, isoniazid, rifamycin, rifampin, tetracycline, minocycline, tigecycline, amadacycline, dalbavancin, teicoplanin,
daptomycin, pleuromutilin, pipemldic acid, cotrimoxazole, linezolid, radezolid, tedizolid, fosfomycin, mupirocin, chloramphenicol, fusulic acid, doxycycline, lincomycin,
clindamycin, erythromycin, oleandomycin, spiramycin, josamycin, dirithromycin, flurithromycin, clarithromycin, midecamycin, telithromycin, azithromycin, cetromicina, oritavancin, moditromicina, solitromicina, trimethoprim, methotrexate, sulfacetamide, sulfisoxazole, sulfadiazine, sulfamethoxazole, sulfamoxole, sulfadimethoxine, sulfamethoxypyridazine, sulfametoxidiazina, phthalsulfatiazole, succinylsulfatiazole, mafenide, sulfadoxine, sulfaguanidine, sulfacetamide and future members of any of these families.
- The term "antifungal" refers to substances of biological or synthetic origin that act against pathogenic fungi, among which we can select, by way of illustration and without limiting the scope of the invention, to the following: amphotericin B, nystatin, natamycin, griseofulvin, miconazole, ketoconazole, itraconazole, fluconazole, voriconazole, posaconazole, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, flutrimazole, omoconazole, sulconazole, thioconazole, terconazole, flucytosine, caspungung, naphthin, cyclophyrine tolnaftate or future members of any of these families.
- The term "antineoplasic" refers to cytostatic and cytotoxic substances of biological or synthetic origin that are substances that act on one or several phases of the cell cycle or on the mechanisms of control of the proliferation of tumor cells in a characteristic way, inhibiting their cell growth, among which
5
10
fifteen
twenty
25
30
35
40
Four. Five
we can select, illustratively tltuio without limiting the scope of the invention, to the following: cyclophosphamide, chlorambucil, ifosfamide, melphalan, trofosfamide, carmustine, estramustine, fotemustine, dacarbazine, temozolomide, bleomycin, mitomycin, doxorubicin, daunorubicin, 4- epirubicin, cytarabine, gemcitabine, 5-fluorouracil, fludarabine, pentostatin, methotrexate, cisplatin, carboplatin. oxaliplatin, topotecan, irinotecan, trastuzumab, rituximab, mitoxantrone, etoposide, teniposide, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, tamoxifen, cyproterone, flutamide, leuprolide or future members of any of these families.
- The term "antigen" refers to substances capable of inducing a specific immune response that trigger events that may manifest as allergic, immunological and pyrogenic reactions.
- The term "antiprotozoal" refers to substances of biological or synthetic origin that act against protozoan parasites, among which we can select, by way of illustration and without limiting the scope of the invention, to the following: metronidazole, tinidazole, ornidazole secnidazole benzinidazole, nifurtimox, diloxanide furoate, iodoquinol, sulfamethoxazole, trimethoprim, paromomycin, dehydroemetine, pyrimethamine, quinine, quinidine, etofamide, teclozan, clefamide, meglumine antimoniate, stiboglutaminemethamateamine, sodium dihydroxymethamateamine, sodium dihydroxymethamateamide sodium, chloroquine, amodiaquine, mefloquine, artemisin, artemether, artesunate, sulfadoxine, pyrimethamine, lumefantrine, doxycycline, proguanil, polyhexamethylene biguanide, furazolidone, albendazole, nimorazole, melarsoprol, salinomycin, lasalocide or any of these family members.
- The term "aryl" refers to a substituted or unsubstituted phenyl, furyl, thienyl or pyridyl group, or a condensed ring system of any of these groups, such as naphthyl.
- The terms synonymous "fluorescence attenuation" or "quenching" refer to a process capable of decreasing the fluorescence intensity of a given fluorogenic molecular probe, without changing the emission spectrum.
- The term “calibration” refers to the set of operations that determine, under specific conditions, the relationship between the values indicated by an instrument or measurement system and the known values corresponding to a reference pattern;
- The term "effective amount" of a compound, product or composition refers to a sufficient amount of the compound, product or composition to generate the desired results, since, although the exact amount required may vary slightly from batch to batch, in the manner in which it will be administered later, of the specific results sought, etc., can always be determined by a person skilled in the art using only routine methods of experimentation.
- The term "colloid" refers to solid particles with a diameter of 1-100 nanometers where the gravitational force is negligible and the interaction is dominated by short-range forces, such as van der Waals attraction and surface charge.
- The term "minimum inhibitory concentration" refers to the lowest concentration of the antibiotic that results in inhibition of visible growth under standard conditions. and the term "minimum bactericidal concentration" refers to the lowest concentration of the antibiotic that is capable of killing 99.9% of the original inoculum in a given period of time.
5
10
fifteen
twenty
25
30
35
40
Four. Five
- The term "crosstalk" refers to interference between wells, due to light cross contamination between said wells during fluorescence tests.
- The term "calibration curve" refers to the set of concentrations that describe the range in which the compound to be analyzed is quantified.
- The term "specificity" refers to the ability of the method to identify the wells where there is no significant variation of the analyte and is calculated as the proportion of wells without biological load that give negative in the test: Specificity = Positive False / [False Positive + True Negative].
- The term "stable" refers to not react no modify your characteristics or does not undergo changes in its structure, due to the action of external agents, either physical (temperature, radiation, etc.), well chemical (solvents, acids, etc.), or biological (fluids, enzymes, etc.), keeping their position, distribution, shape, composition, state or situation unchanged or unchanged, at least for the time required to carry out The desired studies.
- The term "reliability" refers to the probability that the device performs the functions for which it has been designed under given specifications and for a certain period of time.
- The term "gel" is used to refer to a rigid network interconnected with pores of dimensions smaller than a micrometer and with polymeric chains which are much longer than a micrometer. A gel can be formed by the growth of a network due to a discrete arrangement of colloidal particles or by a three-dimensional network interconnected by hydrolysis and simultaneous polycondensation of an organometallic precursor.
- The terms "immunosuppression" and "immunodeficiency disorder" refer to the decrease or absence of an organism's immune response, as insufficient antibodies are produced or due to a dysfunction of any part of the immune system, such as T lymphocytes or B and derivatives of inherited immunodeficiencies, acquired, immunosenescence, or as a side effect of some treatments.
- The term "plate reader" refers to any device capable of performing the intended analysis of samples contained in "microtiter plates", also called "well plate", "microtiter plate" or "microplate", from 6 to 3456 wells, by detecting signals produced as a result of biological, physical and chemical events, either well to well, or jointly, such as fluorescence readers based on plate detection by a first optical system capable of illuminating the sample with a specific wavelength and a second system in charge of collecting the emitted light and separating it (basically consisting of monochromators, photomultiplier tubes, filters and suitable analytical software).
- The term “controlled liberation” refers to a system of administration of bioactive substances in a slow and continuous way during extended periods of time, in which, the bioactive substance is incorporated into a support that is generally a polymeric material or a combination several, so that the rate of release of the bioactive substance from said system to the surrounding environment, is determined by the properties of the polymer itself and, to a lesser extent, depends on environmental factors, such as pH, temperature, etc.
5
10
fifteen
twenty
25
30
35
40
Four. Five
- The term "quantification limit" refers to the lowest concentration of the compound that can be quantified in compliance with the precision and accuracy established in the method.
- The term "detection limit" refers to the minimum concentration of a compound in a sample which can be detected, but not necessarily quantified, under the established operating conditions.
- The term "leaching" refers to the process in which one or several solutes are extracted from a solid, by using a liquid solvent, when both phases come into intimate contact, allowing the solute or solutes to diffuse from the solid to the liquid phase.
- The term “biological matrix” refers to the whole of the medium and biological sample, of prokaryotic or eukaryotic origin (eg, bacteria, protozoa, plants, insects, birds, fish, reptiles, mammals, etc.), in which the substance of interest is generated and / or found, for in vitro or ex vivo study.
- The term "polymeric matrix" refers to a solid polymeric material that contains multiple units chemically bonded and that are joined together to form a solid, by a polymerization process, so that small molecules are joined to create other molecules and aggregates much larger, either of an inorganic nature, with macromolecules formed from covalent bonds, without the intervention of hydrocarbon molecules in its composition, either of an organic nature, formed from hydrocarbons or their derivatives, or mixed.
- The term "biological medium" refers to systems and mixtures, such as water (including drinking water, wastewater, cooling and processing tower), salt solutions, culture media (understood as a mixture of components that may include , although it is not limited to, inorganic salts, vitamins, amino acids, carbohydrates and other nutrients dissolved in water), biological fluids and any other compatible with the life of various biological agents such as higher organisms (animals and plants), microorganisms (virus , bacteria, yeasts, microalgae, etc.), plant cells, animal cells, genetically modified, or not, and parts derived from any of them.
- The terms "metalophthalocyanine" and "metalloporphyrin" are used to refer to two groups of compounds that are fluorogenic probes, capable of detecting an analyte selectively, reversibly and in real time and report its recognition by issuing an optical signal , constituted by a central macrocycle formed by a closed system of 16 carbon and nitrogen atoms, linked through a cycle or a hydrocarbon chain by means of single and double bonds, forming a resonant system, capable of housing a metal atom or ion, chosen from cobalt, copper, iron, magnesium, manganese, zinc, antimony, nickel, vanadium, europium, terbium, gadolinium, samarium and more preferably, palladium, platinum and ruthenium, which generally forms four metal-nitrogen bonds, covalent and coordinated covalent In the case of the present invention, metalophthalocyanine and metalloporphyrin always carry two to four polar carboxylate, sulphonate, phosphate or phenol groups and which are linked through a cycle or a hydrocarbon chain.
- The terms synonymous “microplate, microtiter plate. well plate or microtiter plate ”refers to a test plate that has a plurality of wells in the form of open receptacles on its upper face, made of a rigid material selected from glass, polystyrene, polycarbonate, polypropylene, polyvinylchloride, polyethylene , polytetrafluoroethylene, cyclo-olefin polymers, or the like, in colors
5
10
fifteen
twenty
25
30
35
40
Four. Five
appropriate to each study (normally, transparent, black and white) and with different geometry (bottom in the form of C, F, U or V), well capacity (from 1 microliter to 16 milliliters) and with a standard format of 6, 12, 24, 48, 96, 384.1536 and 3456 wells, depending on the experiment to be carried out with the plate, the amount of biological material, the means used in the experiment (manual, semi-automatic or automatic), etc.
- The term "mitogen" refers to substances that stimulate mitosis and cell proliferation and, more specifically, the transformation of lymphocytes into undifferentiated lymphoblasts with the ability to divide. There are nonspecific mitogens, such as the phytolac mitogen (PWM), which is both a T and B lymphocyte mitogen, and specific T lymphocytes such as concanavalin A (conA) and phytohemagglutinin (PHA) and other B lymphocytes such as lipopolysaccharide (LPS) of Gram negative bacteria.
- The term "biological sample" refers to any physiological or pathological sample obtained from a biological subject, including liquids, secretions, etc. that are part of or produced from a living organism: biological fluids (including, but not limited to , serum, plasma, blood, urine, saliva, sweat, milk, vaginal exudate, semen, gastric juices, duodenal fluid, clastic fluid, ascetic fluid, intraocular fluid, pericardial fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, exudates from lesions, stool or manure extracts, or any of its isolated components), organs, tissues, including biopsy tissue samples or portions in sections of an organ or tissue, subcellular fractions, isolated cells, including of humans and extracts from a biological sample, including, but not limited to antigens, antibodies, metabolites, etc. isolated from a biological fluid. The sample can, therefore, comprise a chained sample, a fluid from a sample, a fluidized sample or a preparation from a sample that is. The sample may also include complete aquatic organisms, such as algae (eg those of the Tetraselmis genus), invertebrates (eg those of the Artemia genus or those of the genus Caenorhabditis), or fish (eg those of the genus Danio).
- The terms synonymous "control samples" and "controls" refer to samples of known concentration that are quantified during the analysis to corroborate the validity of the method.
- The term "oligomer" refers to molecular species consisting of repetitive units that have an intermediate size between the basic unit of the monomer that forms them and a polymer, that is, it contains monomers in a finite number (called the degree of oligomerization) and, therefore, its molecular mass has failed to reach such a high value as to be considered a polymer.
- The term "one-pot" refers to a strategy to increase the efficiency of chemical processes, both economically and technically, according to which said process is carried out in a single reactor, through additions successive reagents, without intermediate stages of isolation or purification.
- The term “patient” refers to any living being that suffers or may suffer from a disease, characterized by a harmful, real or potential alteration of their state of health.
- The term "porphyrin" is used to refer to a heterocyclic macrocycle consisting of four pyrrole subunits joined by the opposite faces (positionD) by four methine bridges (= CH-), being able to be mono- or polysubstituted with various functional groups (alkyl , alkenyl, carboxylate, sulphonate, phosphate, amino, etc.). Sometimes
5
10
fifteen
twenty
25
30
35
40
Four. Five
the terms porfin and porfina are used interchangeably, with the same meaning as specified above.
- The term "protocol" refers to the document that establishes the objectives, procedures and methods used to conduct a study and analyze the data obtained.
- The term "range" refers to the validity range of the analytical method, defined by the concentrations between the upper and lower levels of the analyte, in which it has been shown that the method is accurate, accurate and linear.
- The term "reproducibility" refers to the precision of an analytical method and expresses the variation obtained between independent determinations made with the same analytical measurement system, but under different analysis conditions, such as dlas, equipment, or analysts.
- The term "sensitivity" refers to the ability of the method to identify wells where there is a significant variation of the analyte, calculated as the proportion of wells with biological load that test positive: Sensitivity = Positive True / [Positive True + False Negatives].
- The term "significant" refers to a statistical expression that depends on the size of the sample and indicates the probability that a series of results obtained were due to the random, chance, fate.
- The term "sun" refers to a colloidal suspension of particles dispersed in a liquid.
- The term "sol-gel" refers to a process of synthesis and manufacturing, generally in solution and at a temperature below 100 ° C, of a polymeric solid material, prepared by hydrolysis of monomers of metal alkoxides or of metalloids, selected from the group consisting of silicon, aluminum, zirconium, titanium, tin, vanadium, iron and any combination thereof, which form a porous network with metal / metalloid and oxygen bonds, to which other derivatives can be added, such as the finished hydroxy polydimethylsiloxane (PDMS), which also contains a main chain of alternating silicon and oxygen atoms, but with radicals of an organic nature, thereby obtaining a polymeric matrix of a hybrid (organic-inorganic) nature; in this way In both cases, when the liquid from the pore is removed at a pressure and temperature close to the ambient temperature, a network contraction occurs and the resulting product is called xerogel.
