apparatus and scanning methods useful for detecting chemical and biological analytes

专利摘要:
An apparatus may include a container, a reference surface, a preload, a scanning actuator and a transmitter. The reference surface can form a structural circuit with a detector. The preload can be configured to urge the container to contact an area on the reference surface. The scan actuator can be configured to slide the container along the reference surface in a scan dimension. The transmitter can be configured to direct signal from the container to a detector and / or direct energy from an energy source to the container, when the container is propelled by the preload to contact the reference surface.
公开号:BR112020003229A2
申请号:R112020003229-8
申请日:2018-08-15
公开日:2020-08-18
发明作者:Dale Buermann；Michael John Erickstad；Rebecca McGinley；Alex Nemiroski；Scott Harry Rapoport；Arnold Oliphant
申请人:Omniome, Inc.；
IPC主号:

专利说明:

[001] [001] This order claims priority for US Provisional Order 62 / 545,606, filed on August 15, 2017 and entitled “SCANNING
[002] [002] The present disclosure generally relates to the detection of chemical and biological analytes and has specific applicability in nucleic acid sequencing.
[003] [003] The determination of nucleic acid sequence information is important in biological and medical research. Sequence information is used to identify gene associations with diseases and phenotypes, identify potential drug targets and understand the mechanisms of disease development and progress. Sequence information is an important part of personalized medicine, where it can be used to optimize diagnosis, treatment or disease prevention for a specific individual.
[004] [004] Many scientists and doctors struggle to take advantage of modern sequencing technology due to the prohibitive costs of operating and maintaining complex instrumentation in today's commercial offerings. These platforms favor centralized laboratories in which expensive “factory scale” instruments are operated by highly trained specialists and samples are batched to obtain economies of scale. This centralized system offers very little flexibility in terms of performance specifications - users are forced into ecosystems that are unnecessarily limited in scope and variety of use. When it comes to clinical applications, the centralized model is expensive for doctors and their patients in terms of both the time and money needed to transport patient samples from local clinics to remote sequencing laboratories. Additional delays can be incurred when a centralized sequencing laboratory waits to receive a sufficient number of samples to batch together in an economical operation. Other applied markets, such as forensics, veterinary diagnosis, food safety, agricultural analysis and environmental analysis, suffer similar limitations.
[005] [005] Therefore, there is a need for a sequencing platform that is more suitable for use in local laboratories in support of a decentralized system of research and clinical care. The present invention satisfies this need and provides related advantages as well. BRIEF SUMMARY
[006] [006] The present disclosure provides a detection apparatus which may include (a) a container having a lumen and a wall, the wall having an inner surface and an outer surface, the inner surface contacting the lumen; (b) a reference surface that forms a structural loop with a detector; (c) a preload configured to urge the outer surface of the container to contact an area on the reference surface; (d) a scan actuator configured to slide the container along the reference surface in a scan dimension; and (e) a transmitter configured to direct a signal from the inner surface or the lumen to the detector when the outer surface of the container is driven by the preload to contact the reference surface.
[007] [007] A method of sweeping a container is also provided. The method may include (a) translating a container along a reference surface of a detection apparatus, where the container comprises a lumen and a wall, where the lumen comprises analytes, where the reference surface contacts at least a portion of the container during translation and where the reference surface forms a structural circuit with the detector; and (b) detecting the analytes at different locations along the container using the detector, where the container is propelled to the reference surface by a preload during detection, thereby sweeping the container.
[008] [008] In some embodiments, a method for sweeping a container may include (a) examining a first subset of analytes in a container while applying a preload to a first portion of the container, where the preload positions the first subset analytes to occupy an xy plane in a detection zone, where the preload is not applied to a second portion of the container; (b) moving the container to position a second subset of analytes on the xy plane of the detection zone; and (c) examining the second subset of the analytes in the container while applying the preload to a second portion of the container, where the preload positions the second subset of the analytes to occupy the xy plane of the detection zone, where the a preload is not applied to the first portion of the container, thereby sweeping the container.
[009] [009] This disclosure provides reactor equipment. A reactor apparatus may include (a) a container having a lumen and a wall, the wall having an inner surface and an outer surface, the inner surface contacting the lumen; (b) a reference surface that forms a structural circuit with an energy source; (c) a preload configured to urge the outer surface of the container to contact an area on the reference surface; (d) a scan actuator configured to slide the container along the reference surface in a scan dimension; and (e) a transmitter configured to direct energy from the energy source to the inner surface or the lumen, when the outer surface of the container is propelled by the preload to contact the reference surface.
[0010] [0010] A method for carrying out reactions in a vessel is also provided. The method may include (a) translating a container along a reference surface of a reactor apparatus, in which the container comprises a lumen and a wall, in which the lumen comprises reagents, in which the reference surface contacts at least a portion of the container during translation and where the reference surface forms a structural circuit with the energy source; and (b) directing energy from the energy source to the reagents, in which the container is driven to the reference surface by a preload directing the energy to the reagents, thereby carrying out reactions in the container.
[0011] [0011] A method for carrying out reactions in a container may include (a) distributing energy from a reactor apparatus to a first subset of reagents in a container while applying a preload to a first portion of the container, where the pre- load positions the first reagent subset to occupy an xy plane of a reaction zone, where the preload is not applied to a second portion of the container; (b) translate the container to position a second reagent subset in the xy plane the reaction zone; and (c) distributing energy from the reactor apparatus to the second subset of the analytes in the container while applying the preload to a second portion of the container, where the preload positions the second subset of the analytes to occupy the xy plane, in that the preload is not applied to the first portion of the container, thereby carrying out reactions in the container.
[0012] [0012] In particular embodiments, the present disclosure provides a detection apparatus that includes (a) a container having a lumen and a wall, where the wall has an inner surface and an outer surface, where the inner surface contacts the lumen and where the outer surface has length ℓ in a scan dimension x; (b) a reference surface; (c) a preload configured to urge the outer surface of the container to contact an area on the reference surface, optionally the contact area can have a maximum length in the scan dimension x that is shorter than the length ℓ; (d) a scanning actuator configured to slide the container along the reference surface in the scanning dimension x; (e) a detector; and (f) a lens configured to direct radiation from the container to the detector when the outer surface of the container is propelled by the preload to contact the reference surface.
