Method for transdermal nucleic acid sampling

专利摘要:
Minimal invasive transdermal nucleic acid sampling methods include puncturing into the lower epidermis through the outermost layer of skin 20 with a plurality of microprojections 4. The rupture of live skin cells in the lower epidermis releases the contents, including nucleic acids (ie, DNA, RNA, fragments thereof or other polynucleic acid materials found in the nucleus and / or mitochondria of the cell). Nucleic acids are collected on microprojections or in separate nucleic acid collection reservoirs. The collected nucleic acids are then analyzed using standard polymerase chain reaction (PCR) techniques. Optionally, the suction device 10 partially vacuums the microcutting in the skin 20 through an opening in the microprojection member 6 to increase the outflow of intracellular and extracellular (ie body) body fluids containing nucleic acid. Add.
公开号:KR20030068127A
申请号:KR10-2003-7002654
申请日:2001-08-20
公开日:2003-08-19
发明作者:마트리아노제임스에이.；코미어마이클제이.엔.
申请人:알자 코포레이션；
IPC主号:

专利说明:

Method for transdermal nucleic acid sampling
[1] Field of invention
[2] The present invention relates to nucleic acid (eg, DNA or RNA) sampling. More particularly, the present invention relates to transdermal nucleic acid sampling. The present invention uses skin-piercing microprojection to puncture skin cells and sample percutaneous nucleic acids.
[3] Background of the Invention
[4] DNA testing is currently performed by blood sampling or tissue swabbing, followed by amplification of the polymerase chain reaction (PCR) of DNA followed by analysis of the amplified DNA. Blood sampling using syringes is invasive and painful. Tissue swabbing makes many patients uncomfortable and uncomfortable. One common method for sampling DNA uses an oral mucosal tissue sample obtained by vigorously rotating the cell collection brush for 30 seconds along the inside of the cheek.
[5] Many attempts have been made to increase transdermal flux by mechanically puncturing the skin prior to transdermal drug administration. See, for example, US Pat. No. 5,279,544 to Gross et al., US Pat. No. 5,250,023 to Lee et al. And US Pat. No. 3,964,482 to Gerstel et al. These devices use solid and hollow microprojections to perforate the outer layers of the skin.
[6] Attempts have been made to sample body samples (eg, glucose) contained in interstitial fluid using devices with similar skin-perforated microprojection. The sample contents contained in the interstitial fluid are then correlated with the contents in the blood. See, eg, WO 97/48441, Cormier et al .; Joseph, US Pat. No. 5,161,532; US Patent No. 5,582,184 to Ericson et al .; US Patent No. 5,682,233 to Brinda; See US Pat. No. 5,746,217 to Ericson et al. And US Pat. No. 5,820,570 to Erickson et al. US Pat. No. 6,091,975 to Dadona et al. Provides a diagnostic device with stratum corneum-perforated microprojection. An electrochemical sensor is placed directly above the microprojection to detect / measure body sample concentrations, such as blood glucose levels. One advantage of sampling the interstitial fluid is that the wound on the skin is not as deep as the wound needed for blood sampling. Thus, interstitial fluid sampling using such stratum corneum-perforated microprojections is generally considered less invasive than blood sampling.
[7] However, genetic tests (e.g. paternity tests, blood / semen sample matching for individuals in policing / crime detection work), medical diagnostics (e.g., the presence of disease and / or predisposition to diseases such as heart disease) Less invasive nucleic acid sampling is still desired for such purposes as patient investigation.
[8] Description of the invention
[9] The present invention provides a percutaneous DNA sampling method using reproducible, highly productive, low cost devices. The present invention includes perforating into the lower epidermal layer or both the epidermal and dermal layers through the outermost layer of the skin (eg, the stratum corneum) with a plurality of microprojections. Individual skin cells in the epithelial / epidermal layer are punctured to release the cell contents, including the nucleus and nucleic acid. The nucleic acid is coated onto the microprojection surface and / or absorbed into the absorbent coating on the microprojection. Microprojections typically have a length of less than about 0.4 mm, the width and thickness of which are smaller.
[10] The method of the present invention can also be used to extract and sample the released nucleic acid into skin interstitial fluid. As noted above, the outermost stratum corneum of the skin is perforated to form a pathway through which interstitial fluid containing nucleic acid is taken out (sampled). Optionally, the sampling device used in conjunction with an embodiment of the present invention may apply a partial vacuum (also referred to herein as "negative pressure") to the microcut skin. Negative pressure drains the interstitial fluid from the microcuts. The interstitial fluid is collected and the nucleic acid contained therein is amplified using standard polymerase chain reaction techniques and analyzed for the amplified nucleic acid.