- The terms synonymous "fluorogenic molecular probe", "fluorogenic probe" and "fluorogenic sensor" refer to systems that contain a coordination center attached to an indicator unit, which transforms the chemical information into a measurable signal and is characterized by its high sensitivity and due to its high specificity, by specifically recognizing the analyte of interest by means of the appropriate selection of the excitation and emission wavelengths.
- The term "bioactive substance" refers to a chemical or biological compound that can exert "an interaction with" or "produce effects on" any living being (animal or plant) or any of its parts, generally producing an improvement in its health and well-being, or reducing a risk of disease by inhibition of growth and survival of pathogenic biological agents, and includes terms such as antibiotics, antifungals, antiprotozoals, antineoplasics, mitogens or specific antigens.
5
10
fifteen
twenty
25
30
35
40
Four. Five
- The term "substituted" is generally used to refer to a carbon atom or a heteroatom or suitable in which a hydrogen atom is replaced by another atom or a chemical group such as aryl, cyano, carboxylate, sulfonate, phosphate, amino, (C1-10) alkyl, nitro, mercapto, (C1-10) alkylthio, halo, (C1-6) alkylamino, (C1- 6) dialkylamino, (C1-6) alkoxy, tri (C1-4 ) alkoxysilyl, amino (C1-4) alkyltrialkyloxysilane, etc., and their possible combinations within the molecule, which confer new physicochemical properties, which improve the fundamental and novel utilities of the compounds, products or compositions of the present invention.
- The terms synonymous "non-ionic surfactant" and "surfactant" refer to a series of chemical compounds with a polar-non-polar structure, but with non-dissociable groups, which are capable of decreasing the value of the surface tension of an interface, such as Triton X-100, Brij 30, Brij 35, Brij 56, Brij 58, Brij 700 and Tween 20.
- The term "validation" refers to the documented experimental evidence that a method meets the purpose for which it was designed, by comparison or not, with other methods established and known in the state of the art.
-The term "xerogel" refers to the rigid material formed by an interconnected three-dimensional network of an inorganic oxide, usually containing silicon and optionally of a hybrid material that can be synthesized through the in situ formation of the polymer matrix with organic species or inorganic, consisting of submicrometer pores and polymer chains, which generally use one or more alkoxides as suitable monomers, which, mixed with water and other solvents, are capable of hydrolyzing to form intermediate silanol (Si-OH) groups, which condense to produce siloxane bonds (Si-O-Si) After a process of drying under atmospheric pressure, the volatile solvents and volatile by-products are eliminated, which leads to a contraction of the final volume of the polymeric material.
General description of the invention
The object of the present invention is an oxygen (optical) detection device and m edition of its concentration variations in a sample, said device being a low cost polymeric homopollmeric and / or copolymeric microsensor, comprising:
- a support that is a test plate, also called a microplate or microtiter plate, which has a plurality of wells in the form of open receptacles on its upper face and whose number commonly varies between 6, 12, 24, 48, 96, 384, 1536 or 3456 wells, and variable geometry and well capacity; coated with
- an inert and stable polymer matrix comprising a porous structure xerogel, formed by inorganic polymers consisting of a main chain of alternating silicon and oxygen atoms, selected from a three-dimensional network of silica, developed from monomers with alkoxy groups hydrolysable, and / or a polysiloxane, said matrix being deposited in the form of a homogeneous film on the inner wall of the support wells, and being sensitive to oxygen, as defined in the previous section, thanks to
- one or several fluorogenic probes anchored to the matrix, soluble in water and selected from derivatives of metalophthalocyanine and / or metalloporphyrin.
The test plates are also referred to in the scope of the present invention as "well plate", "microtiter plate", "microtiter plate" or "microplate", all these terms referring to a plate that can be manufactured from various rigid materials, typically glass or plastic (polystyrene, polycarbonate,
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
polypropylene, polyvinylchloride, polytetrafluoroethylene, polyethylene, cyclo-olefin polymers, or the like) and having multiple wells, usually 6, 12, 24, 96, 384, 1536 or even 3456, arranged in a rectangular matrix of 2: 3 format, which are routinely used in the laboratory for research applications, drug discovery, test validation, quality controls and monitoring of various biological processes, among others. In these microplates biological and chemical reactions take place between the biological agents that are immobilized on the substrate of the plate, or dispersed in the biological medium, and the analytes produced or consumed in the biological matrix containing said plate. they can have different geometry in their base, and they are arranged in a geometric pattern that simplifies the organization and execution of operations.Therefore, it is important to emphasize that the device object of the present invention can have various shapes, geometries, capacities, manufacturing materials , thicknesses of the deposited sensing film, etc., in order to adapt to the technical specifications necessary in each biochemical study, in which they are used and for which they are specifically designed.
When selecting the microplate, the expert must adapt on the one hand the materials from which it is manufactured (polystyrene, polycarbonate, polypropylene, polyvinylchloride, polyethylene, polytetrafluoroethylene, cyclo-olefin polymers, or similar and in colors suitable for each transparent study , white and, preferably, black), on the other, the geometry and capacity of the well (bottom in the form of C, F, U or V and capacity from 10 microliths to 16 milliliters) and finally the suitable format, depending on the experiment, quantity of biological material, manual, semi-automatic or automatic means, with 6, 12, 24, 48, 96, 384, 1536 and 3456 wells all without the need for the addition of additional reagents, complex manipulations or long incubation periods required in other methodologies , which represents a clear technical advantage over current commercialized systems.
For its part, the fluorogenic molecular probe, also known as "fluoroforo" or "fluorescent signal generator" is the term that commonly refers to a molecule that, based on its fluorescent properties, is capable of converting a particular external chemical signal or stimulus. , in another measurable specific macroscopic signal or response and may have various chemical structures, such as natural products (tryptophan), monocyclic compounds (pyridoxal phosphate), bicyclics (dansyl chloride), tricyclics (fluorescent, rhodamine B. BODIPY) and polycyclic ( Phenantrolins, porphyrins and phthalocyanines). Some of these known probes are soluble in water, but logically said solubility varies and depends to a greater or lesser extent on the functional group, its number and its distribution on the periphery of the molecule.
The (micro) sensors described in their essential form act as high performance miniaturized devices based on optical transduction processes, for which xerogel-shaped polymeric materials are used to configure the film that covers the test plate that contains a main chain of alternating atoms of silicon and oxygen, and may contain other functional organic groups, which is preferably obtained by sol-gel technology, to which an oxygen-sensitive additive such as the fluorogenic molecular probe is added. In this way, the polymeric matrix / oxygen sensitive additive set (probe) is capable of measuring kinetic, ultra fast, fast or slow, of the oxygen concentration present in a biological matrix, such as water, saline solutions, culture media , serum, blood, urine and others defined in the previous section. The fluorogenic probe allows to measure variations in the concentration of the analyte, by converting a certain signal or external chemical stimulus, more specifically the concentration of dissolved oxygen, into another signal
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
or measurable specific macroscopic response, more specifically a fluorescent signal.
The chemically inert and stable homogenous film that covers the plate is conveniently a polymer matrix of a biocompatible material, specifically with a silicon-based structure, since the microstructures of said material can be easily developed using different manufacturing techniques as described. In the present memory. In this way, an ideal situation is produced regarding the fixation of the probes to the film or polymer matrix that covers the plate: strong bonds are formed between the probe and the silicon-derived matrix. Thus, taking into account that the sensitivity of the optical oxygen microsensor depends on the ability for the oxygen to reach the molecule that acts as a transducer, which is determined by the permeability of the encapsulating matrix towards the oxygen, the proposed device allows diffusion and physical contact suitable for the "quenching" of the probe.
The introduction into the polymer matrix of fluorogenic molecular probes, carriers of polar groups through which they are anchored to the functional groups existing in the matrix polymer, created by hydrolysis of each alkoxy group (understood as any organic radical derived from an alcohol due to the loss of hydroxyl hydrogen, as defined in the previous section) present in the monomers used, they showed a high resistance against leaching phenomena by the liquid medium under study, adequate to avoid dissolution in said medium and, consequently , minimize the possible harmful effects of the probe discussed in the section on the state of the art. Thus, the strong bonds that form between the polar groups of the probe and the functional groups of the matrix keep said probe attached to the film in such a way that its cytotoxicity is reduced (since it is proven that these probes are cytotoxic when they are added without anchoring, for example in dissolution to the biological medium inside a plate), and what is not less important: it keeps the concentration of fluorogenic probe constant over time. This immobilization of the probes of interest described here, which, as will be seen below, increases when the polymer matrix is obtained by a sol-gel process, does not decrease its fluorescence, which will be explained by postulating the possible reaction of the peripheral groups (Trytek M. et al., Biomimetic Based Applications, 2011, Prof. Marko Cavrak (Ed.); Trytek M. et al., J. Catalysis, 2012, 286, 193-205), as in the present case the polar groups will be present in the probe, such as carboxylates, with the silanol moieties of the matrix, forming covalent bonds.
The pores of the polymer matrix deposited on the film-shaped plate play an essential role in the process of measurement and interaction between the fluorogenic molecular probe and the medium in which the biological phenomena subject to the study are produced (Garcla-Sanchez, MA et al., J. Non-Crystalline Solids 2009, 355 (2), 120-125) and therefore, it is essential to control many of the variables involved in hydrolysis and condensation during the sol-gel process, to achieve an adequate balance between the non-leaching of the adsorbed probe in the matrix and its accessibility to the reagents. The configuration of the polymer matrix allows the following advantages a) that the oxygen containing the solvent or solvents is diffused through the pores, since these are wide enough to allow its passage, or that of the solution containing it; b) retain the probe by anchoring and prevent its release to the medium; c) that the film formed is homogeneous at the macroscopic level, the pores being interconnected with each other and with the outside; and d) release the solvents in the drying stage (and the bioactive substances during the test when the microsensor understands them, as will be seen later).
5
10
fifteen
twenty
25
30
35
40
Four. Five
These optical fluorescence attenuation sensors show a series of technical advantages over other known methods of measuring dissolved oxygen concentration, among which those that refer to the minimum amount of maintenance work required and the quality of the measured values; in this sense, it should be stressed that the sensitivity of the measurement effect (change in the duration of the luminescence / change in the oxygen concentration) increases as the oxygen concentration decreases; hence, the measurement principle offers an extremely good resolution even in the low measurement range, with wide limits of detection and quantification.
It is also the object of the present invention the manufacturing process of the microsensor described here that, although supported by the generic sol-gel techniques for the preparation of the film or polymeric matrix that covers the plate, includes certain essential modifications that allow to achieve improvements both in the generic properties (chemical and mechanical resistance, prevention of its environmental degradation, adequate pore size, etc.) and in the specific properties for use as microsensors in this type of devices (formation of strong links with the probe, capacity of adsorption and selective and controlled release of added bioactive substances, biocompatibility with the biological matrices subject to study, etc.), said process being characterized by obtaining a polymer matrix of porous structure formed by chains of alternate silicon and oxygen atoms selected between sllice and polysiloxanes from a sol-gel process, which com turn on the following stages:
(a) forming a precursor sol of silicon compounds selected from a polysiloxane and / or silica, from the mixture of at least one monomer consisting of at least one silicon alkoxide of formula
If (OR) n (R ’) 4-n,
where n varies between 2 and 4; R is an alkyl, as defined in the previous section, and each R 'is independently selected from alkyl or alkenyl, as defined in the previous section, together with a proportion comprised between 2 and 30 moles of water per mole of alkoxide; said water being used as a hydrolysis reagent, together with a catalyst promoting the reaction which is an organic acid, an inorganic acid or a base in a molar ratio catalyst: alkoxide between 0.000001: 99.999999 and 0.0001: 99.9999;
(b) add to the precursor sun resulting from step (a) at least one oxygen sensitive additive consisting of the fluorogenic molecular probe described above, in a probe molar ratio: alkoxide between 0.00001: 99.99999 and 0.001: 99.999 ;
(c) deposit a film of the precursor sun obtained in step (b) on the inner surface of the wells of the microplate that homogeneously supports, in a volume between 0.25% and 25% of the volume total well to be coated;
(d) gel the precursor sol resulting from step (c) by polycondensation of the alkoxide at a temperature between 15 ° C and 70 ° C for a time between 1 and 72 hours, resulting in a porous xerogel matrix, the which undergoes
(e) syneresis or aging to evaporate the dissolution medium of the precursor sun, by processes known in the state of the art as curing and drying, for a period of 2 to 5 days.
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
The term "xerogel" refers to the rigid material formed by an interconnected three-dimensional network formed by alternating chains of silicon and oxygen atoms, and, optionally, a hybrid material that can be synthesized through the in situ formation of the matrix polymer with organic or inorganic species, consisting of submicrometer pores and polymer chains, which generally use as suitable monomers one or more alkoxides, which mixed with water and other solvents, established and known in the state of the art, are capable of hydrolyzing to form intermediate silanol groups (Si-OH), which condense to produce siloxane bonds (Si-O-Si). After an atmospheric pressure drying process, liquid volatile solvents and by-products are removed, which leads to a contraction of the final volume of the polymeric material, resulting in a humidity typically less than 1% by weight of the total polymer composition and preferably less than 0.1% by weight.
The sol-gel technique is commonly used for the manufacture of metal and metalloid oxides from a stable suspension in a liquid medium (normally aqueous or hydroalcoholic), of colloidal solid particles between 2 and 200 nanometers, with about 103-109 atoms per particle, which, through a series of chemical reactions, acts as a precursor to a three-dimensional gel formed by interconnecting solid particles in the liquid medium; said gel has the property of creating a film with the desired shape when deposited on the surface to be coated, forming a porous network of discrete particles or polymers, all depending on the reaction conditions (pH, water ratio, temperature, etc.). ). This network is formed by micro-, meso- and macropores, with sizes ranging from 17 to 3000 angstroms. Therefore, not only is the physical accommodation of the metalophthalocyanines and metalloporphyrins used as fluorogenic molecular probes (including their aggregates H and J) possible, which favors the interaction between the analyte and the probe, but also their possible leaching from the matrix sillcea towards the solution in which the experiment is carried out.