[0013] [0013] A method of optically scanning a container is also provided. The method may include (a) providing a container having a lumen and a wall, where the lumen contains optically detectable analytes and where the wall is transparent to optically detectable analytes; (b) translating a length of the container along a reference surface and detecting the optically detectable analytes at different locations along the length, where the reference surface contacts only a portion of the length of the container at any time during translation, where the container is propelled to the reference surface by a preload during detection, where detection includes transmitting radiation through the wall, then through a lens and then to a detector, thereby optically scanning the container.
[0014] [0014] The present disclosure further provides a detection apparatus that includes (a) a container having a lumen and a wall, in which the wall has an internal surface and an external surface, in which the wall has a plurality of discrete contacts between the inner surface and the outer surface, where the inner surface contacts the lumen and where the plurality of discrete contacts occupies a length ℓ in a scan dimension x; (b) a transmissive surface; (c) a preload configured to urge discrete contacts on the outer surface of the container to contact the transmissive surface, optionally the area of the transmissive surface may have a maximum length in the scan dimension x that is shorter than the length ℓ; (d) a scanning actuator configured to slide the container along the transmissive surface in the scanning dimension x; and (e) a detector configured to acquire signals from the discrete contacts via the transmissive surface. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] [0015] FIG. 1 shows dimensions and axes of rotation used to describe the relative orientation of components in optical systems and other devices established here.
[0016] [0016] FIG. 2A shows an exploded profile view of a flow cell and a detection apparatus; FIG. 2B shows a profile view of the flow cell in contact with a detection device; FIG. 2C shows a perspective view of the flow cell in contact with the detection apparatus; and FIG. 2D shows an exploded perspective view of the flow cell in contact with the detection device.
[0017] [0017] FIG. 3A and FIG. 3B show front and rear perspective views of a film pinion mechanism for translating a flow cell in relation to a detection device.
[0018] [0018] FIG. 4A shows a flow cell cartridge; FIG. 4B shows a film pinion and guide interacting with the flow cell cartridge; FIG. 4C shows a flow cell; and FIG. 4D shows a perspective view of the film pinion, guide, flow cell cartridge, flow cell and a motor for the film pinion.
[0019] [0019] FIG. 5A and FIG. 5B show front and rear perspective views of a cylindrical gear mechanism for translating a flow cell in relation to a detection apparatus.
[0020] [0020] FIG. 6A and FIG. 6B show front and rear perspective views of a ball screw mechanism for translating a flow cell in relation to a detection device.
[0021] [0021] FIG. 7A shows a perspective view of a heating plate and film pinion scanner and FIG. 7B shows a perspective view of a lens and heater plate and film pinion scanner.
[0022] [0022] FIG. 8A shows a perspective view of a fluidic box with a fixed flow cell; FIG. 8B shows an expanded view of the attachment points for the flow cell to the housing; FIG. 8C shows a front view of the fluidic box with a fixed flow cell; FIG. 8D shows a side view of the fluidic box with a fixed flow cell; FIG. 8E shows a top view of the fluidic box with a fixed flow cell; and FIG. 8F shows a perspective view of the fluidic box emptied of various fluidic components.
[0023] [0023] FIG. 9A shows a perspective view of a fluidic box and flow cell interacting with a detection device; FIG. 9B shows a top view of the fluidic box and the flow cell interacting with the detection apparatus; and FIG. 9C shows a perspective view of the fluid box disengaged from the detection apparatus.
[0024] [0024] FIG. 10A shows a side view and an expanded view of section c, for a fluidic box with fixed flow cell; FIG. 10B shows the expanded view of the flow cell after being released from the fluidic box; FIG. 10C shows a top view of a fluidic box engaged with components of a detection apparatus; FIG. 10D shows a sectional view of the fluidic box (along line m) engaged with components of a detection apparatus; and FIG. 10E shows an expanded view of the fluidic box engaged with components of a detection apparatus.
[0025] [0025] FIG. 11 shows a cross-sectional view of a rigid support aligned with a flow cell and an immersion objective. DETAILED DESCRIPTION
[0026] [0026] This disclosure provides apparatus and methods for detecting analytes, such as chemical or biological analytes. Detection can occur for analytes that are consumed, modified or produced as part of a reaction of interest. Various types of apparatus and methods are well suited for detecting repetitive reactions, such as those used to characterize or synthesize polymers. There is a wide variety of polymers in nature and an infinite variety of polymers can be made by natural processes or synthetic processes, which, however, use a relatively small number of monomeric building blocks. For example, DNA is synthesized in nature from four different nucleotides, so is RNA. Protein, another ubiquitous polymer, is made up of 20 different genetically encoded amino acids. Apparatus and methods of the present disclosure can be configured to sequentially detect monomeric building blocks, thereby providing an ability to identify any sequence. In particular modalities, the apparatus and methods can be configured to detect analytes that are consumed, produced or modified during a repetitive multi-cycle reaction process. For example, intermediate products can be detected in each individual cycle. For a more specific example, nucleic acids can be sequenced by serially distributing reagents that specifically react with, or bind to, the four different types of nucleotide monomers and components of each reaction (for example, labeled nucleotides or labeled polymerases) can be detected during or after each cycle. Alternatively, nucleic acids can be synthesized by serially distributing one of four different nucleotide monomers, or precursors thereof, in a predefined order for a growth polymer and then products (for example, blocking fractions released during deprotection) can be detected for each cycle. Protein sequencing or synthesis can also be detected cyclically using the apparatus and methods established here.
[0027] [0027] Several aspects of the present invention are exemplified in relation to scan detection. It will be understood that the apparatus and the methods set forth herein can be used for spatially resolved manipulation needs reagents or substrates in a container, whether or not the reagents or substrates are detected. For example, light energy can be distributed to a container to perform photoreactions at spatially resolved locations in a container or to manufacture light-responsive materials in a spatially resolved manner.
[0028] [0028] This disclosure provides apparatus and methods that can be used to observe a container by translational movement of the container in relation to a detector. Apparatus and methods for treating a container are also provided, for example, by localized energy distribution, by translational movement of the container in relation to an energy source. When detecting analytes, this scanning movement allows the detector to collect signals from sequential subsections of the container. The collective signal combination adds up to a total detection field that is greater than the static detection field of the detector. Taking, for example, a container having an internal surface on which a matrix of optically marked analytes, the translation of the container relative to an optical detector can provide an image of the matrix that is larger than the field of view of the detector. Likewise, sweep-based energy distribution can allow sequential reactions to be carried out in a container.