[11] In one aspect of the invention, an apparatus for puncturing the skin includes a sheet having a plurality of openings, and a plurality of microprojections extending and integrated downwardly (ie, towards the skin). Any negative pressure drive device applies a partial vacuum (i.e., suction) to the microcut through the opening in the seat.
[12] In another aspect of the present invention, there is provided the use of an apparatus for puncturing the stratum corneum of a skin for analyzing nucleic acids of an animal. The use includes disrupting or dislodging skin cells containing nucleic acids by perforating through the stratum corneum into at least the lower epidermal layer with skin-perforated microprojection. Nucleic acid is collected from ruptured or isolated skin cells and analyzed.
[13] Brief description of the drawings
[14] 1 is an enlarged perspective view of an adjacent skin side of a microprojection array device according to one embodiment of the invention.
[15] 2 is a partial plan view of a microprojection array pattern according to one embodiment of the invention.
[16] 3 is a cross-sectional side view of a microprojection DNA-sampling array properly attached to the skin by an adhesive overlay.
[17] 4 is a side view of any negative pressure drive device with a microprojection DNA-sampling array shown in cross section.
[18] Mode for carrying out the invention
[19] The present invention provides a method for obtaining nucleic acid samples from the skin without bleeding and without pain. The term “nucleic acid (s)” as used herein refers to DNA (ie, deoxyribonucleic acid), DNA fragments, RNA (ribonucleic acid), RNA fragments, chromosomes, genes and any that are found in the nucleus and / or mitochondria of a cell. It is widely used to include other polynucleic acid sequences, or portions thereof.
[20] The skin is perforated by at least one, preferably plural and more preferably plural micro stratum corneum perforated microprojections. Although the present invention is not limited in size, shape or configuration of the microprojection, the microprojection has a depth of about 25 μm to about 400 μm, preferably about 50 μm to about 300 μm for sampling without pain and without bleeding. Should perforate the skin. Perforation to this depth using multiple microprojections results in a small amount of bleeding and little feeling (pain), even with bleeding, and through the outermost stratum corneum, where the blade is dead, into the epidermal or epidermal and dermal layers containing living skin cells. It is sure to penetrate. Perforation can be carried out using conventional spring-loaded devices of the type used to inject lancets into the skin for blood droplet sampling or in FIGS. 22 and 23 of WO 01/41863 by Trautman et al. It can be achieved by pushing microprojection onto the skin surface using the device of the disclosed type.
[21] When punctured into the epidermal layer, typically for 20 μm diameter living epidermal skin cells, large microprojections typically puncture and / or destroy multiple skin cells in the epidermal layer or in both the epidermal and dermal layers so that these cells destroy the contents. Will release. This allows the nucleic acid originally contained in the nucleus of the skin cells and / or the nucleus of the skin cells to be released into the extracellular fluid (also called interstitial fluid) of the skin. This body fluid is either coated on the surface of the skin-perforated microprojection or absorbed into the absorbent coating on the microprojection. As a result, the nucleic acid of the cell is coated or absorbed onto the skin perforation microprojection. Skin perforation microprojections are typically made from metals, plastics or silicones, which are materials that can be appropriately coated with intracellular (from ruptured skin cells) and extracellular fluids, including nucleic acids of ruptured cells. Once the nucleic acid-containing body fluid is coated on the surface of the microprojection, the microprojection is removed from the skin and the nucleic acid is collected for PCR amplification and analyzed. When using conventional metal, plastic or silicon based microprojections, nucleic acid samples can be simply collected by immersing / incubating the microprojections in sterile water, optionally with surfactants, buffers and proteases. Target gene (s) in DNA and / or RNA containing solutions are amplified using known polymerase chain reaction techniques. These techniques are disclosed, for example, in Saiki et al., Primer-directed Enzymatic Amplification of DNA with a Thermosable DNA Polymerase, Science, 239, 487, 1988.
[22] After amplification of the target gene (s), standard DNA such as those known from Saiki et al., Analyss of Enzymatically Amplified Beta-globin and HLA-DQA1 with Allele Specific Oligonucleotide Probes, Nature, 324, 163, 1986. Analyze using analytical techniques.
[23] DETAILED DESCRIPTION OF THE DRAWINGS An embodiment of a skin perforated microprojection array device 2 for sampling nucleic acids in accordance with the present invention is generally shown in FIG. 1. The device 2 comprises a plurality of microprojections 4 (ie microprojection arrays) extending downward from one surface of the sheet or plate 6 (the device 2 is inverted to show microprojection). 1 in position). Microprojection 4 perforates through the stratum corneum into at least the epidermal layer of the skin when pressure is applied to the device to sample the nucleic acid therethrough. As used herein, the term "skin" refers to the skin of a living or dead animal, especially a human, and specifically excludes mucosal membranes (eg oral mucosa). Device 2 preferably has a microprojection density of at least about 10 microprojections / cm 2, more preferably at least about 50 microprojections / cm 2. Similarly, the number of openings per unit area of plate 6 is typically at least about 10 openings / cm 2, more typically at least about 100 openings / cm 2.