The use of a “one-pot” synthesis strategy was shown technically and economically more feasible than the other known sequential processes of formation of strong bonds, such as covalent bonds, making use of the properties that some porphyrins substituted with groups loaded on the periphery of the porphyrin ring, such as carboxylic and sulfonic acids, which give them, on the one hand, a water solubility that allows to obtain aqueous solutions with a concentration of at least 10 micromolar (avoiding the use of organic solvents such as chloroform or N, N-dimethylformamide, incompatible with most plastics) and, on the other, give them an reactivity and electrostatic characteristics capable of anchoring more potently in the porous matrix of the xerogel, avoiding that Desorption mode of the fluorescent molecular probe throughout the biological process studied.
In short, the manufacturing process of devices object of the present invention takes advantage of the properties of the amorphous silica, which combines excellent chemical stability with good mechanical properties and wear resistance, together with a high adsorption capacity on its porous surface of numerous molecules, through electrostatic attractions, Van der Waals forces, hydrogen bonds or covalent bonds with some of the reactive groups that it presents on its surface, which makes it the material of choice for the coating of different types of components, both plastic and metal.
In addition, unlike other substances of a ceramic nature, in which it is very difficult to deposit using physical processes and without using temperatures that exceed the
5
10
fifteen
twenty
25
30
35
40
Four. Five
melting point of the substrates to be coated, sol-gel processes can be used that consist of the use of a chemical route for the deposition of the coating in a homogeneous way and using at all times processing temperatures lower than the melting points of most of the plastics of structural interest and the decomposition temperatures of a large number of organic molecules. To this, it would be necessary to add the advantage of the sol-gel processes, to have under control, depending on the materials and conditions of the manufacturing process, possible modifications of the structural, textural, electronic and morphological properties in the microstructure of the layer deposited, modifying, depending on our technical needs, its inertia or its reactivity, its resistance to thermal shock or its capacity for retention by adsorption on its surface of molecules of interest, etc.
The present invention is also directed to the multiple uses of the described microsensors, which also allow the addition of bioactive substances such as antibiotics, antifungals, antiprotozoals, antineoplasics, mitogens, specific antigens, etc., offering multiple uses in useful optical applications in assays. related to eukaryotic cell proliferation (such as lymphoblastic transformation test) and prokaryotes (such as antibiograms for the selection of antimicrobials in in vitro susceptibility studies), in cell cytotoxicity and senescence and in processes linked to the breathing chain mitochondrial Microsensors are generally suitable for those operations of the biochemical laboratory that require the handling of multiple small volume samples, for example, immunological techniques or cell-based assays, such as those mentioned here among other applications, and may also contain and release controlled bioactive substances.
Brief description of the Fiquras
Figure 1. Detection and measurement in time of the growth of E. coli cells in the wells of the microsensor prepared according to Example 1, with 0.002 ml / well, as a function of the measured oxygen consumption, as described in Example 5, with different
dilutions: (without cells), _ (dilution 1/1024), A (dilution 1/254), ▼ (dilution 1/64), ♦
(1/16 dilution), • (1/4 dilution), ■ (no dilution). The graph shows the average measured value of four wells of the same plate.
Figure 2. Detection and measurement in time of the growth of E. coli cells in the wells of the microsensor prepared according to Example 1, with 0.005 ml / well, as a function of the measured oxygen consumption, as described in Example 6, with different
dilutions: (without cells), _ (dilution 1/1024), A (dilution 1/254), ▼ (dilution 1/64), ♦
(1/16 dilution), • (1/4 dilution), ■ (no dilution). The graph shows the average measured value of four wells of the same plate.
Figure 3. Detection and measurement in time of the growth of E. coli cells in the wells of the microsensor prepared according to Example 1, as a function of the measured oxygen consumption, as described in Example 7, with different dilutions: • ( without cells), ■ (dilution 1/1024), _ (dilution 1/254), _ (dilution 1/64), _ (dilution 1/16), • (dilution 1/4), ■ (no dilution). The graph shows the average measured value of four wells of the same plate.
Figure 4. Detection and measurement in time of the growth of E. coli cells in the wells of the microsensor prepared according to Example 2 with antibiotics as bioactive substances, as a function of the measured oxygen consumption, as described in Example 8: (without cells), ■ (without cells + antibiotics), A (with cells at dilution 1/16), _ (with cells adilution 1/16 + antibiotics). The graph shows the average measured value of four wells of the same plate.
5
10
fifteen
twenty
25
30
35
40
Four. Five
Figure 5. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 9: ( 1: 4 dilution with vehicle), ■ (1: 4 dilution with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 6. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 10: ( 1: 5 dilution with vehicle), ■ (1: 5 dilution with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 7. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the consumption of oxygen measured, as described in Example 11: ( 1: 8 dilution with vehicle), ■ (1: 8 dilution with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 8. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the consumption of oxygen measured, as described in Example 12: ( dilution 1:10 with vehicle), ■ (dilution 1:10 with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 9. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 13: ( dilution 1:20 with vehicle), ■ (dilution 1:20 with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 10. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 14: ( 1:30 dilution with vehicle), ■ (1:30 dilution with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 11. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 15: ( dilution 1:40 with vehicle), ■ (dilution 1:40 with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 12. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 16: ( dilution 1:50 with vehicle), ■ (dilution 1:50 with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
5
10
fifteen
twenty
25
30
35
40
Four. Five
Figure 13. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the consumption of oxygen measured, as described in Example 17: ( 1: 100 dilution with vehicle), ■ (1: 100 dilution with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 14. Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 18: ( 1: 200 dilution with vehicle), ■ (1: 200 dilution with
vehicle + phytohemagglutinin). The graph shows the average measured value of four wells of the same plate.
Figure 15. Detection and measurement in cytotoxicity time in an in vitro sample of THLE-2 humid cells extracted from tissues and deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, such as It is described in Example 19. The graph shows the average measured value and its typical six-well deviation of the same plate.
Figure 16: Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, depending on the consumption of oxygen measured, as described in Example 20. The graph shows the average measurement value and its typical four-well deviation of the same plate, with vehicle and with phytohemagglutinin (PHA), measured with its lid, without the addition of mineral oil.
Figure 17: Detection and measurement in time of lymphoproliferation in an in vitro sample of human blood deposited in the wells of the microsensor prepared according to the present invention, as a function of the measured oxygen consumption, as described in Example 20. The graph shows the average measurement value and its typical four-well deviation of the same plate, with vehicle and with phytohemagglutinin (PHA), measured with its lid and with the addition of 0.1 ml of mineral oil.
Detailed description of the invention
The device described above may have various shapes, geometries, capacities, manufacturing materials, thicknesses of the deposited sensor film, number of layers of sensor and reflective films, etc., in order to adapt to the technical specifications necessary in each biochemical study, in which are used and for which they can be specifically designed.
Each well in which the sensing film is deposited (which is the polymer matrix with the fluorogenic probes anchored, as described above) is constituted by a receptacle whose walls and bottom are suitable and suitable for the in vitro laboratory experiment to perform. Especially and preferably suitable are the wells in which the walls are opaque, black in color and the bottom is flat and transparent and within these, more preferably, the so-called F-shaped ones, which are useful for precise optical measurements, such as fluorimetric and colorimetric determinations and for cell cultures. Another particular object of the invention is the use of flat-bottomed wells with the deposited sensing film, which allow biological substrates to be immobilized or adhered thereto, allowing its use in tests in which biological material anchored or adhered to the material is used. flat bottom or well base, as! as for tests in which the biological material and / or analytes are in solution and / or in suspension.
5
10
fifteen
twenty
25
30
35
40
It should be noted that without departing from the invention, the plate on which the xerogel film is deposited can be chosen from inorganic materials that include, without limitation, glass and quartz, or between organic materials such as for example polymers that may include, but not limited to, polystyrene, polycarbonate, polypropylene, polyvinylchloride, polytetrafluoroethylene, polyethylene, cyclo-olefin polymers, or the like.
Apart from silicon, the polymeric matrix may include in its structure other different atoms such as preferably metals or metalloids selected from the group consisting of aluminum, zirconium, titanium, tin, vanadium, iron and any combination thereof. Normally, in these cases the polymer is formed from alkoxides of these metals and / or metalloids, which are surrounded by several reactive ligands that have a high reactivity towards water, preferably selected from metal alkoxides, such as aluminum and of titanium, and non-metal alkoxides such as alkoxysilanes, with alkoxy groups preferably chosen from methoxy, ethoxy, propoxy, butoxy or other long chain alkoxy groups, more preferably tetramethoxysilane and tetraethoxysilane.
Among the most preferred polysiloxanes to form part of the matrix polymer structure, is the finished hydroxy polydimethylsiloxane of formula I
image 1
which is a compound also formed by chains of alternating atoms of silicon and oxygen but with radicals of an organic nature, where n describes the degree of oligomerization, which indicates the number of silicon units per oligomer molecule; In this way, stable inert matrices of a xerogel are obtained that are hybrid in nature (organic-inorganic) in terms of their radicals.
In short, there are three types of polymer matrix that the sensor device can contain and that are of special interest: a totally inorganic polymer matrix (of “pure” silanes as precursors), which forms a “pure silicon dioxide” film ”(That is, it is a polymer matrix film whose structure is composed of 100% silica); a second polymeric matrix, of an organic-inorganic hybrid nature, whose structure, of the type known as hybrid materials, contains in a combined form silica and polysiloxanes, as is preferably the hydroxy-terminated polydimethylsiloxane of formula I and which may appear most preferably between 10% and 99% of the total matrix. And since it is possible that the polysiloxane, such as the hydroxy-terminated polydimethylsiloxane of formula I appears up to 100% of the total weight of the polymer matrix, then this matrix can also be of "pure" polysiloxane, that is, a porous matrix formed by chains of alternating silicon and oxygen atoms but with radicals of an organic nature, with organic groups, also attached to the metal or metalloid atoms, preferably to the silicon atoms that comprise said chain of alternate silicon atoms and of oxygen.
On the other hand, when the fluorogenic molecular probe is a metalated porphyrin, it can be selected from the group consisting of hematoporphyrin, coproporphyrin, uroporphyrin, chlorophyllin, sulfonatoporphyrins and all its positional isomers; hydroxyphenyl porphyrins and all their positional isomers; carboxyphenyl porphyrins and all its isomers
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
positional; sulfonatophenyl porphyrins and all their positional isomers; and phosphatophenyl porphyrins and all their positional isomers; and any combination thereof. When said probe is a metaphthalocyanine, it can be selected from the group consisting of sulfonatophthalocyanines, carboxyphthalocyanines and any combination thereof. In the most preferred case, the fluorogenic probe is at least one metalloporphyrin comprising a central nitrogen cycle containing a metal atom or ion preferably selected from the group consisting of palladium, platinum and ruthenium, substituted at its periphery with one, two, three or four groups of the carboxylate type. More preferably, the probe is 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (ll). In the most preferred case of all, the 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (ll) probe is present in the polymer matrix in a proportion comprised between 10% and 100% by weight of the total mixture of metalloporphyrins and / or metalphthalocyanines present in the device; that is, the 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (ll) probe can be present in at least 10% by weight of the total probe present, combined with other probes, or it can reach Be the only one present.
Additionally, in a very preferred case the polymers that constitute the matrix or film of the microsensor can contain one or more bioactive substances adsorbed on the porous surface of the xerogel that, by a process of gradual desorption and leaching, is slowly released from the xerogel that it contains it towards the biological matrix subject of the study. These bioactive substances may be antibiotics, antifungals, antiprotozoals, antineoplasics, mitogens or specific antigens, etc., or any combination thereof so that the plates or microplates may be appropriate for all types of in vitro and ex-vivo chemosensitivity related studies. with cell proliferation of eukaryotes (for example, the lymphoblastic transformation test) and prokaryotes (for example, antibiograms for the selection of antimicrobials in in vitro susceptibility studies), cytotoxicity and mitochondrial breathing chain, both point final, as in real time, by using a plate / microplate reader capable of measuring the fluorescence or fluorescence in resolved time, both in lower and upper reading mode, adapting, on the one hand, the geometry and capacity of the well and, on the other, the format in 6, 12, 24, 48 6 96 wells (useful for standard analysis of immunology and cell cultures), or in mato of 384, 1536 and 3456 wells (necessary for high capacity miniaturized studies), all without the need for the addition of additional reagents, complex manipulations or long incubation periods required in other methodologies, which implies a clear technical advantage over the current commercialized systems.
Since a bioactive substance, such as a drug dissolved, embedded or adsorbed on the pores of a solid polymer, tends to diffuse towards its surface, releasing itself continuously in the surrounding liquid medium, the proposed device has several advantages from the Bioavailability point of view (Saez, V., Hernaez E. and Sanz-Angulo, L .; Iberoamerican Journal of Polimeros, 2004, 5 (1), 55-70), as they would be: the protection of bioactive substances susceptible to degradation in dissolution, greater efficiency in the use of the bioactive substance and, therefore, with a lower cost, which is important when it comes to a high-price active agent, etc. The release kinetics of the bioactive substance is determined by its physical properties, particularly by its molecular weight and its partition coefficient between the polymeric matrix and the aqueous biological medium contained in the well, along with other particular characteristics such as the physical properties of the matrix and its geometry and, ultimately, by the amount of drug incorporated. The amount of bioactive substance released and the rate of release from the polymeric film is proportional to the square root of time (Roseman, T. and Higuchi, WI, J. Pharm. Sci., 1970, 59, 353) and its velocity of diffusion depends on various factors such as the surface area and the