[0029] [0029] A difficulty that plagues many scanning detectors is that mechanisms to move the container in relation to the detector are coupled with mechanisms to adjust the rotational coincidence of the container in relation to the detector. As such, the scan detector is overloaded with a tolerance stack that includes not only translational tolerances, but also rotational tolerances. Relatively small amounts of roll rotation or tilt rotation (ie rotation around the x axis and rotation around the y axis, respectively, as diagrammed in FIG. 1) can have significant adverse impacts on a matrix's high resolution image analyte. This adverse impact is exacerbated in optical scanning applications, as a small tilt deviation (ie rotation around the y axis) will manifest itself as an increasing out of focus deviation when the optical detector sweeps a container along the x dimension . The longer the scan, the greater the focus shift.
[0030] [0030] A common solution to the problem of high tolerance stacks in optical scanners has been to employ mobile stages having high precision actuators that are adjustable in a variety of translational and rotational directions. High-precision actuators add cost and complexity to a scanner and these platforms typically require highly trained technicians for routine maintenance. Particular modalities of the apparatus and methods established herein avoid these problems by uncoupling the mechanism that is used to move a container in relation to a detector from the mechanism that is used to rotate the container in relation to the detector. Uncoupling translation from the rotational coincidence reduces the tolerance stack for the translation mechanism in the detection device and other devices of the present disclosure.
[0031] [0031] An additional advantage of replacing a typical stage with a container translation apparatus of the present disclosure is that the container can be scanned more quickly. The increase in sweep speed is largely a function of the container translation apparatus being configured to move a mass that is less than a typical stage. A small mass takes less time to settle compared to a larger mass that is moved the same distance. For example, the time spent waiting for a container to settle before acquiring an image becomes increasingly significant as the desired detection resolution increases, because the movement of the container must cushion to a point where the average displacement experienced by object characteristics under observation is small enough to prevent substantial distortion in the image. Taking a typical nucleic acid sequencing apparatus as an example, DNA is present in sites in a matrix that are only a few microns apart and are observed in low-micron resolution. A typical stage used to move the matrix for sequencing requires settling times of several hundred milliseconds to cushion to the point that the displacements are less than a few microns. Avoiding a typical stage using a device of the present disclosure allows settling times of the order of a few tens of milliseconds. Milliseconds can result in up to hours for a nucleic sequencing protocol or other repetitive scanning operation. For example, saving 500 hundreds of milliseconds per image results in savings of about 4 hours in settling time for a sequencing protocol that acquires 200 images per cycle and performs 150 cycles per run. Similar improvements in processing speed can be obtained for other scanning applications, such as photochemistry, photolithography, microfabrication or nanofabrication (for example, via laser engraving), laser ablation or the like.
[0032] [0032] Although the apparatus and methods set out in this document provide advantages in reducing settling time, it will be understood that the uses need not be limited to processes that include a settling step. Therefore, the apparatus and the methods established here in the context of the so-called “step and trigger” scanning procedures can be applied to continuous scanning operations, such as time-delayed integration scanning (TDI). For example, the apparatus and methods set forth herein can be modified for use in TDI line scan operations, such as those set out in the US Patent
[0033] [0033] As set out in more detail in this document, the rotational match of a container to a detector can be achieved by physically contacting the container with a reference surface, the reference surface being rotatively fixed with respect to the detector. In particular embodiments, as exemplified below, a container can be compressed to the reference surface by a preload. Separately, translation can be achieved by a sweeping actuator (for example, a gear) that interacts directly with another surface of the container (for example, a rail that complements the gear). The preload and sweep actuator does not need to interact to obtain movement and matching of the container. For example, preload does not need to be applied to the container while the container is being moved. However, the interaction between the preload and the sweep actuator can occur for certain applications of the device and the methods established here. Therefore, preload can be applied to the container while the container is being moved.
[0034] [0034] In some embodiments, a container that will be detected can be a component of a cartridge. The cartridge can provide a convenient mechanism for distributing the container to a detector. For example, a detector can be kept inside an analytical instrument to protect the detector from environmental factors, such as moisture, dust or light. A cartridge can be inserted into the analytical instrument via a door or opening, so that the container is contacted with the detector. In some embodiments, the analytical instrument will remove the container from the cartridge and move the container through the detector in a way that does not necessarily involve movement of the cartridge. Alternatively, the container can maintain contact with the cartridge, so that both the cartridge and the container are moved to obtain translation or scanning. In a further alternative, the cartridge can be a component of the analytical instrument and the container can be introduced into the instrument by placing the container in the cartridge.
[0035] [0035] Alternatively and / or in addition, the container may be a component of a box that also includes reservoirs and fluidic components that deliver reagents to the container during the course of a reaction that is detected, such as a nucleic acid sequencing reaction. In some embodiments, the box includes sufficient fluidic components that it functions as a “wet” component and the analytical instrument housing the detector functions as a “dry” component. An advantage of having separate wet and dry components is that the box and container can be dedicated to a particular sample or reaction and, when the reaction is complete, the box and container can be removed from the analytical instrument and replaced with a new box and a new container dedicated to a second sample or reaction. Since samples, reagents and reaction products for each of these two reactions are physically separated from the analytical instrument, cross-contamination between reactions, which would otherwise cause detection artifacts, is avoided.
[0036] [0036] The physical separation of the components provides an additional advantage of avoiding unnecessary downtime for the analytical instrument if the fluidic component experiences mechanical difficulties. Specifically, unlike many commercially available analytical instruments that have fluid permanently integrated, a failure in the fluid system can be conveniently overcome by simply removing a defective fluid case and replacing it with another one, so that the analytical instrument experiences little or no time of inactivity. In some embodiments, the box is disposable, for example, being made of relatively inexpensive components. The box can be configured in a way that reagents are sealed in the box, thereby avoiding unwanted contamination of the environment and unwanted exposure of laboratory personnel and equipment to reagents. Alternatively, the fluidic box can be emptied, refilled and reused, if desired, for a particular application.