[24] Microprojection 4 can be formed using a photo-etching process disclosed in WO 97/48440 by Cormier et al., Which is incorporated herein by reference. This process allows the microprojection 4 to be formed reproducibly on a very small (ie tens of microns) scale.
[25] A plurality of microprojections 4 for puncturing the stratum corneum is a group of microprojections spaced in any desired arrangement, for example any desired number of rows, or in a state in which the microprojections 2 are spaced apart from one another per microprojection. Present on surface 48. The device 2 of the embodiment shown in FIG. 1 is produced by the pattern shown in FIG. 2. Each microprojection has a width and thickness that allow penetration of the stratum corneum without bending. Since one of the main features of the present invention is that microprojection penetrates the stratum corneum and enters the epidermis, the required length of the microprojection is susceptible to changes in the skin to be perforated, which corresponds to the original thickness of the stratum corneum. Since microprojection often does not penetrate the skin to the same depth as the microprojection length due to the elasticity of living animal skin, another factor affecting the length of microprojection is the method of application of microprojection. Typically, the length of the microprojection is between about 25 μm and about 500 μm, with the most typical length being between about 100 μm and about 450 μm.
[26] The pattern for any microprojection array device used in the present invention can be produced by a photo-etching process. Thin sheets or plates 6 of metal, such as stainless steel or titanium, are photo-lithographically etched in a pattern with a microblade like structure. Generally, a thin sheet dry or wet resist is applied on the sheet to a thickness of about 7 μm to about 100 μm, preferably from about 25 μm to about 50 μm. This resist is developed after contact exposure using a mask having a desired pattern. These operations are performed in the same manner as the operations for the manufacture of a printed circuit board. Thereafter, the sheet is etched using an acidic solution. After the pattern is etched through the sheet, the sheet is placed on a die having a plurality of openings corresponding to the openings 8 in the sheet. Initially, a punch having a plurality of protrusions corresponding to the die and the opening in the sheet is located above the sheet and the die. In the initial stage, the microprojection 4 is coplanar with the remaining sheets 6. Thereafter, the punched protrusion is pressed into the opening, thereby causing the microprojection 4 to be bent downward so as to have an angle (for example, substantially perpendicular) to the plane of the sheet 6 as shown in FIG. do.
[27] In general, the microprojection 4 makes an angle of about 90 ° with respect to the surface 48 of the sheet 6 after being perforated, but may be arranged at different front and rear angles from the vertical position as long as penetration of the stratum corneum is achieved. .
[28] Another embodiment of the invention extracts DNA-containing intracellular and extracellular body fluids from the microcuts formed by skin perforation microprojection, wherein the body fluids are extracted with a device that applies partial vacuum to the surface of the skin. Referring to FIG. 4, there is shown a negative pressure drive device 10 having a microprojection array device 2 on the skin proximal side of the device 10. Device 10 and device 2 together are used for transdermal sampling of interstitial fluid containing nucleic acids released from skin cells punctured or individually destroyed by skin puncture microprojection. Device 10 is a known sound pressure (ie, suction) application device, such as that disclosed in Ishibashi U.S. Patent No. 5,320,607, which is incorporated herein by reference. The device 10 is mounted on the skin distal surface of the device 2. The skin proximal side of the device 2 is in contact with the surface of the skin 20, and the microprojection 4 extends at least through the stratum corneum of the skin 20. By properly sealing the device 10 on the skin distal side of the device 2, the negative pressure exerted by the device 10 causes the suction to be applied through the opening 8 in the seat 6. In this manner, the nucleic acid-containing interstitial fluid is extracted from the microcuts cut in the skin 20 and discharged into the device 10.
[29] Although it is preferable to use an uncoated microprojection array made of sterile metal, such as stainless steel or titanium, the skin proximal side of the microprojection 4 and / or plate 6 absorbs nucleic acid-containing body fluids when puncturing the stratum corneum. It is then possible to coat with a thin coating of an acceptable body fluid absorbent material. It is also possible to use a nucleic acid transfer / receiving layer that coats the skin proximal side of the plate 6. Such transport / receive layers are disclosed in WO 98/28037 to Theeuwes et al., Which is incorporated herein by reference.