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
membrane density, as well as the solubility and diffusion coefficient of the bioactive substance, although, if there is enough drug to maintain an internal concentration greater than that of the external medium, the rate of diffusion of the drug through the membrane is keeps constant (Saez, V., Hernaez E. and Sanz-Angulo, L .; Ibero-American Magazine of Pollmeros, 2002, 3 (3), 1-20).
Its applications and advantages over the sensors currently known in the state of the art for the monitoring of metabolic studies related to the proliferation and chemosensitivity of eukaryotes and prokaryotes, with cytotoxicity and cellular senescence and with the mitochondrial breathing chain, are based in the different affinity for the matrix, between the fluorescent molecular probe (a metalophthalocyanine and / or a metalloporphyrin substituted with at least one polar group of the phenol, carboxylate, sulfonate and / or phosphate type, including all its positional isomers) and the bioactive substance to be used in the assay (preferably antibiotics, antifungals, antiprotozoals, antineoplasics, mitogens, specific antigens and their possible combinations).
Preferably, the amount of bioactive substance in the microsensor is between 0.001% and 50% by weight with respect to the total weight of the deposited film of xerogel.
In a particular embodiment of the invention, the device comprises in its composition at least a second film or polymer matrix containing an inert material suitable to reflect and disperse the light emitted by the probe. This inert material is preferably a metal oxide such as tin dioxide, zinc oxide, titanium dioxide or mixtures thereof, more preferably titanium dioxide, to take advantage of the properties of these oxides to reflect and scatter ("scattering") the light . This second film, placed below the sensor film (that is, deposited first directly on the test plate, and before the first matrix that is deposited in the form of a layer later), acts as a light reflecting layer. emitted by the probe, so that, when said film that contains the chosen metal oxide acts, the intensity of the luminescence emitted by the main sensor film (which contains the fluorogenic probe) increases, upon reading the fluorescent emission higher . Similarly, in the lower reading devices, when the inert layer of metal oxide is placed above the sensor xerogel, it can be achieved that the metal oxide layer reflects the emission of radiant energy, preventing the loss of light, resulting in light loss an increase in the sensitivity of the device.
As for the sol-gel process for preparing the patented devices, said process is well known to any person skilled in the art, who can carry it out using only routine experimentation methods, using at least one silicon alkoxide in the presence of water as a hydrolysis reagent and suitable catalysts, preferably acids. The amount of water can vary from 1 to 30 moles per mole of alkoxy, preferably between 2 and 4 moles of water per mole of alkoxide, and the amount of catalyst typically added to the reaction mixture ranges from 1 to 0.0000001 moles per mole of alkoxide, preferably between 0.000001 and 0.0001 moles of catalyst per mole of alkoxide, all at a temperature typically between 15 ° C to 70 ° C, preferably between 20 ° C and 40 ° C, during a reaction period that can vary between 1 hour and 4 days, until the reaction is complete. In a preferred embodiment, the mixture of alkoxide and water is carried out in a dissolution medium selected from an alcohol, a hexane and a cyclic ether to carry out the reaction in a homogeneous medium; more preferably, an additional 2 to 20 moles of this medium are still used as cosolvent. During step a) of mixing the components, a drying and retarding agent, preferably n-butanol, can optionally be added in an alkoxide ratio / dissolution medium molar
5
10
fifteen
twenty
25
30
35
40
Four. Five
(preferably water) / butanol of 1 / 1-30 /> 4. After having incorporated the fluorogenic probes, in step b), the suspension (sol) is distributed by deposition in the wells of the microplate in amounts that may vary depending on the volume of the well and the sensing film to be deposited, preferably volumes of the precursor solution (sol) ranging from 0.25% and 25% of the total volume of the well to be coated (more preferably between 0.5% and 20%) and, more preferably, from 0.001 milliliter, to 0.020 milliliters in each well of the 96-well plates (and maintaining this proportion depending on the number of wells that make up the plate), after which there is an evolution of the solid network and the drying of the hydroalcoholic solvent, together with all volatiles produced in the process, with an appropriate speed to achieve a gel, in the form of a solid or semi-solid with an ideal porosity that allows the subsequent release of the bioactive substance for the purposes for which prepares, preferably between 1 and 20 days and more preferably between 3 and 7 days.
The sol-gel process begins with the mixture of precursors, catalysts and reaction media, in solution and in its early stages they form colloidal dispersions (sol) that subsequently gel. This process depends on the speed of formation of the components acting as precursors of the gel and is based on the hydrolysis of the previously defined alkoxides contained in the precursor monomer and the polycondensation of the products of said hydrolysis.
The hydrolysis of the alkoxide or of the mixture of several alkoxides is usually carried out in a solution of water with a second cosolvent, which allows generating a polymeric structure, by means of a simple hydrolysis-polycondensation mechanism, preferably in the presence of acids, both inorganic (which are more preferred, such as hydrochloric acid, nitric acid, sulfuric acid, etc.) and organic (acetic acid, propionic acid, maleic acid, oxalic acid, citric acid, etc.) or bases, these acting as catalysts for said reaction, and that, by way of illustration and never limiting the scope of the present invention, is outlined below:
Yes (OR) 4 + H2O ___________ ^ HO-Yes (OR) 3 + HOUR
HO-Si (OR) 3 + H2O _______ ^ (HO) 2-Si (OR) 2 + HOUR
(HO) 2-Si (OR) 2 + H2O ______ (HO) 3-Si-OR + HOR
(HO) 3-Si-OR + H2O ______> Yes (OH) 4 + HOUR
Yes (OH) 4 + Yes (OH) 4 _____________> (HO) 3-Si-O-Si- (OH) 3 + H2O
In the case of carrying out the hydrolysis of the mixture of two or more alkoxides, a double alkoxide may be formed before the hydrolysis occurs, which generally results in a more stable compound than the alkoxides separately and, by therefore, longer hydrolysis times should be observed. Similarly, one or more of the alkoxy radicals (understood as any organic radical derived from an alcohol by loss of hydroxylic hydrogen), represented in the above scheme as -OR, where R is an alkyl group as defined above, may be substituted by another type of radical, preferably a substituted or unbranched substituted alkoxy, a substituted or unsubstituted aryl, a substituted or not substituted alkyl, or a substituted or not substituted alkenyl.
The hydrolysis-condensation reaction between the alkoxides in stage a) of mixing leads to a homogeneous solution, which leads, on a molecular scale, to a homogeneous distribution of several particles and therefore a final homogenous stoichiometric distribution.
5
10
fifteen
twenty
25
30
35
40
Four. Five
Consequently, to obtain a polycrystalline oxide, a slow polymerization of an alkoxide solution with traces of water dissolved in a cosolvent, such as an alcohol, is necessary, which means a slow hydrolysis, so that the gel is formed by a reaction of Two stages: one of hydrolysis and a subsequent of polycondensation, taking advantage of the fact that in general the alkoxides used are very sensitive to moisture, so that the hydrolysis for the formation of the gel can be carried out using a cosolvent, which is selected from a alcohol (preferably methanol, ethanol, propanol), a hexane (preferably cyclohexane) or a cyclic ether that is preferably tetrahydrofuran or dioxane, in a proportion that varies between 1 and 20 moles of solvent per mole of alkoxide, which are added to the moles of water.
As for the specific stage of application of the sol-gel solution on the test plate of step (c), it can be chosen at will between spin coating (dip-coating), dip coating (dip dip coating) ), or any other suitable to the characteristics of the materials used and more preferably by centrifugation, at a rotation speed of between 300 and 2000 rpm, for a time that can vary from 1 minute to 1 hour, other speeds and times may be used centrifugation to control the quality and thickness of the desired layer.
The conditions of the gelation or gelation process of the previously formed sun influence the structure, volume and gel pore size, so these properties depend on factors such as water / alkoxide ratio, pH, concentration and chemical nature of alkoxides, the most suitable general conditions being those indicated in the previous section and as more specifically illustrated in the examples, by way of illustration and never limiting the scope of the invention.
The process of formation of gels and their deposition on the microplate described in an essential way in the previous section of the present report thus entails! the following stages:
a) Mixing stage: it consists of the incorporation of the chosen monomers, whose mixing will give rise to the structure of the polymer matrix, formed by chains of alternate silicon and oxygen atoms, on the mixture of solvents in which they will be carried Perform all subsequent steps and containing, at least, water, which acts as a reagent and as a solvent, together with a catalyst, acid or base, and more preferably other cosolvents, as defined above, creating a reaction medium. homogeneous.
In a particular embodiment, in addition to the silicon alkoxides mentioned hereinabove, of formula Si (OR) nR’4-n, where n varies between 2 and 4; R is an alkyl, as defined in the preceding sections, and each R 'is independently selected from alkyl or alkenyl, as defined in the preceding sections, other metal alkoxides and / or metalloids may be added to the mixture, which are formula alkoxides
M (OR) n
and any of its binary and ternary mixtures with stoichiometers in a molar ratio between 0.1 and 0.9 of each of the alkoxides of the mixture; where M is selected from the group consisting of aluminum, zirconium, titanium, tin, vanadium and iron; (RO-) represents an alkoxy group, understood as any organic radical derived from an alcohol due to the loss of hydroxyl hydrogen, as defined in the previous sections, where R is more preferably an alkyl group and more preferably even methyl, ethyl, propyl, butyl or other long chain alkyls; and n varies between 2 and 4. In the most preferred cases, these metal alkoxides and / or metalloids
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
they are selected from the group consisting of aluminum, titanium and silicon alkoxides, these alkoxyls being more preferably tetramethoxysilane or tetraethoxysilane.
Preferably, the alkoxides are selected from the group consisting of: tetramethoxysilane, tetraethoxysilane, octyltriethoxysilane, ethyltrimethoxysilane, hexyltriethoxysilane, hexyltrimethoxysilane, cyclohexyltrimethoxysilane, phenyltriethoxysilane, phenyltrimethoxysilane, benzyltriethoxysilane, dimethyldimethoxysilane, chloromethyltriethoxysilane, tetra (1,1,1,3,3,3- hexafluoroisopropoxy) silane, polyfluorooctyltriethoxysilane, tetra-n-propoxysilane, tetra-n-butoxysilane, tetrakis (2-methoxyethoxy) silane, methyltriethoxysilane, dodecyltriethoxysilane, dodecyltrimethoxysilane, hexadecyltrimethoxysilane, octadecyltriethoxysilane,
octadecyltrimethoxysilane, methyltrimethoxysilane, (3-aminopropyl) triethoxysilane, (3- aminopropyl) trimethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, triethoxyvinylsilane, [3- (2- aminoethylamino) propll] trimethoxysilane, (3-chloropropyl) trimethoxysilane, (3-
bromopropyl) trimethoxysilane, 3-chloropropyldimethoxymethylsilane, 3-chloropropyltriethoxysilane, 2-cyanoethyltriethoxysilane, (3-mercaptopropyl) triethoxysilane, (3-mercaptopropyl) trimethoxysilane, cyclohexyl (dimethoxy) methylsilane, diethoxydimethylsilane, diethoxydiphenylsilane, diethoxy (3- glycidyloxypropyl) methylsilane, diethoxymethylvi nilsilane, dimethoxymethylphenylsilane, trimethoxy (2- phenylethyl) silane, N- [3- (trimethoxysilyl) propyl] aniline, tetraethyl titanate, tetraisopropyl titanate and tetrabutyl titanate and its possible binary and ternary mixtures, with variable stoichiometry, in a molar ratio from 0.1 to 0.9 of each of its components and more preferably binary mixtures with 1: 1 stoichiometry, which may be dissolved in the medium of solution which is water and a second cosolvent, of an organic nature, preferably selected from the group consisting of methanol, ethanol, propanol, cyclohexane, tetrahydrofuran, dioxane or mixtures thereof, to form the colloidal solutions or precursor soles.
The previous monomers useful in the present invention have one or more alkoxy groups of the formula -OR, some of their hydrogen atoms can be substituted by another type of radical, as defined in the previous sections, preferably when the group - is not substituted. OR represents methoxy and ethoxy. It is preferred that the monomers have two or more alkoxy groups, in order to provide adequate cross-linking to the resulting xerogel, effectively increasing the control of the release of the bioactive substances used in the present invention; In addition, mixtures of alkoxy monomers containing different numbers of alkoxy groups are useful in the present invention, since, in this way, variable rates of release of bioactive substances can be obtained.
In a particular case, 100% of alkoxides that are added to the polymer obtaining mixture are alkoxides of formula Si (OR) n (R ') 4-n, such that they are all precursors of inorganic polymers consisting of a main chain of alternate silicon and oxygen atoms, giving rise to a three-dimensional network of alternate silicon and oxygen atoms. In another particular case, as explained in defining the structure and composition of the device, the polysiloxanes are added to the mixture in an amount of 100% of the mixture, preferably the finished hydroxy polydimethylsiloxane. Between one particular case and another, there is one in which the chain of alternating atoms of silicon and oxygen is obtained by the mixture of alkoxides and aggregates of the polysiloxane segments, in a proportion between 10% and 99 % of polysiloxanes, preferably of the finished hydroxy polydimethylsiloxane of formula I, with respect to the total weight of the deposited polymer matrix film. The hybrid combination improves the physical, rheological or biocompatibility properties of the coatings obtained. A more preferred process of manufacture of the device in question is still one in which in step a) a polysixolane consisting of finished hydroxy polydimethylsiloxane is added to the solution, in which case at least one non-ionic surfactant is additionally added ,
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
as defined above. This process, and the device obtained from it, is of great importance and is an advantage in the field of application of the device, thanks to the adherence of the biological sample under study to the surface of the well covered by the matrix. polymeric, specifically in the case of media formed by cells, this quality of the device being very advantageous in studies with adherent cells, which are suitable for most types of cells, including primary cultures.
b) add to the precursor sun resulting from step (a) at least one oxygen sensitive additive consisting of the fluorogenic molecular probe described above, in a probe molar ratio: alkoxide between 0.00001: 99.99999 and 0.001: 99.999.
c) Stage of distribution by deposition: it consists of the distribution of allcuotas of the sun obtained in the previous stage in a suitable mold, which is the test plate, taking advantage of its liquid texture of low viscosity, once mixed in stage (b) with the chosen fluorogenic probe (and optional bioactive substances, in the most preferred case), in a volume between 0.25% and 25% of the total volume of the well to be coated, solidifying the gel from the colloidal solution (sun), resulting from stages a) and b). The mold in which the sun is deposited is the bottom of each of the wells of the microplate chosen according to the test to which it is going to be subjected, allowing, once dry, it adapts perfectly to the shape of the well that contains The method of application of the solution can be chosen at will, between immersion coating, jet printing or centrifugation at a rotation speed of between 300 and 2000 rpm for 1 to 3 minutes, with other coating speeds and times being used by centrifugation to control the quality and thickness of the desired layer.