[0037] [0037] In some embodiments, a fluidic box of this disclosure includes not only reagent reservoirs, but also includes one or more waste reservoirs. Reagent that is not consumed in a reaction and / or unwanted products from a reaction can be collected in the waste tank. The advantages of retaining pre- and post-reaction fluids in a box include user convenience in handling a single fluid component before and after a reaction is performed, minimizing user contact with chemical reagents, providing a compact footprint for the device and avoiding unnecessary proliferation of fluid containers.
[0038] [0038] Fluidic housings, reaction vessels and exemplary fluidic components that can be modified, in accordance with the teachings of this document, for use in combination with detection components of the present disclosure are described in Commonly Owned US Patent Application 15 / 922,661, which claims the benefit of Provisional Order US 62 / 481,289, each of which is incorporated herein by reference. Other fluidic components that are useful, particularly for cyclic reactions, such as nucleic acid sequencing reactions, are set out in US Patent Application Publication 2009/0026082 A1; 2009/0127589 A1; 2010/0111768 A1; 2010/0137143 A1; or 2010/0282617 A1; or US Patents
[0039] [0039] The details of one or more modalities are established in the attached drawings and in the description below. The drawings and description are provided as examples for the purpose of explanation and are not necessarily intended to limit the scope of the invention. The invention is susceptible to changes in methods and materials, as well as changes in manufacturing methods and equipment. Such modifications will be apparent to those skilled in the art from a consideration of the drawings and description below.
[0040] [0040] The present disclosure provides a detection device. The detection apparatus may include (a) a container having a lumen and a wall, the wall having an inner surface and an outer surface, the inner surface contacting the lumen; (b) a reference surface that forms a structural loop with a detector; (c) a preload configured to urge the outer surface of the container to contact an area on the reference surface; (d) a scan actuator configured to slide the container along the reference surface in a scan dimension; and (e) a transmitter configured to direct a signal from the inner surface or the lumen to the detector when the outer surface of the container is driven by the preload to contact the reference surface.
[0041] [0041] In particular embodiments, a detection apparatus may include (a) a container having a lumen and a wall, where the wall has an inner surface and an outer surface, where the inner surface contacts the lumen and where the outer surface has length ℓ in a scan dimension x; (b) a reference surface; (c) a preload configured to urge the outer surface of the container to contact an area on the reference surface, optionally the contact area can have a maximum length in the scan dimension x that is shorter than the length ℓ; (d) a scanning actuator configured to slide the container along the reference surface in the scanning dimension x; (e) a detector; and (f) a lens configured to direct radiation from the container to the detector when the outer surface of the container is propelled by the preload to contact the reference surface.
[0042] [0042] The present disclosure also provides a method of sweeping a container. The method may include (a) translating a container along a reference surface of a detection apparatus, where the container comprises a lumen and a wall, where the lumen comprises analytes,
[0043] [0043] In some embodiments, a method for sweeping a container may include (a) examining a first subset of analytes in a container while applying a preload to a first portion of the container, where the preload positions the first subset analytes to occupy an xy plane in a detection zone, where the preload is not applied to a second portion of the container; (b) moving the container to position a second subset of analytes on the xy plane of the detection zone; and (c) examining the second subset of the analytes in the container while applying the preload to a second portion of the container, where the preload positions the second subset of the analytes to occupy the xy plane of the detection zone, where the a preload is not applied to the first portion of the container, thereby sweeping the container.
[0044] [0044] A method of optically scanning a container is also provided. The method may include (a) providing a container having a lumen and a wall, where the lumen contains optically detectable analytes and where the wall is transparent to optically detectable analytes; (b) translating a length of the container along a reference surface and detecting the optically detectable analytes at different locations along the length, where the reference surface contacts only a portion of the length of the container at any time during translation, where the container is propelled to the reference surface by a preload during detection, where detection includes transmitting radiation through the wall, then through a lens and then to a detector, thereby optically scanning the container.
[0045] [0045] FIG. 2 shows an exemplary arrangement for scanning a container with respect to a detector. As shown in the profile views of FIG. 2A and FIG. 2B, the container is a flow cell 101 that is aligned with the objective 110 via a rigid body 100. The rear side of the rigid body 100 has a conical depression 116 that complements the shape of the objective 110. Therefore, objective 110 can be moved near the flow cell to a desired focus or resolution. Any of a variety of forms of depression can be used as desired to accommodate forms for various objectives or other optical components. The front side of the rigid body 100 has a reference surface 117 which will contact a flat face of the flow cell 101. The flow cell 101 is held in contact with the reference surface 117 by a preload that applies positive pressure to the side the flow cell 101 which is opposite the reference surface 117. The preload is formed by the compression foot 102 which contacts the flow cell 101 under spring force 103.
[0046] [0046] Generally, the reference surface 117 and the compression foot 102 create low-friction contacts with the flow cell 101. This allows the flow cell to slide across the reference surface 117 and slide through the compression foot 102 while under under preload compression force. This compression provides alignment of flow cell 101 with objective 110 via the rigid body throughout the scanning course of flow cell 101 through the objective
[0047] [0047] In the example shown in FIG. 2, reference surface 117 is polished aluminum, which provides rigidity for aligning flow cell 101 to objective 110 and a low-friction surface for sliding the glass surface of flow cell 101. Any of a variety of materials can be used to provide stiffness and low friction for the reference surface including, for example, acetal resins (eg, Delrin ® available from DuPont, Wilmington, DE), diamond like carbon or polished metals. The compression foot 102 provides a low-friction surface for the sliding translation of the glass surface of the flow cell 101 and also provides compressibility to form a compliant contact with the flow cell 101 under the spring force 103. Either of variety of materials can be used that provide low friction to the compression foot including, for example, those set out above for reference surface 117. Optionally, a low friction material used in an apparatus here can also be compressible, examples of which include , without limitation, polytetrafluoroethylene (PTFE, Teflon®), perfluoroalkoxy alkane (PFA), fluorinated ethylene propylene (FEP), silicone foam, nitrile rubber, Buna-N, Perbunan, acrylonitrile butadiene rubber or nitrile butadiene rubber (NBR) . Alternatively or additionally, low friction can be achieved using ball bearings, rollers and / or lubricating fluids. Typically, the lubricating fluid is used on the side of the flow cell that is not between the analytes and the detector or a fluid is used that does not interfere with detection. In some embodiments, lubricating fluids are not present at the interface between the reference surface and the outer surface of the container wall. For example, lubricating fluids can be avoided to avoid interference caused when the fluid enters the area between the detector and the container.