[30] The main barrier properties of the skin, such as resistance to nucleic acid efflux, are first in the individual skin cell walls. In other words, the microprojection in the device 2 should be dimensioned, shaped and configured to rupture the cell wall of living skin cells in the skin layer just below the stratum corneum, without perforation into the capillary in the skin so that there is little bleeding. . The microprojection should then create openings (ie, cutouts) in the stratum corneum, particularly for embodiments that govern the collection of nucleic acid released from the skin cells into the interstitial fluid.
[31] Since microprojection punctures the skin almost simultaneously with nucleic acid-containing intracellular and extracellular fluids coating the skin-perforation microprojection, the microprojection array does not need to be fixed or attached to the skin for a significant amount of time. That is, although not necessary, it is possible to provide a conventional adhesive overlay to leave the microprojection array on the skin for a short time. An embodiment of such a system with a peripheral adhesive overlay 3 supporting the device 2 in puncturing skin 20 is disclosed in FIG. 3. In some cases, the microprojection array should preferably be sterilized in advance to avoid any possible secondary contamination, for example by collection of tissue or other nucleic acid-containing material from a source other than the test subject. Most preferably, the microprojection array and any device used to apply the microprojection to the skin should be manufactured as a disposable device that is sterilized and placed in a sealed package before use.
[32] Sheets and microprojections can be made of materials that have sufficient strength and manufacturability to produce microprojections, such as glass, ceramics, hard polymers, metals and metal alloys. Examples of metals and metal alloys include titanium, stainless steel, iron, steel, tin, zinc, copper, platinum, aluminum, germanium, nickel, zirconium, and titanium alloys consisting of nickel, molybdenum and chromium, nickel, gold, rhodium, Metals plated with iridium, titanium, platinum, and the like include, but are not limited to these. Examples of glass include opaque glass and silicon, such as "Photoceram" available from Corning, Corning, NY. Examples of hard polymers include polystyrene, polymethylmethacrylate, polypropylene, polyethylene, "Bakelite", cellulose acetate, ethylcellulose, styrene / acrylonitrile copolymers, styrenebutadiene copolymers, acrylonitrile / butadiene / styrene (ABS ), But are not limited to acrylic acid polymers including copolymers, polyacrylates and polymethacrylates and polyvinyl chloride. Most preferably, the sheet and microprojection are made of titanium or stainless steel.
[33] The number of apertures and microblades in the device 20 of any embodiment varies with the desired DNA sample size, and other factors will be apparent to those skilled in the art whether or not a negative pressure driven sampling device is used. The following examples illustrate the uses and advantages of the present invention.
[34] Example 1
[35] Two motives and four non-kinetic individuals were studied. In each subject, the treatment site (dorsal forearm) was washed with a 70% sterile isopropyl alcohol pad. The skin area was then dried with a sterile gauze pad. The skin was punctured by pushing a microprojection device onto the skin surface using a spring-loaded applicator. The microprojection device comprises a 1 cm 2 disc-shaped microprojection array with a density 320 microprojections / cm 2 and a 200 μm length microprojection adhered to the low density polyethylene backside with polyisobutyrene adhesive. After application, the system was immediately removed with sterile forceps and transferred into sterile vials and stored frozen (-20 ° C.) until analysis. In the same subjects, the inside of the cheek was swipped for 30 seconds with a cell collection brush to obtain additional DNA samples and immediately transferred into sterile vials and stored frozen (-20 ° C.) until analysis.
[36] For analysis, the vials were sent to contract laboratories that specialize in DNA profiling. DNA was extracted using standard methods. Polymorphism for gene DQA1 was evaluated by PCR and reverse dot blot hybridization procedure. The results demonstrated that the two motives were actually kinetic and that the other individuals were not kinetic. The same result was obtained by the brush for collecting cells.
[37] These results demonstrate the justification of the new nucleic acid sampling method. Applications include forensic science, paternity analysis, genetic testing for predisposition of genetic diseases, and detection of infectious diseases.
[38] Although the present invention has been described in conjunction with specific preferred embodiments, it is to be understood that the above description as well as the above examples are intended to illustrate, but not limit, the scope of the invention. Modifications, advantages and other aspects within the scope of the invention will be apparent to those skilled in the art.