d) Gelation or gelation stage: it consists in the production of colloidal particles that join together to form a three-dimensional structure, as polycondensation of silicon alkoxide and / or segments of polysixolane, preferably polydimethylsiloxane, occurs finished hydroxy. In this process, the catalyst, including both inorganic acids (hydrochloric acid, nitric acid, sulfuric acid, etc.) and organic acids (acetic acid, propionic acid, maleic acid, oxalic acid, citric acid, etc.) or bases plays a role of extreme importance, due to its direct influence on the polycondensation speed and, therefore, the size of the particle and the number of links.
e) Syneresis or aging stage: encompasses the evolution time of the solid network, still immersed in the solvent, used both for the solubilization of the alkoxides, and of the additives useful in the present invention (fluorogenic probe, bioactive substances or precursors organic, in the case of obtaining organic-inorganic hybrid gels), during which, on the one hand, the polymerization of free hydroxyl groups continues, so that the network connectivity increases and, on the other, a reduction is observed irreversible gel volume (syneresis), due to the progressive expulsion of the liquid stored in the pores. Finally, at the macroscopic level, a strengthening of the gel and a progressive drying is observed. At this stage there is a no less risk of gel fracture, due to the tensions caused by the capillary forces associated with the liquid-vapor interfaces. Therefore, evaporation of the solvent must be carried out very slowly, combining the good results obtained through this practice, with the technical inconveniences posed by excessively long drying times (from weeks to months, to form a dry monolithic xerogel). Therefore, in the scope of the present invention, it is optionally proposed to use at least one chemical additive that modifies the surface tension of the solvent encompassed in the pores, allowing its fastest evaporation; it can be both acidic and acidic
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
oxalic, or basic, such as formamide, which favors the nucleation and growth of aggregates, increasing the microhardness of the wet gel and the corresponding pore size of the dry gel, while helping to maintain a homogeneous distribution of pore size. Finally, during this stage there is a stabilization which consists in the reduction of the concentration of silanol groups (-SiOH) on the surface of the xerogel by forming strong bonds with other silanoles or reactive groups present in the medium, such as phenols, carboxylic, sulfonic and / or phosphonic acids present in the phthalocyanine and porphyrin structure previously added.
In a preferred embodiment, the method comprises the deposition of a second film or polymeric matrix as described when defining the detailed embodiments of the microsensor. Preferably, this second layer is deposited on the inner surface of the wells that make up the test plate that serves as support, before depositing the first polymer matrix layer in step (c). This second film, of composition described above, can be deposited well by adding the metal oxide to the mixture of alkoxides, during the sol-gel process of step (a), by forming a hybrid or mixed xerogel, preferably from of silicon alkoxides, such as tetraethoxysilane and titanium, such as tetrabutyl titanate or by dispersion of the metal oxide in a silicone, preferably of commercial origin such as Loctite® 5091 ™ Nuva-Sil®.
Regardless of the number of polymeric films that are deposited in the microplate in question, a preferred and particular embodiment of the manufacturing method comprises adding, preferably in step (b), one or more bioactive substances such as antibiotics, antifungals, antiprotozoals, antineoplasics, specific mitogens or antigens, etc., or in a combination thereof at the same stage b) in which the fluorogenic probes are added to the precursor sun that will form the polymer matrix. In the case of using a suitable combination of more than one bioactive substance, said substances are mixed in any proportion, as it is in a weight ratio between the two that can vary between 0.1: 99.9 to 99.9: 0, 1, so that the resulting sensor plates are directly appropriate for all types of chemosensitivity studies related to cell proliferation of eukaryotes (for example, the lymphoblastic transformation test) and prokaryotes (for example, antibiograms for the selection of antimicrobials in in vitro susceptibility studies), cytotoxicity and mitochondrial breathing chain, through the use of a plate reader capable of measuring fluorescence or fluorescence in resolved time, both in lower and upper reading mode, all without the need for addition of supplementary reagents, complex manipulations or long incubation periods required in other methodologies, which represents a clear technical advantage over the actions The same commercialized systems.
The amount of bioactive substance useful in the present invention is between 0.001% and 50% by weight, based on the total weight of the polymeric film deposited on the well, preferably an amount of bioactive substance between 0.01 and 30% by weight.
Another particular object of the invention is the use of the device object of interest for the direct in vitro measurement of kinetic, ultra-fast, fast or slow, of the oxygen concentration present in any biological medium, as defined in previous sections and of Preferred mode in biological fluids, including, but not limited to: serum, plasma, blood, urine, saliva, sweat, milk, vaginal exudate, semen, pericardial fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, pleural fluid and peritoneal fluid, preferably urine and blood, complete or, more preferably diluted with water, physiological serum, calf serum, fetal bovine serum,
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
horse serum, human serum, Hank's balanced saline solution, RPMI 1640, Eagle's basal medium (BME), Eagle's minimum essential medium (MEM), Dulbeco-modified MEM medium (DMEM), Iscove modification of DMEM medium (IMDM) ), McCoy 5A, half L-15 of Leibovitz, half F-10 of Ham, half F-12 of Ham, half 199 and any of its variants, known in the state of the art.
Particularly and preferably, the laboratory use of the resulting device, as set forth herein, is directed to research, detection and monitoring in vitro and ex vivo in assays with enzymes and enzymatic oxygen-dependent systems; or cultures of adherent cells or cell cultures in suspension of cellular organs and animal tissues, genetically modified or not, or of eukaryotic organisms formed by a single cell, or by a colony of cells equal to each other, without differentiation of tissues and that they live in aqueous media or in internal Kids of higher organisms, such as protozoa, which include, but are not limited to the genera Giardia, Enteromonas, Nosema, Naegleria, Trichomonas, Trypanosoma, Leishmania, Toxoplasma, Sarcocystis, Plasmodium, Balantidium,
Acanthamoeba, Entamoeba, or other genera of the same orders, and complete aquatic organisms, such as algae (eg those of the Tetraselmis genus), invertebrates (eg those of the genus Artemia or those of the genus Caenorhabditis), or fish (eg those of the genus Danio). The use of the microsensor in research includes, but is not limited exclusively to in vitro and ex vivo research fields in stimulation of lymphoblastic transformation with nonspecific mitogens; the cellular immune response caused by immunological, pharmacological, surgical, nutritional, hormonal, environmental treatments, etc., including the cellular immune response to vaccines or immunogens, with or without adjuvants, in development or already existing in the market; the immunological profile and the state of the subject's immune system for the routine control of risk groups; the suspicion of immunomediated diseases, including immunosenescence; immunodeficiencies that may be suspected against recurrent or refractory infectious conditions, including, but not limited to infectious diseases, such as those with latent forms at high risk of progressing to the disease; the deficit of the immunological capacity in autoimmune pathological states, such as degenerative myelopathy, or genetic, such as agammaglobulinemia; the consequences of immunosuppressive therapies or immune enhancement therapy, of cellular hypersensitivity reactions reacting against environmental allergens or antigens; the response to mitogens, including disorders that show a variable blastogenic response to selective B cell mitogens; mucocutaneous candidiasis and other chronic fungal infections; beryllium-induced blastogenesis, detection of previous exposure to various pathogens, for example malaria, hepatitis or other infections including Mycoplasma pneumoniae, Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum; periodontal diseases and certain viral infections, for example dengue or lymphoproliferative diseases associated with the Epstein-Barr virus; the detection of previous exposure to the corresponding antigen in individuals without response to antibodies; the presence of antigens responsible for allergies that also stimulate specific lymphoproliferative reactions in vitro (immediate allergic reactions, contact reactions and drug hypersensitivity); allergic states; immunological reactions to pathogens, allergens and autoantigens; the autoimmune conditions in which antigens specifically stimulate lymphoblastic transformation only in patients suffering from this state; the states of genetic and acquired immunodeficiency, with a depressed lymphocyte function (even in the absence of lymphocytopenia); the therapeutic effects, including, but not limited to, those resulting from antibiotic, antiprotozoal, antifungal, antineoplastic, immunostimulatory or immunosuppressive therapies, as well as methods to optimize drugs,
5
10
fifteen
twenty
25
30
35
40
Four. Five
fifty
to identify effective doses of chemotherapy and methods of phenotypic characterization; monitoring the degree of deterioration / improvement of lymphocyte reactivity in cancer patients; cellular behavior in mitochondrial disorders; monitoring the degree of deterioration and / or improvement of lymphocyte reactivity in cancer patients; cellular behavior in mitochondrial disorders; the in vitro study of enzymatic reactions of oxygen consumption or production and of models of diseases associated with oxidative stress; the functional link between neurological disorders (Alzheimer's, schizophrenia, autism, ALS, etc.) and the mechanisms of bioenergetic regulation of peripheral cells; diabetic complications and inflammation; manipulation techniques for reproductive, regenerative and therapeutic cell therapy; the effect of formulations containing nanoparticles and other therapeutic systems on the basic immunological function of animal lymphocytes and other applications for research in the food, dairy and microalgae industries, or in vitro and ex vivo uses in the toxicological, diagnostic, prognostic and Therapeutic of any of the aforementioned applications, taking into account that although, by itself, the microsensor of the present invention does not generate the results of an in vitro diagnostic method, for the detection of diseases, conditions or infections, it can be useful as a key component of a diagnostic system, when used in combination with other procedures, that validate the device and determine its suitability, optimization and standardization, with other materials and with other systems that can be designed by a person skilled in the art.
It should be borne in mind that the present report, when contemplating these uses of the device, and that they are always carried out in vitro and / or ex vivo, also covers the corresponding methods to carry out the aforementioned tasks (in vitro lymphoblastic transformation monitoring with non-specific mitogens, of the possible optimization of substances with therapeutic potential and the identification of effective doses of chemotherapy and methods of phenotypic characterization, etc.) using the device described here, by means of miniaturized microplate type systems, which can have 6 formats, 12, 24, 48, 96, 384.1536 or 3456 wells and variable well geometry and capacity, which allows reducing the volume of reagents and medium to be used in the tests, as well as studying in a manageable format the effect of a composed on a large number of isolates or that of a series of compounds on a given isolate; said methods generally consisting of depositing a sample of one or more biological matrices, which refer to the set consisting of a medium and biological sample, of prokaryotic or eukaryotic origin (eg, bacteria, protozoa, plants, insects, birds, fish , reptiles, mammals, genetically modified or not, etc.) as defined above, in one or several wells of the device, and thus measuring by means of fluorescence and fluorescence specimens in fixed time (TRF), real-time variations in the concentration of oxygen produced in said biological matrix throughout the experiment.
In general, the use of the device described herein is carried out on any commercially available standard platform of time-resolved fluorescence (TRF), commonly used in biochemical, medical, chemical or research analysis and capable of accepting any type of standard microplates (including 96, 384 well or higher formats),
In this way, the reading of each well can be taken several times and involves exciting the fluorescent marker with a short pulse of light, then waiting a certain time (from 50 to 200 microseconds) after the excitation and before measuring the signal fluorescent remaining long life. In this way, the short-lived fluorescent background signals and the radiation dispersion are eliminated.
5
10
fifteen
twenty
25
30
35
40
Four. Five
For a better understanding of this descriptive report, the following are illustrative and never limited to the impact of the invention, some examples of the essence of the same.
Examples
Example 1: fabrication of a sensor film and deposition on the microplate to obtain the oxygen detection device and measurement of the variation in its concentrations object of the present invention
32 miiimoies (mmoies) of tetraethoxysian and 32 mmoies of octiitriethoxysian are mixed in 12.5 miiiiiters (mi) of etanoi absoiuto and 4 mi (0.0004 mmoies) of 0.1 N ciorhldrico acid are added to that dissociation.
The mixture is stirred vigorously for 1 hour and 34 ml of ethane are added, stirring for an additional 30 minutes. After that time, 6 ml of a 0.01 minute (mM) hydroaicohoic dissociation (100 etanoi: 10 v / v water) of ia are added dropwise
5,10,15,20-tetrakis (4-carboxyphene) porphyrin Pt (II) (0.06 mmol).
The resuitant dissociation is pipetted and 0.010 ml / well is deposited on a black piaca piaca, with 96 wells and transparent piano bottom, to allow a good visualization of the content of the well, and, therefore, the possibility of measurements by transmission of light , next to a "crosstaik" reduced between wells.
Finally, the resuscitating piaca is allowed to geiify, dry and settle at room temperature for 5 to 7 days.
Example 2: manufacture of a sensor film with antibiotics and deposition on the microplate according to a particular embodiment of the present invention
The piacas are manufactured following a similar procedure described in Example 1, in which in some wells the sensor dissociation is deposited as described in the example 1, which are used as a bianco, together with other wells containing a dissociation in the that ios 6 ml of 0.01 mM hydroaicohoic dissociation (100 etanoi: 10 water v / v) containing 0.06 mmoies of ia 5,10,15,20-tetrakis (4-carboxyphene) porphyrin Pt (II), are formed from 0.4 ml of 10mM probe in dimethisuifoxide, diiuide with the corresponding volume of water in which peniciiin and streptomycin have been dissolved in an adequate amount to contain the final concentrations in the sensorinectomy of 100 U / ml and 0.1 mg / mi, respectively and tested with etanoi to obtain 1/10 of the probe base dissociation. Finally, the piaca generated with both types of wells (peilcuia sensora and peilcuia sensora with antibiotics) is allowed to geiify, dry and stabilize at room temperature for 5 to 7 days.
In this way, it is expected that when bacteria are planted in a bioiogic medium suitable for growth, in the piaca of a non-antibiotic piaca (manufactured according to example 1) an iogarmalmic bacterial growth will take place, which will deplete the oxygen depletion in the middle; as the level of oxygen decreases further, bacterial growth could be hindered and partial pressure of oxygen can be imimitant, until partial pressure of oxygen reaches almost zero, point at which ceiiar growth can cease and said growth pattern it will be reflected in a sigmoid curve. On the contrary, adding an antibiotic agent that covers the restoring potion of the previous process will suppress bacterial growth, oxygen will not be consumed and the emission measurements of fiuorescence will give a constant line, next to the wells with a medium Bioiogenic without bacteria.
5
10
fifteen
twenty
25
30
35
40
Four. Five
Example 3: fabrication of a sensor film with a mitogen and deposition on the microplate