[0048] [0048] In particular modalities, a container (or cartridge containing a container) is positioned on an xy plane without contacting a reference surface. For example, a container (or cartridge) can be propelled, by a preload, towards a fluid bearing or magnetic bearing, so that the combination of forces provided by the preload and the bearing results in a desired positioning . A fluid bearing can be a gas bearing, whereby gas pressure provides a force to position the container (or cartridge). Another useful type of fluid bearing is a liquid bearing, by which liquid pressure provides a force to position the container (or cartridge). The liquid can be selected for its ability to index correspondence with optical components of the system, such as the container wall, in order to minimize aberrations when detecting optical signals or distributing radiation.
[0049] [0049] As shown in FIG. 2C and in FIG. 2D, reference surface 117 has a planar surface that forms a flat ring on the front face of rigid body 100. The ring is raised compared to the front face of rigid body 100. Raising the reference surface helps prevent unwanted contact between the flow cell 101 and the rigid body 100, which may otherwise create friction that makes translation difficult. Raising the reference surface 117 also isolates the area of the flow cell that will be detected and prevents unwanted warping that could otherwise occur if the flow cell contacted other regions of the rigid body 101. In the example of FIG. 2, the reference surface has an area that is smaller than the flow cell surface and thus only contacts a portion of the flow cell surface. However, in alternative embodiments, the reference surface can be substantially the same size or larger than the flow cell surface, and thus can substantially contact the entire flow cell surface (optionally, except the cell surface area. flow that is juxtaposed with a detection window, objective or other transmitter).
[0050] [0050] In the example shown, the reference surface 117 surrounds circular window 118, this window being a hole through rigid body 100. Alternatively, circular window 118 may include material that is capable of transmitting a signal that will be detected. For example, the window can be made of quartz, glass or plastic that facilitates the transmission of signals that will be detected. In some configurations, the window may contain a combined indexing immersion fluid which contacts the surface of the flow cell to facilitate detection, as set out in more detail below in relation to FIG. 11. The circular window 118 is aligned with the front lens 115 of objective 110, so that objective 110 can view flow cell 101 through window 118. Compression foot 102 has a flat ring shape providing a grip on the flow cell 101 which is complementary to the flat ring footprint 117 on the opposite side of the flow cell. In this example, the preload (via foot 102) has a contact area with the container (flow cell 101) which is the same as the contact area between the reference surface 117 and the container. Alternatively, the preload can have a contact area with the container that is less than the contact area between the reference surface and the container. In fact, the preload can have a contact area with the container that is no larger than the contact area between the reference surface and the container.
[0051] [0051] Generally, the complementarity between the preload footprints and the reference surface can be configured to result in the compression foot 102 having a contact area in the flow cell 101 that excludes the surface area of the opposite flow cell to the circular window 118 and which also excludes the surface area of the flow cell opposite the region of the rigid body surrounding the reference surface 117. The complementarity between the footings of the compression foot 102 and the reference surface 117 helps to maintain the flatness of the portion of the flow cell surface that is seen through window 118. This complementarity can be beneficial for detecting analytes on the internal surface of the flow cell, especially at high magnification and high resolution. Complementarity can also facilitate transillumination, so radiation can pass forwards or backwards through a path defined by the hollow space in spring 103, compression foot 102 and window 118. The circular shape of the reference surface and the preload it's exemplary. Other shapes may be used, including, without limitation, square, rectangular, polyhedral, elliptical, triangular or the like. Moreover, the form does not have to be continuous. Instead, the reference surface and / or the contact surface for the preload can be a discontinuous area, such as that formed by two parallel bands or by interruptions in the shapes above. Particularly useful applications are nucleic acid micro array detection and nucleic acid sequencing. The shapes and orientations for the preload and the reference surface can be used for apparatus that distributes energy to a container or that detects non-optical signals.
[0052] [0052] As exemplified by FIG. 2, a particularly useful container for use in a detection apparatus or other apparatus of the present disclosure is a flow cell. Any of a variety of flow cells can be used including, for example, those that include at least one channel and openings at each end of the channel. The openings can be connected to fluidic components to allow reagents to flow through the channel. The flow cell is generally configured to allow detection of analytes within the channel, for example, in the lumen of the channel or on the inner surface of a wall that forms the channel. In some embodiments, the flow cell may include a plurality of channels, each having openings at its ends. For example, the flow cell shown in FIG. 2 has three channels 120, 121 and 122, each having openings at both ends. Several channels can interact with a fluidic system via a collector.
[0053] [0053] In particular embodiments, a flow cell will include a solid support to which one or more analytes or target reagents are attached. A particularly useful solid support is one having a variety of sites. Matrices offer the advantage of facilitating multiplex detection. For example, different reagents or analytes (e.g., cells, nucleic acids, proteins, small molecule candidate therapies, etc.) can be attached to a matrix via attachment of each different analyte to a particular site in the matrix. Examples of matrix substrates that may be useful include, without limitation, a BeadChipTM Matrix available from Illumina, Inc. (San Diego, CA) or matrices such as those described in US Patents 6,266,459;
[0054] [0054] Other useful matrix substrates include those that are used in nucleic acid sequencing applications. For example, matrices that are used to create fixed amplicons of genomic fragments (often referred to as clusters) can be particularly useful. Examples of substrates that can be modified for use here include those described in Bentley et al., Nature 456: 53-59 (2008), PCT Publication WO 91/06678; WO 04/018497 or WO 07/123744; US patent 7,057,026; 7,211,414; 7,315,019;
[0055] [0055] A matrix can have sites that are separated by less than 100 µm, 50 µm, 10 µm, 5 µm, 1 µm or 0.5 µm. In particular modalities, the sites of a matrix may have an area that is greater than about 100 nm 2, 250 nm2, 500 nm2, 1 µm2, 2.5 µm2, 5 µm2, 10 µm2, 100 µm2 or 500 µm2. Alternatively or additionally, the sites of a matrix may have an area that is less than about 1 mm 2, 500 µm2, 100 µm2, 25 µm2, 10 µm2, 5 µm2, 1 µm2, 500 nm2 or 100 nm2. In fact, a site can have a size that is in a range between an upper and lower limit selected from those exemplified above. A matrix can have sites in any of a variety of densities including, for example, at least about 10 sites / cm2, 100 sites / cm2, 500 sites / cm2, 1,000 sites / cm2, 5,000 sites / cm2,
[0056] [0056] Several modalities use optical detection of analytes in a flow cell. Therefore, a flow cell can include one or more channels, each having at least one transparent window. In particular embodiments, the window may be transparent to radiation in a particular spectral range including, but not limited to, x-ray, ultraviolet (UV), visible (VIS), infrared (IR), microwave and / or radio waves. In some cases, the analytes are attached to an internal surface of the window (s). Alternatively or additionally, one or more windows can provide a visualization for an internal substrate to which the analytes are attached. Exemplary flow cells and physical characteristics of flow cells that may be useful in a method or apparatus set forth herein are described, for example, in US Patent Application 2010/0111768 A1, WO 05/065814 or US Patent Application 2012 / 0270305 A1, each of which is incorporated herein by reference in its entirety.