权利要求:
Claims (26)
[1" claim-type="Currently amended] Perforating through the stratum corneum of skin 20 into at least the lower epidermal layer with skin-piercing microprojection 4 to disrupt or dissociate skin cells containing nucleic acids;
Collecting nucleic acid from ruptured or isolated skin cells;
A method for transdermally sampling a nucleic acid, including analyzing the nucleic acid.
[2" claim-type="Currently amended] The method of claim 1, wherein the collecting step comprises coating the microprojection (4) with nucleic acid-containing bodily fluid and withdrawing the microprojection (4) from the skin (20).
[3" claim-type="Currently amended] 3. A method according to claim 2 comprising absorbing body fluids into absorbent material coating the microprojection (4).
[4" claim-type="Currently amended] The method of claim 1, wherein the collecting step comprises applying negative pressure to the perforated skin (20) to collect the nucleic acid released into the body fluid from the ruptured or isolated skin cells.
[5" claim-type="Currently amended] The method of claim 1, wherein the microprojection (4) perforates the skin (20) to a depth of less than about 500 μm.
[6" claim-type="Currently amended] The method of claim 1 comprising perforating the skin (20) with a plurality of microprojections (4).
[7" claim-type="Currently amended] 7. A method according to claim 6, comprising perforating the skin (20) with a plurality of microprojections (4).
[8" claim-type="Currently amended] 7. The method of claim 6, wherein the microprojection (4) punctures the skin (20) to a depth of at least about 25 μm.
[9" claim-type="Currently amended] 7. The method of claim 6, wherein the microprojection (4) punctures the skin (20) at a density of at least about 10 microprojections / cm 2.
[10" claim-type="Currently amended] 7. A method according to claim 6, wherein the nucleic acid is collected on the surface of the microprojection (4).
[11" claim-type="Currently amended] The method of claim 1 wherein the nucleic acid is collected in an absorbent matrix.
[12" claim-type="Currently amended] The method of claim 1, wherein the nucleic acid is analyzed by polymerase chain reaction (PCR) assay.
[13" claim-type="Currently amended] The method of claim 1, wherein the skin (20) is human skin.
[14" claim-type="Currently amended] The method of claim 1 wherein the nucleic acid is selected from the group consisting of DNA, DNA fragments, RNA, RNA fragments, genes, chromosomes and polynucleic acid sequences.
[15" claim-type="Currently amended] Skin-perforated microprojection 4 perforates through the stratum corneum of skin 20 into at least the lower epidermal layer to rupture or separate skin cells containing nucleic acids; Collecting nucleic acid from ruptured or isolated skin cells; Use of an apparatus for puncturing the stratum corneum of skin (20) for analyzing nucleic acids of animals by analyzing nucleic acids.
[16" claim-type="Currently amended] Use according to claim 15, wherein collecting the nucleic acid comprises coating the microprojection (4) with nucleic acid-containing bodily fluid and withdrawing the microprojection (4) from the skin (20).
[17" claim-type="Currently amended] 17. The method according to claim 16, wherein the body fluid is absorbed into an absorbent material coating the microprojection (4).
[18" claim-type="Currently amended] Use according to claim 15, wherein collecting the nucleic acid comprises applying a negative pressure to the perforated skin (20) to collect body fluids containing the nucleic acid released from the ruptured or isolated skin cells.
[19" claim-type="Currently amended] Use according to claim 15, wherein the microprojection (4) perforates the skin (20) to a depth of less than about 500 μm.
[20" claim-type="Currently amended] Use according to claim 15, comprising perforating the skin (20) with a plurality of microprojections (4).
[21" claim-type="Currently amended] 21. Use according to claim 20, comprising perforating the skin (20) with a plurality of microprojections (4).
[22" claim-type="Currently amended] 21. Use according to claim 20, wherein the microprojection (4) punctures the skin (20) to a depth of at least about 25 μm.
[23" claim-type="Currently amended] 21. Use according to claim 20, wherein the microprojection (4) punctures the skin (20) at a density of at least about 10 microprojections / cm 2.
[24" claim-type="Currently amended] Use according to claim 20, wherein the nucleic acid is collected on the surface of the microprojection (4).
[25" claim-type="Currently amended] The use of claim 15, wherein the nucleic acid is collected in an absorbent matrix.
[26" claim-type="Currently amended] Use according to claim 15, wherein the nucleic acid is selected from the group consisting of DNA, DNA fragments, RNA, RNA fragments, genes, chromosomes and polynucleic acid sequences.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-08-24|Priority to US22767500P

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2000-08-24|Priority to US60/227,675

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2001-08-20|Application filed by 알자 코포레이션

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2001-08-20|Priority to PCT/US2001/026102

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2003-08-19|Publication of KR20030068127A

优先权:

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申请号 | 申请日 | 专利标题

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US22767500P| true| 2000-08-24|2000-08-24|

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US60/227,675|2000-08-24|

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PCT/US2001/026102|WO2002015800A1|2000-08-24|2001-08-20|Method for transdermal nucleic acid sampling|