The plates are manufactured following a procedure similar to that described in Example 1, in which in some wells the sensor solution is deposited as described in example 1, which are used as blank, together with other wells containing a solution in the that the 6 ml of 0.01 mM hydroalcoholic solution (100 ethanol: 10 water v / v) containing 0.06 mmol of 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II), are formed from 0.4 ml of 10 mM probe in dimethylsulfoxide, diluted with the corresponding volume of water in which phytohemagglutinin has been dissolved in adequate quantity to contain the final concentrations in the 0.02 mg / ml sensor film and completed with ethanol to obtain 1/10 of the probe base solution. Finally, the plate generated with both types of wells (sensor film and mitogen sensor film) is allowed to gel, dry and stabilize at room temperature for 5 to 7 days.
Thus, it is expected that when mononucleated cells of peripheral blood are sown or biological samples containing them are added, in the wells of a plate without mitogen (manufactured according to example 1), cell growth will not be observed, it will not be consumed Oxygen and fluorescence emission measurements will give a constant line, close to the value of the wells with a biological medium without peripheral blood mononuclear cells. On the contrary, adding a suitable mitogen to the xerogel that covers the well resulting from the previous process will produce a logarithmic cell growth, which will deplete the dissolved oxygen in the medium; as the oxygen level decreases further, cell growth could be hindered and the partial pressure of oxygen may become limiting, until the partial pressure of oxygen reaches almost zero, at which point cell growth can cease and said pattern of Growth will be reflected in a sigmoid curve.
Example 4: manufacture of a hybrid sensor film and deposition on the microplate
On a solution of 44 mmol (10 ml) of tetraethoxysilane in 40 ml of absolute ethanol and 37 ml of water, 0.4 ml (0.38 g) of finished hydroxyl poly (dimethylsiloxane) and 0.02 are added dropwise ml of polyoxyethylene (23) lauryl ether (Brij-35).
The mixture is stirred vigorously for 1 hour and 4 ml (0.06 mmol) of a 0.01 mM hydroalcoholic solution (100 ethanol: 10 water v / v) of water is added dropwise
5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II).
The resulting solution is sonicated for 30 minutes, in an ultrasonic bath, after which it is stirred for an additional 30 minutes and deposited with a 0.04ml / well pipette on a black polystyrene plate, with 96 wells and bottom transparent plane. Finally, the resulting plate is allowed to gel, dry and stabilize at room temperature for a period of 2 days.
Example 5: E. Coli detection protocol with 0.002 ml of sensor / well film
To each well of a microplate obtained according to example 1, in which 0.002 ml of precursor solution of the sensor film has been deposited, 0.1 ml of a suspension of standard culture medium (Difco Laboratories) is added, to which a bacterial colony of E. coli has been transferred. Thus, the resulting plate, either frozen at -20 ° C, for later use, or placed, covered with 0.1 ml / well of mineral oil, in a plate reader (Envision, Perkin-Elmer) , so that the reader captures two signals: a first excitation signal at 340 nanometers and an emission at 615 nanometers, after a delay time of 70 microseconds and a second signal at the same excitation and emission lengths, but with a delay of 30
5
10
fifteen
twenty
25
30
35
40
Four. Five
microseconds The relationship between the first and second reading is considered the final signal that appears in the graph, which establishes a comparative advantage over traditional fluorescence measurements, by eliminating some methodological problems, such as autofluorescence.
After continuous reading (between 40 and 80 readings every 15 minutes) of the plate at 37 ° C and 5% carbon dioxide in the cell incubator, a graph of the bacterial growth in each well is obtained, through the representation of oxygen consumption, as expected (Figure 1).
Example 6: E. Coli detection protocol with 0.005 ml of sensor / well film
To each well of a microplate obtained according to example 1, in which 0.005 ml of precursor solution of the sensor film has been deposited, 0.1 ml of a suspension of standard culture medium (Difco Laboratories) is added, to which a bacterial colony of E. coli has been transferred. Thus, the resulting plate, either frozen at -20 ° C, for later use, or placed, covered with 0.1 ml / well of mineral oil, in a plate reader (Envision, Perkin-Elmer) , so that the reader captures two signals: a first excitation signal at 340 nanometers and an emission at 615 nanometers, after a delay time of 70 microseconds and a second signal at the same excitation and emission lengths, but with a delay of 30 microseconds. The relationship between the first and second reading is considered the final signal that appears in the graph, which establishes a comparative advantage over traditional fluorescence measurements, by eliminating some methodological problems, such as autofluorescence.
After continuous reading (between 40 and 80 readings every 15 minutes) of the plate at 37 ° C and 5% carbon dioxide in the cell incubator, a graph of the bacterial growth in each well is obtained, through the representation of oxygen consumption, as expected (Figure 2).
Example 7: E. Coli detection protocol with 0.01 ml of sensor / well film
To each well of a microplate obtained according to example 1, in which 0.01 ml of precursor solution of the sensor film has been deposited, 0.1 ml of a suspension of standard culture medium (Difco Laboratories) is added, to which a bacterial colony of E. coli has been transferred. Thus, the resulting plate, either frozen at -20 ° C, for later use, or placed, covered with 0.1 ml / well of mineral oil, in a plate reader (Envision, Perkin-Elmer) , so that the reader captures two signals: a first excitation signal at 340 nanometers and an emission at 615 nanometers, after a delay time of 70 microseconds and a second signal at the same excitation and emission lengths, but with a delay of 30 microseconds. The relationship between the first and second reading is considered the final signal that appears in the graph, which establishes a comparative advantage over traditional fluorescence measurements, by eliminating some methodological problems, such as autofluorescence.
After continuous reading (between 40 and 80 readings every 15 minutes) of the plate at 37 ° C and 5% carbon dioxide in the cell incubator, a graph of the bacterial growth in each well is obtained, through the representation of oxygen consumption, as expected (Figure 3).
Example 8: Detection protocol E. coli at 0.01 ml of sensor film + antibiotics / well
5
10
fifteen
twenty
25
30
35
40
Four. Five
To each well of a microplate obtained according to example 2, in which 0.01 ml of precursor solution of the sensing film has been deposited, 0.1 ml of a suspension of standard culture medium (Difco Laboratories) is added, to which a bacterial colony of E. coli has been transferred. Thus, the resulting plate, either frozen at -20 ° C, for later use, or placed, covered with 0.1 ml / well of mineral oil, in a plate reader (Envision, Perkin-Elmer) , so that the reader captures two signals: a first excitation signal at 340 nanometers and an emission at 615 nanometers, after a delay time of 70 microseconds and a second signal at the same excitation and emission lengths, but with a delay of 30 microseconds. The relationship between the first and second reading is considered the final signal that appears in the graph, which establishes a comparative advantage over traditional fluorescence measurements, by eliminating some methodological problems, such as autofluorescence.
After continuous reading (between 40 and 80 readings every 15 minutes) of the plate at 37 ° C and 5% carbon dioxide in the cell incubator, a graph of the bacterial growth in each well is obtained, through the representation of oxygen consumption, as expected (Figure 4).
Example 9: Protocol for detecting proliferation in diluted whole human blood (1: 4) in the presence of phytohemagglutinin
The blood is collected in 10 ml tubes with sodium heparin as an anticoagulant and diluted with RPMI 1604 commercial culture medium supplemented, in a v / v ratio of blood to 1: 4 medium and distributed in allots of 0.188 ml / well of said blood solution in the multiwell plates obtained according to Example 3.
The plate is incubated at 37 ° C and 5% carbon dioxide in the cell incubator for 72 hours, covered with 0.1 ml / well of mineral oil, in a plate reader (Envision, Perkin-Elmer), so that the reader captures two signals: a first excitation signal at 340 nanometers and an emission at 615 nanometers, after a delay time of 70 microseconds and a second signal at the same excitation and emission lengths, but with a delay of 30 microseconds The relationship between the first and second reading is considered the final signal that appears in the graphs, which establishes a comparative advantage over traditional fluorescence measurements, by eliminating some methodological problems, such as autofluorescence and allowing blood measurements. complete, for 40-80 times, every 15 minutes, so that a graph of lymphoproliferation is obtained in each well, through the representation of oxygen consumption, as expected (Figure 5).
Example 10: proliferation detection protocol in diluted whole human blood (1: 5) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at half of 1: 5 a graph of lymphoproliferation is obtained in each well, through the representation of oxygen consumption, as expected (Figure 6).
Example 11: proliferation detection protocol in diluted whole human blood (1: 8) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at a mean of 1: 8 a graph of lymphoproliferation is obtained in each well, by representing the oxygen consumption, as expected (Figure 7) .
Example 12: proliferation detection protocol in diluted whole human blood (1:10) in the presence of phytohemagglutinin
5
10
fifteen
twenty
25
30
35
40
According to a procedure similar to that of Example 9, with a v / v ratio of blood at a mean of 1:10 a graph of lymphoproliferation is obtained in each well, by representing the oxygen consumption, as expected (Figure 8) .
Example 13: proliferation detection protocol in diluted whole human blood (1:20) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at a mean of 1:20 a graph of lymphoproliferation is obtained in each well, by representing the consumption of oxygen, as expected (Figure 9) .
Example 14: Proliferation detection protocol in diluted whole human blood (1:30) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at half past 1:30 a graph of lymphoproliferation is obtained in each well, by representing the oxygen consumption, as expected (Figure 10) .
Example 15: proliferation detection protocol in diluted whole human blood (1:40) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at a mean of 1:40 a graph of lymphoproliferation is obtained in each well, by representing the consumption of oxygen, as expected (Figure 11) .
Example 16: proliferation detection protocol in diluted whole human blood (1:50) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at a mean of 1:50 a graph of lymphoproliferation is obtained in each well, by representing the consumption of oxygen, as expected (Figure 12) .
Example 17: proliferation detection protocol in diluted whole human blood (1: 100) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at a mean of 1: 100 a graph of lymphoproliferation is obtained in each well, by representing the oxygen consumption, as expected (Figure 13) .
Example 18: Protocol for detecting proliferation in diluted whole human blood (1: 200) in the presence of phytohemagglutinin
According to a procedure similar to that of Example 9, with a v / v ratio of blood at half of 1: 200 a graph of lymphoproliferation is obtained in each well, by representing the consumption of oxygen, as expected (Figure 14) .
Example 19: Cytotoxicity protocol of 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II), against THLE-2 cells
To each well of a black 96-well microplate, for tissue culture, 10,000 THLE2 cells / well are added, suspended in the medium recommended by the supplier. Thus, the resulting plate is stored in the cell incubator at 37 ° C and 5% carbon dioxide, overnight, after which the medium is aspirated and 0.18 milliliters / well of medium is added. whole new, followed by 0.02 milliliters / well of varying concentrations of 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II), previously dissolved in dimethylsulfoxide, until a final concentration, in each well, 1% dimethylsulfoxide.
5
10
fifteen
twenty
25
30
35
40
Four. Five
The plate is kept in the cell incubator at 37 ° C and 5% carbon dioxide, for 72 hours, after which the medium is aspirated and replaced with 0.02 milliliters / well of the lysis buffer (“Ready to use lysis buffer 'of the Cambrex Vialight Plus kit, Cat # LT07-121). After an additional 45 minutes of incubation, 0.05 milliliters / well of the ATP detection reagent ("ATP monitoring reagent-Plus" of the Cambrex Vialight Plus kit, Cat # LT07-121) is added and kept at room temperature for 5 minutes , after which a luminescence reading is done on a plate reader (Envision, Perkin-Elmer).
Thus, the graph of Figure 15 is obtained, which represents the content of ATP, whose data is the mean ± error of 6 different wells for each concentration of the
5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II), resulting in a mean inhibitory concentration (IC50) of 0.07 millimolar.
Example 20: proliferation detection protocol in whole human blood in the presence of phytohemagglutinin
The blood is collected in 10 ml tubes with sodium heparin as an anticoagulant and diluted with 100 ml of a modified RPMI 1604 commercial culture medium, in which phytohemagglutinin has been dissolved in adequate quantity to contain a final concentration of 0.02 mg / ml The resulting mixture is incubated at 37 ° C and 5% carbon dioxide in the cell incubator for 72 hours, after which it is distributed in 0.180 ml / well allotots of said blood solution in the multiwell plates obtained according to Example 1
Said plate, containing the blood and cover with its corresponding lid, is introduced into the cell incubator at 37 ° C and 5% carbon dioxide, after adding 0.1 ml / well of mineral oil to half of the wells , to prevent any exchange of oxygen with the interior, while the rest of the wells remain without oil. After three hours, the plate is inserted into a plate reader (Envision, Perkin-Elmer), so that the reader captures two signals: a first excitation signal at 340 nanometers and an emission at 615 nanometers, after a while of delay of 70 microseconds and a second signal at the same lengths of excitation and emission, but with a delay of 30 microseconds. The relationship between the first and second reading is considered the final signal with which the graphs are created, so that lymphoproliferation is obtained in each well, through the representation of oxygen consumption and whose data are the mean ± 4 different wells error for the given concentration (Figures 16 and 17).
Example 21: leaching protocol of 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II), towards the medium
On 24 wells of a microplate generated according to Example 1 (rows AH and columns 7-9), 0.2 ml of RPMI 1640 culture medium is added. Thus, the medium of three of the wells (row A) is Remove immediately (called day 0) or remain on the plate for different days up to 8 days (called day 8, corresponding to rows F-H), after which the medium is also aspirated.
The plate, once completely vacla, is processed by adding 0.1 ml of vehicle (RPMI 1640 culture medium) on the well of row A and column 7 (n = 1) and 0.1 ml of a standard solution 10% sodium sulphite in water on each well of row A and columns 8 and 9 (n = 2), all of them corresponding to day 0. Similarly 0.1 ml of vehicle (RPMI culture medium are added) 1640) on each well of rows F-H and column 7 (n = 3) and 0.1 ml of a 10% solution of sodium sulphite in water on the wells of rows FH and columns 8 and 9 (n = 6), all of them corresponding to day 8.
The wells containing sodium sulphite vehicle or solution are covered with 0.15 ml of mineral oil, to avoid any oxygen exchange with the outside and taken to a plate reader (Envision, Perkin-Elmer) and each well is read 10 times, with an interval of 1 one hour (10 hours of reading), similar to what is described in the 5 previous examples, it supplies each of the final signals collected in Table 1, where the average values of each group and the standard deviation (□).
Table 1
 Mean □ Mean □
 19.3 - 25.29 14.48
 18.8 - 14.93 1.54
 21.4 - 17.80 2.35
 Vehicle  Day 0 23.1 - Day 8 16.97 1.75
 25.1 - 17.60 2.88
 28.9 - 18.77 2.87
 28.9 - 18.83 5.46
 27.8 - 18.33 2.61
 29.3 - 18.17 2.73
 30.9 - 18.47 3.17
 29.1 - 18.90 2.43
 28.2 - 19.73 2.24
 44.85 25.53 71.57 35.52
 182.30 111.16 222.88 73.57
 Positive Pattern    212.10 51.34 244.85 110.11
 Day 0  242.65 36.70 Dfa 8 259.25 122.31
 262.30 33.80 260.43 118.87
 275.90 37.90 247.30 111.65
 284.10 39.74 254.08 128.49
 284.05 36.42 242.75 117.29
 286.50 31.25 249.87 112.58
 290.95 35.43 238.97 106.50
 289.90 33.66 241.85 102.44
 296.50 38.75 238.62 91.32