[0057] [0057] Several examples of this document are demonstrated for a rectangular flow cell 101 having elongated channels. In these examples, the contact area between the flow cell 101 and the reference surface 117 has a maximum length in the scan dimension x that is shorter than the length of the flow cell strip in the scan dimension x. More specifically, the diameter of ring 117 is shorter than the length of strips 120, 121 or 122. Alternatively or additionally, the contact area between flow cell 101 and reference surface 117 can have a maximum width w in dimension y that is shorter than the width of the flow cell strip in the y dimension. Specifically, the diameter of the ring 117 may be shorter than the width of any of the bands 120, 121 or 122.
[0058] [0058] Likewise, the maximum diameter or length of window 118 in the scan dimension x may be shorter than the length of the flow cell strip in the scan dimension x. Alternatively or in addition, the maximum diameter or width of window 118 in dimension y may be shorter than the width of any of the bands 120, 121 or 122. In this configuration, the complete width of the band can be observed by translation in the y direction . In some embodiments, the window area 118 and the strip width can be configured so that translation in the y dimension is not necessary to observe the entire strip width. For example, window area 118 may have a maximum diameter or width w in dimension y that is equivalent to or longer than the width of the flow cell track in dimension y.
[0059] [0059] In particular modalities, a container, such as a flow cell, can be moved in an arched path during all or part of a sweeping operation. Looking at the orientation of the flow cell in FIG. 1, the arcuate path may result from rotation about the yaw axis. The arched path can be a circle, spiral or other path that is desirable for sweeping a container. Optionally, the contact area between a container and the reference surface can have a length or an area that is less than the length or area, respectively, of the arcuate path. For a more specific example, a ring-shaped reference surface may have a diameter that is shorter than the length of the arcuate path or shorter than the length of a track in a flow cell that is moved along the path arched. Likewise, the maximum diameter or area of a window on the reference surface, through which detection occurs, may be less than the length or area, respectively, of the arcuate path; or the window may be smaller than a flow cell track that is scanned along an arched path.
[0060] [0060] A flow cell does not have to be rectangular in shape. Alternative shapes that can be used include, but are not limited to, a disc, square, polygon, or irregular shape. The strips of a flow cell can follow a linear path, arcuate path, winding path or the like. Other types of containers can also be used. For example, a well from a multi-well strip or multi-well plate can be detected using an apparatus or method of the present disclosure. The bottom surface of a well can be propelled towards a reference surface by a preload applied to the top of the container (for example, by contacting a compression foot with the top side of a multi-well plate or range of multiple wells ). Optionally, the well may have a flat bottom that contacts the reference surface. As an additional option, the well will be larger than the detector's field of view. For example, the well may be circular in shape and may have a diameter ℓ in the scan dimension x that is longer than the length of the reference surface in the scan dimension x.
[0061] [0061] Another type of exemplary container is a cylindrical or tube-shaped container, such as a capillary tube. The body of a tube can be retained on a reference surface under the force of a preload, as exemplified here for flat containers. In an exemplary configuration, the length of the tube can be parallel to the scan axis, so that scanning the tube along x will result in relative movement of the reference surface along the length of the tube. For a tube that is configured in this orientation, it can also be useful to rotate the tube on the scroll axis. This rotation will result in relative movement of the reference surface around the circumference of a section of the tube. The combination of translation along x and rotation along the scroll axis can allow a substantial surface area of the tube to contact the reference surface. For example, the tube and the reference surface can move in a helical or spiral path in relation to each other. The reference surface can be flat, as exemplified here for flow cells having a flat outer wall. Alternatively, the reference surface may have a curved shape (e.g., u-shaped or saddle cross section) that accommodates and guides a cylindrical or tube-shaped container that it contacts.
[0062] [0062] Typically, the container wall is made of a rigid material that is not readily flexible under the conditions used. In alternative embodiments, a container is made of a flexible material, for example, forming a sheet, tape, strap or tape that can be passed along a reference surface and detected while the container is under the preload . For example, a plurality of analytes, such as a matrix of nucleic acids, can be attached to the surface of the flexible material and detected when in contact with the reference surface. Exemplary flexible materials having fixed analytes are described, for example, in US Patent 9,073,033 and US Patent Application Publication 2016/0076025 A1, each of which is incorporated herein by reference.
[0063] [0063] When using a container having a flexible wall, it may be advantageous to pull the wall material over a reference surface, for example, to stretch or straighten the portion of the wall material that is observed by a detector. For example, the reference surface can be a raised rim surrounding a detection window and the flexible material can be pulled over the rim to apply a tensile force through the window. Traction can be achieved, for example, by applying suction to the flexible material via a vacuum mandrel surrounding the raised rim. Suction can be applied as an alternative or supplement to other preload mechanisms established here.