权利要求:
Claims (21)
[1]
5
10
fifteen
twenty
25
30
35
40
1. Polymeric chemical oxygen sensing microsensor characterized by comprising:
- a support that is a test plate that has a plurality of wells in the form of open receptacles on its upper face and whose number varies between 6, 12, 24, 48, 96, 384, 1536 or 3456 wells, being of geometry and variable capacity; coated with
- an oxygen sensitive film homogeneously deposited on the inner surface of the support wells, comprising:
• an inert and stable polymer matrix comprising a porous structure xerogel, formed by inorganic polymers consisting of a main chain of alternate silicon and oxygen atoms selected from a three-dimensional network of silicon dioxide, developed from monomers with alkoxy groups hydrolysable, and / or a polysiloxane, said matrix being sensitive to oxygen thanks to
• at least one fluorogenic molecular probe anchored to the polymer matrix, which is soluble in water and selected from derivatives of metalophthalocyanine and / or metalloporphyrin.
[2]
2. Microsensor according to claim 1, wherein the fluorogenic molecular probe is at least one metalloporphyrin comprising a central nitrogen cycle containing an atom or metal ion selected from the group consisting of palladium, platinum and ruthenium, substituted at its periphery with one , two, three or four groups of the carboxylate type.
[3]
3. Microsensor according to claim 2, wherein one of the molecular probes is 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin Pt (II), and is present in the polymer matrix in a proportion comprised between 10% and 100% by weight of the total metalloporphyrins and / or metalphthalocyanines of the microsensor.
[4]
4. Microsensor according to any one of claims 1 to 3, wherein the xerogel of the polymeric matrix includes in its structure at least a second metal or metalloid atom different from the silicon selected from the group consisting of aluminum, zirconium, titanium, tin , vanadium, iron and any of its combinations.
[5]
5. Microsensor according to any one of claims 1 to 4, wherein the polymer matrix is selected from the group consisting of:
- an inorganic matrix with a structure composed of 100% silicon dioxide;
- an organic-inorganic hybrid matrix containing in a combined manner silicon dioxide and polysiloxanes, said polysiloxanes being contained in a range between 10% and 99% of the total weight of the matrix; Y
- an organic matrix with a structure composed of 100% polysiloxane.
[6]
6. Microsensor according to any one of claims 1 to 5, wherein the polysiloxane is finished hydroxy polydimethylsiloxane of formula I
5
10
fifteen
twenty
25
30
35
image 1
where n corresponds to the number of silicon units per oligomer molecule, and represents the degree of oligomerization.
[7]
7. Microsensor according to any one of the preceding claims, which comprises a second film of a polymer matrix containing an inert material suitable for reflecting and dispersing the light emitted by the probe consisting of a metal oxide selected within the group consisting of stannous dioxide, zinc oxide, titanium dioxide and any combination thereof, said layer being deposited directly on the inner surface of the microplate wells and between said inner surface and the first polymeric matrix layer.
[8]
8. Microsensor according to any one of claims 1 to 7, further comprising at least one bioactive substance adsorbed on the surface of the porous polymer matrix of the xerogel.
[9]
9. Microsensor according to claim 8, wherein said bioactive substance is selected from the group consisting of antibiotics, antifungals, antiprotozoals, antineoplasics, mitogens and specific antigens, as well! as any of its combinations, and is present in the polymer matrix in an amount between 0.001% and 50% by weight with respect to the total weight of the deposited polymer matrix.
[10]
10. Process for the manufacture of the microsensor defined in any one of claims 1 to 6, by preparing a matrix of porous structure formed by alternating chains of silicon and oxygen atoms selected from silica and polysiloxanes from a solid process. gel, which comprises the steps of:
(a) forming a precursor sol of silicon compounds selected from silicon dioxide and / or polysiloxane, from the mixture of at least one carrier monomer of at least one silicon alkoxide of formula
If (OR) n (R ’) 4-n,
where n varies between 2 and 4; R is an alkyl, and each R 'is independently selected from alkyl or alkenyl, in a proportion comprised between 2 and 30 moles of water per mole of alkoxide, used as a hydrolysis reagent, and a reaction precursor catalyst which is an organic acid , an inorganic acid or a base in a catalyst molar ratio: alkoxide between 0.000001: 99.999999 and 0.0001: 99.9999;
(b) add to the precursor sun resulting from step (a) at least one oxygen sensitive additive consisting of the fluorogenic molecular probe described above, in a probe molar ratio: alkoxide between 0.00001: 99.99999 and 0.001: 99.999 ;
(c) depositing a film of the precursor sun obtained in step (b) on the inner surface of the microplate wells that acts as a support
5
10
fifteen
twenty
25
30
35
40
Four. Five
homogeneous, in a volume between 1% and 25% of the total volume of the well to be coated
(d) gel the precursor sol resulting from step (c) by polycondensation of the alkoxide at a temperature between 15 ° C and 70 ° C for a time between 1 and 72 hours, resulting in a porous xerogel matrix, the which undergoes
(e) syneresis or aging to evaporate the dissolution medium of the precursor sun, for a period of 2 to 5 days.
[11]
11. Process according to the preceding claim, wherein at least a second metal and / or metalloid alkoxide is added to the mixture of step a), which is an alkoxy of formula
M (OR) n
and any of its binary and ternary mixtures with stoichiometers in a molar ratio between 0.1 and 0.9 of each of the alkoxides of the mixture; where M is selected from the group consisting of aluminum, zirconium, titanium, tin, vanadium and iron; (RO-) represents an alkoxy group which is an organic radical derived from an alcohol upon loss of hydroxyl hydrogen, where R is an alkyl group; and n varies between 2 and 4.
[12]
12. Process according to any one of claims 10 or 11, wherein the alkoxides
are selected from the group consisting of: tetramethoxysilane, tetraethoxysilane, octyltriethoxysilane, ethyltrimethoxysilane, hexyltriethoxysilane, hexyltrimethoxysilane, cyclohexyltrimethoxysilane, phenyltriethoxysilane, phenyltrimethoxysilane, benzyltriethoxysilane, dimethyldimethoxysilane, chloromethyltriethoxysilane, tetra (1,1,1,3,3,3-
hexafluoroisopropoxy) silane, polyfluorooctyl triethoxysilane, tetra-n-propoxysilane, tetra-n-butoxysilane, tetrakis (2-methoxyethoxy) silane, methyltriethoxysilane, dodecyltriethoxysilane, dodecyltrimethoxysilane, hexadecyltrimethoxysilane, octadecyltriethoxysilane,
octadecyltrimethoxysilane, methyltrimethoxysilane, (3-aminopropyl) triethoxysilane, (3- aminopropyl) trimethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, triethoxyvinylsilane, [3- (2- aminoethylamino) propll] trimethoxysilane, (3-chloropropyl) trimethoxysilane, (3-
bromopropyl) trimethoxysilane, 3-chloropropyldimethoxymethylsilane, 3-
chloropropyltriethoxysilane, 2-cyanoethyltriethoxysilane, (3-mercaptopropyl) triethoxysilane, (3- mercaptopropyl) trimethoxysilane, cyclohexyl (dimethoxy) methylsilane, diethoxydimethylsilane,
diethoxydiphenylsilane, diethoxy (3-glycidyloxypropyl) methylsilane, diethoxymethylvinylsilane, dimethoxymethylphenylsilane, trimethoxy (2-phenylethyl) silane, N- [3- (trimethoxysilyl) propyl] aniline, tetraethyl titanate, tetraisopropyl titanate and tetrabutyl titanate and their possible binary and ternary mixtures, with variable stoichiometry, in a molar ratio from 0.1 to 0.9 of each of its components.
[13]
13. Process according to any one of claims 10 to 12, wherein the mixture of alkoxide and water is carried out in a dissolution medium selected from an alcohol, a hexane and a cyclic ether; said dissolution medium being present in an amount comprised between 2 and 20 moles.
[14]
14. Process according to any one of claims 10 to 13, wherein:
- 100% of the alkoxides added in the mixture of step a) are alkoxides of formula Si (OR) n (R ’) 4-n, giving rise to a three-dimensional network of alternate silicon and oxygen atoms;
5
10
fifteen
twenty
25
30
35
40
- 100% of the alkoxides added in the mixture of step a) are polysiloxanes, in which case at least one non-ionic surfactant is additionally added; or
- between 10% and 99% of the total precursors added in the mixture of step a) are polysiloxanes, in which case at least one non-ionic surfactant is added.
[15]
15. Process according to any one of claims 10 to 14, wherein the polysiloxane is finished hydroxy polydimethylsiloxane of formula I
, 0.
H '
CH,
'Yes'
I
CH,
, 0.
CH,
fn Yes
CH,
, 0,
ch3
~ sK
CH,
, 0,
where n corresponds dl I lull ICI U UC Ul NUdUCS UC SIIIOIU [JUI represents the degree of oligomerization.
H
oligomer molecule, and
[16]
16. Process according to any one of claims 10 to 15, wherein the deposition of step (c) is carried out by centrifugal coating at a rotation speed of between 300 and 2000 rpm for a time between 1 and 60 minutes, or by immersion.
[17]
17. Process for the manufacture of a microsensor as defined in claim 7, characterized in that it comprises adding a second layer of a polymeric matrix, which contains at least one suitable inert material to reflect and disperse the light emitted by the probe consisting of a metal oxide selected from the group consisting of tin dioxide, zinc oxide, titanium dioxide and any combination thereof, before the deposition of the polymeric matrix layer containing the fluorogenic probe, where said metal oxide is added in the process sol-gel of step (a) giving rise to a mixed xerogel, or is added by dispersion in at least one silicone
[18]
18. Process for the manufacture of a microsensor as defined in any one of claims 8 or 9, characterized in that it comprises adding at least one bioactive substance in step (b) in an amount between 0.001% and 50% by weight of the total polymer film deposited on the well.
[19]
19. Use of a microsensor as defined in any one of claims 1 to 9 for the monitoring of in vitro and ex vivo samples by means of a study selected from the group consisting of: study of eukaryotic or prokaryotic cell proliferation, cytotoxicity study and chemosensitivity, study of cellular senescence, study of the mitochondrial breathing chain; study of lymphoblastic transformation with non-specific mitogens; study of cellular immune response caused by immunological, pharmacological, surgical, nutritional, hormonal or environmental treatments; study of immunological profile and state of the immune system of a subject for the routine control of risk groups; study of immunomediated diseases; study of immunodeficiencies that may be suspected against recurrent infectious conditions or refractory to treatment; study of the deficit of the immunological capacity in autoimmune pathological states; study of consequences of immunosuppressive therapies, immune enhancement therapies, of
5
10
fifteen
twenty
25
30
35
40
Four. Five
cellular hypersensitivity reactions reacting against environmental allergens or antigens; study of the response to mitogens; study of mucocutaneous candidiasis and other chronic fungal infections; study of beryllium-induced blastogenesis; study of the detection of previous exposure to various pathogens; study of periodontal diseases and certain viral infections; study of the detection of previous exposure to the corresponding antigen in subjects without response to antibodies; study of the presence of antigens responsible for allergies that also stimulate specific lymphoproliferative reactions; study of allergic states; study of immunological reactions towards pathogens, allergens and autoantigens; study of autoimmune conditions in which antigens specifically stimulate lymphoblastic transformation only in subjects suffering from this state; study of the states of genetic and acquired immunodeficiency, with a depressed lymphocyte function; study of therapeutic effects; follow-up study of the degree of deterioration / mussel of lymphocyte reactivity in subjects with cancer; study of cellular behavior in mitochondrial disorders; study of the degree of deterioration and / or improvement of lymphocyte reactivity in patients with cancer; study of cellular behavior in mitochondrial disorders; study of enzymatic reactions of oxygen consumption or production and models of diseases associated with oxidative stress; study of the functional link between neurological disorders and mechanisms of bioenergetic regulation of peripheral cells; study of diabetic complications and inflammation; study of manipulation techniques for reproductive, regenerative and therapeutic cell therapy; study of the effect of formulations containing nanoparticles and other teranostic systems on the basic immunological function of animal lymphocytes; which are carried out in a culture selected from the group consisting of cultures with enzymes or enzyme systems dependent on oxygen, adherent cell cultures and cell cultures in suspension of: cellular organs, tissues of genetically modified animals or not; Eukaryotic organisms formed by a single cell or by a colony of cells equal to each other without differentiation of tissues and living in aqueous media or in internal liquids of higher organisms, and complete aquatic organisms.
[20]
20. Use of a microsensor as defined in any one of claims 1 to 9, for the direct in vitro measurement of kinetics of the oxygen concentration present in organic liquids, in inorganic liquids, in their mixtures or in at least one matrix Biology selected from a group consisting of serum, plasma, saliva, sweat, milk, vaginal exudate, semen, pericardial fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, pleural fluid and peritoneal fluid, whole urine, whole blood, urine or blood diluted in a medium selected from the group consisting of: water, physiological serum, calf serum, fetal bovine serum, horse serum, human serum, Hank's balanced saline solution, RPMI 1640, Eagle's basal medium, Eagle's minimum essential medium , MEM medium modified by Dulbeco, Iscove modification of DMEM medium, McCoy 5A, Leibovitz L-15 medium, Ham F-10 medium, Ham F-12 medium, 199 medium and any of its variants.
[21]
21. Use of a microsensor as defined in any one of claims 8 or 9, for the controlled release of bioactive substances.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Depagne et al.2011|How to design cell-based biosensors using the sol–gel process