[0064] [0064] As will be evident from the examples set out here, a container can be opened (for example, a well of a multi-well plate, surface of a chip or surface of a leaf) or the container can be closed (for example , a strip of a flow cell). It will be understood that, wells of a multi-well plate can optionally be covered to create a closed container and, similarly, a sheet, belt, ribbon or ribbon can have multiple layers, so that an internal lumen occurs between layers. Alternatively, a container can have one or more open structures, such as a gutter, well or other concave structure that contains a fluid. A container can also have a convex or protruding structure, such as a pole or crest, and, optionally, individual protrusions can each be attached to one or more analytes that will be detected or manipulated.
[0065] [0065] The preload exemplified in FIG. 2 creates a pushing force on the side of the container (e.g., flow cell) that is opposite the side of the container that contacts the reference surface. The pushing force can derive from a spring, clamp, positive air pressure, positive fluid pressure, charge repulsion, charge attraction, magnetic attraction or magnetic repulsion. Alternatively, a preload can be configured to create a pulling force on the container. For example, a magnetic or ferromagnetic material that is in or on the container can be attracted to the reference surface or charges within or on the container can be attracted to the reference surface. In this example, the reference surface or area surrounding the reference surface may contain magnetic or ferromagnetic material that acts as a preload. In another embodiment, the pulling force can result from a vacuum chuck that is configured to apply suction to an area of the container that contacts the reference surface. In an additional embodiment, a magnetic clamping force can be used, whereby the container is sandwiched between a magnetic or ferromagnetic material on or around the reference surface that attracts a magnetic or ferromagnetic body that is external to the opposite side of the container.
[0066] [0066] A detection device or other device of the present disclosure can include a scanning actuator that is configured to slide a container along a reference surface. The container can slide along the reference surface and along the surface of the preload. Generally, the sweep actuator is configured to move the container while the container is in contact with the reference surface under the impulse of a preload. However, it is also possible to move the container without simultaneously applying a preload to the container. It is also possible to move the container through a space defined by a bearing that does not physically contact the container, such as a fluid bearing or magnetic bearing. For example, a container can be positioned using opposite forces from a preload against a bearing. Particularly useful actuators employ one or more gears that interact with perforations or threads in a flow cell or in a cartridge that contains the flow cell. Several examples are set out below.
[0067] [0067] In some embodiments, the sweep actuator may use a film sprocket mechanism. The container to be transferred, or a cartridge that holds the container, can contain a perforation track that engages a gear wheel in a detection device to obtain translation. As shown in the exemplary configuration of FIG. 3, flow cell 101 is housed in cartridge 125, which contains two perforation tracks 130 and 140. Perforation track 130 is located near the top edge of cartridge 125 and runs parallel to the longest dimension ℓ of the flow cell. Drill track 140 is located near the opposite edge of cartridge 125 and also runs parallel to ℓ. The sprockets 150 and 160 are configured to engage the drilling tracks 130 and 140, respectively, when propelled towards the reference surface 117 by the force of the preload spring 103. The flow cell 101 can be translated in the dimension of sweep x, which is parallel to ℓ, by turning the engaged sprockets 150 and 160.
[0068] [0068] FIG. 4A shows a cartridge 400 having an insert 403 for flow cell 430. The insert includes notches 404 and 405 that are placed to facilitate adjustment or removal of flow cell 430. Cartridge 400 has a simple perforation path 401 near of the upper edge 402. As shown in FIG. 4B, the perforations are complementary to the teeth on the gearwheel 420 and the drilling track 401 is inserted into the face of the cartridge 400, thereby providing a track that engages the guide 410. The guide 410 engages the drilling track 401 to prevent rotation of the cartridge 400 on the yaw axis during translation under the action of cog 420, thereby preventing unwanted yaw rotation of flow cell 430 relative to a detector. As shown in FIG. 4C, flow cell 430 includes a lower plate 431 which is sized for pressure fitting with insert 403 and also includes an upper plate 440. A channel 443 is formed between plates 431 and 440 due to the presence of a spacer or a gasket. The top plate 440 also includes holes 441 and 442 that act as an inlet and outlet for channel 443. A perspective view of the cartridge 400 with mounted flow cell 430, sprocket 420 with motor 425 and guide 410 is shown in FIG. 4D.
[0069] [0069] Another useful mechanism for sweeping actuation is a spur gear that engages the teeth on one edge of a flow cell or on the edge of a cartridge retaining the flow cell. FIG. 5A shows the cartridge 200 which is press-fit into the flow cell 101 and which has a serrated lower edge 240 and a smooth upper edge 241. The serrated lower edge 240 engages with the spur gear 230 when the cartridge 200 is driven by the spring of preload 103 to contact a reference surface on rigid body 100. Cartridge 200 and flow cell 101 are translated by rotating spur gear 230. Wheel guides 210 and 220 engage the smooth edge 241 of cartridge 200, when cartridge 200 is positioned to contact flow cell 101 with a reference surface on rigid body 100. The wheel guides work to prevent rotation of cartridge 200 and flow cell 101 about the yaw axis.
[0070] [0070] The sweep actuation can also employ a ball screw that engages a threaded lock in a flow cell or a cartridge retaining the flow cell. FIG. 6A shows the cartridge 300 which is pressed into the flow cell 101 and which has a threaded lock 311 at the top and two guide locks 312 and 313 at the bottom. Threaded lock 311 engages with screw 310 when cartridge 300 is driven by preload spring 103 to contact a reference surface on rigid body 100. Cartridge 300 and flow cell 101 are translated by turning screw 310 against threads of the lock 311. Guide locks 312 and 313 engage with rail 320 when cartridge 300 is positioned to contact flow cell 101 with reference surface 117. Guide lock 312 and 313 works to prevent rotation of cartridge 300 and flow cell 101 around the yaw axis.
[0071] [0071] The sweep actuation can use mechanical contact between the motor and the container (or container cartridge), as exemplified above. Alternatively or additionally, the interaction between motor and container (or container cartridge) can be mediated by magnetic attraction. For example, the container or cartridge may have a magnetic or ferromagnetic material that interacts with a magnetic or ferromagnetic component of the actuator.
[0072] [0072] If using mechanical contact or other interactions to mediate the actuation, a linear motor can be used to trigger the sweeping movement. Exemplary linear motors that can be used include synchronous linear motors, linear induction motors, homopolar linear motors and piezoelectric linear motors.