---

Mansur et al.2005|Biomaterial with chemically engineered surface for protein immobilization

---

Nivens et al.1998|A fiber-optic pH sensor prepared using a base-catalyzed organo-silica sol–gel

---

Bonanno et al.2011|Nanostructured porous silicon–polymer-based hybrids: from biosensing to drug delivery

---

ES2753144T3|2020-04-07|Silica-based fluorescent nanoparticles

---

Dash et al.2008|Organically modified silica: Synthesis and applications due to its surface interaction with organic molecules

---

Yang et al.2010|Photocatalyzed surface modification of poly | with polysaccharides and assay of their protein adsorption and cytocompatibility

---

ES2384745T3|2012-07-11|Analysis chip and analysis method

---

Jayant et al.2009|Polyelectrolyte-coated alginate microspheres as drug delivery carriers for dexamethasone release

---

Kang et al.2012|Design of polydiacetylene-phospholipid supramolecules for enhanced stability and sensitivity

---

Otis et al.2018|Selective labeling and growth inhibition of Pseudomonas aeruginosa by aminoguanidine carbon dots

---

Ronda et al.2015|Immobilization of proteins in silica gel: biochemical and biophysical properties

---

ES2554077B1|2016-09-20|Polymeric chemical microsensor with fluorogenic molecular probe, manufacturing process and use for the controlled release of bioactive substances and other applications

---

Wu et al.2018|Hydrogel-based fluorescent dual pH and oxygen sensors loaded in 96-well plates for high-throughput cell metabolism studies

---

Frankær et al.2018|Biocompatible microporous organically modified silicate material with rapid internal diffusion of protons

---

Marzioch et al.2018|On-chip photodynamic therapy–monitoring cell metabolism using electrochemical microsensors

---

Liu et al.2020|An NIR-light-responsive surface molecularly imprinted polymer for photoregulated drug release in aqueous solution through porcine tissue

---

Xu et al.2020|pH and redox dual-responsive MSN-SS-CS as a drug delivery system in cancer therapy

---

Rounds et al.2007|Microporated PEG spheres for fluorescent analyte detection

---

Skalickova et al.2017|Zinc-modified nanotransporter of doxorubicin for targeted prostate cancer delivery

---

Zhang et al.2019|Gold nanoclusters as a near-infrared fluorometric nanothermometer for living cells

---

O’Connor et al.2020|Polydopamine antioxidant hydrogels for wound healing applications

---

JP6120518B2|2017-04-26|Simple measuring instrument

---

Žid et al.2020|Mesoporous silica as a drug delivery system for naproxen: Influence of surface functionalization

---

Serban et al.2020|Tetraethyl Orthosilicate-Based Hydrogels for Drug Delivery—Effects of Their Nanoparticulate Structure on Release Properties

同族专利:

---

公开号 | 公开日

---

ES2554077B1|2016-09-20|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO1999037998A1|1998-01-21|1999-07-29|Bayer Corporation|Oxygen sensing membranes and methods of making same|

---

US20040171094A1|2001-06-18|2004-09-02|Ingo Klimant|Oxygen sensors disposed on a microtiter plate|

---

US20110086418A1|2009-10-08|2011-04-14|National Institute of Standards and Technology, U.S. Department of Commerce|Highly sensitive oxygen sensor for cell culture|

---

CN109030433A|2018-06-13|2018-12-18|西北师范大学|The preparation and its application in detection hydrogen peroxide and glucose of porphyrin compound fluorescent molecule|

法律状态:
2016-09-20| FG2A| Definitive protection|Ref document number: 2554077 Country of ref document: ES Kind code of ref document: B1 Effective date: 20160920 |

优先权:

---

申请号 | 申请日 | 专利标题

---

ES201430899A|ES2554077B1|2014-06-12|2014-06-12|Polymeric chemical microsensor with fluorogenic molecular probe, manufacturing process and use for the controlled release of bioactive substances and other applications|ES201430899A| ES2554077B1|2014-06-12|2014-06-12|Polymeric chemical microsensor with fluorogenic molecular probe, manufacturing process and use for the controlled release of bioactive substances and other applications|