[0073] [0073] An apparatus of the present disclosure can also include an actuator y configured to change the relative translational position of the detector and the container along the dimension y. Taking as an example the apparatus shown in FIG. 2, an actuator y can operate, for example, by changing the relative translational position of the objective 110 and the reference surface 117. Alternatively or additionally, an actuator y can operate by changing the relative translational position of the flow cell 101 and the reference surface 117. Translation along the y dimension can allow different bands of a flow cell to be treated. When a range is wider than the field of view for the lens, the y translation can be used to detect multiple tracks in the range (ie, a first track can be detected by scanning along x and a second track can be treated) by a step along the y dimension followed by a second scan along x). An actuator y can be configured similarly to the actuators x exemplified here. For example, an actuator y can be configured to translate the flow cell while it is propelled to a reference surface by a preload. Other stepping motors or travel actuators can also be used for x or y translation.
[0074] [0074] In particular embodiments, an apparatus of the present disclosure may include a rotational actuator configured to change the relative translational position of the detector and the vessel along an arcuate path. Taking the exemplary flow cell oriented as shown in FIG. 1, a rotational actuator can rotate the flow cell on the yaw axis. Yaw axis rotation can be particularly useful for sweeping tracks or features that follow an arched path. An additional or alternative rotary actuator can rotate a container along the scroll axis. Rotation on the yaw axis can be particularly useful when the container is a tube or cylinder that is oriented to have its length along the x axis.
[0075] [0075] Several modalities of the present disclosure are exemplified in relation to an objective having several lenses to collect and focus the radiation of an object (for example, a container, such as a flow cell). It will be understood that any one of a variety of optical elements can serve as a lens in an apparatus or method of the present disclosure including, for example, a lens, mirror, optical fiber, fiber bundle, lens array or other optical element that brings together radiation of an object being observed, whether or not the optical element is also capable of focusing the radiation. Lenses or other optical components used in an apparatus or method set forth herein can be configured to transmit radiation in any of a variety of spectral bands including, but not limited to, X-ray, ultraviolet (UV), visible (VIS), infrared bands (IR), microwaves and / or radio waves.
[0076] [0076] A lens that is used in an apparatus established in this document can be placed to direct radiation from the inner surface or from the lumen of a container, through the wall of the container and to a detector, when the outer surface of the container contacts a surface of reference. In particular modes, a lens and other optional components of an optical system can be configured to detect epi-luminescence luminance (ie, epi-luminescence), whereby excitation radiation is directed from a radiation source, through the lens , then through the wall of the container to the inner surface or the lumen of the container; and so that the emission from the inner surface or the lumen of the container is directed back through the wall and through the objective (that is, excitation and emission pass amabs through the objective). Alternatively, objectives and other optional components of an optical system can be configured for transillumination fluorescence, whereby excitation radiation is directed from a radiation source through a first container wall to the inner surface or the container lumen; and so that the emission from the inner surface or the lumen of the container is directed through another wall of the container and through the objective (that is, emission passes through the objective, excitation not). Other useful settings for fluorescence detection include those that excite a vessel via total internal reflection fluorescence (TIRF) or through waveguides. In any of a variety of configurations, the radiation source can form a structural circuit with a reference surface, so that a container that contacts the reference under the impulse of a preload is properly oriented in relation to the radiation source .
[0077] [0077] The objectives shown in FIGs. 2, 3, 5 and 6 are exemplary, having 4 lenses. Any number or type of lens can be included to suit a particular application. Particularly useful objectives will have a numerical opening that is at least 0.1 and at most 0.9. Numerical openings above 0.95 can be obtained using an immersion objective, as set out in more detail below. A lens or other transmitter can be configured to operate with a detection system that resolves characteristics (for example, nucleic acid sites) on a surface that is separated by less than 100 µm, 50 µm, 10 µm, 5 µm, 1 µm or 0.5 µm. The detection system, including objective or other transmitter, can be configured to resolve characteristics having an area on a surface that is less than about 1 mm2, 500 µm2, 100 µm2, 25 µm2, 10 µm2, 5 µm2, 1 µm2, 500 nm2 or 100 nm2.
[0078] [0078] An optical system used in an apparatus or method established here may have a field of view that is at least 0.1 mm2, 0.5 mm2, 1 mm2, 2 mm2, 3 mm2, 4 mm2 or higher. Alternatively and / or in addition, the field of view can be configured to have a maximum of 4 mm2, 3 mm2, 2 mm2, 1 mm2, 0.5 mm2, 0.1 mm2 or less.
[0079] [0079] The objective, or other appropriate component of a detection system used in an apparatus established in this document, can be configured to focus on analytes that are in or on the container. For example, the device may include a focus actuator configured to change the relative position of the objective and the reference surface in the focus dimension z. Physically aligning the container with the reference surface under the force of a preload effectively fixes the position of the container in the z dimension, thus favoring precise and robust focusing during a sweeping operation.
[0080] [0080] An apparatus established herein may employ subsystems or optical components used in nucleic acid sequencing systems. Several of these detection devices are configured for optical detection, for example, detection of fluorescent signals. Examples of detection apparatus and components thereof that can be used to detect a container in this document are described, for example, in US Patent Application 2010/0111768 A1 or US Patent 7,329,860; 8,951,781 or 9,193,996, each of which is incorporated herein by reference. Other detection devices include those marketed for nucleic acid sequencing, such as those provided by IlluminaTM, Inc. (for example, HiSeqTM, MiSeqTM, NextSeqTM or NovaSeqTM systems), Life TechnologiesTM (for example, ABI PRISMTM systems, or SOLiDTM), Pacific Biosciences (for example, systems using SMRTTM Technology, such as SequelTM or RS IITM systems), or Qiagen (for example, GenereaderTM system). Other useful detectors are described in US Patents
[0081] [0081] Generally, a lens is the optical element of the detection device that is proximal (that is, closer) to the container that will be detected (for example, flow cell). In some embodiments, the container does not need to include any optical components. In alternative embodiments, one or more optical components, such as lenses or fiber optics, can be provided by a container or by a cartridge to which the container is attached. For example, the lens of the detection device can be configured to direct excitation,
[0082] [0082] A detection device that is used to observe a container in a method or device set out in this document does not need to be capable of optical detection. For example, the detector can be an electronic detector used for the detection of protons or pyrophosphate (see, for example, US Patent Application 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1, each one of which is incorporated herein by reference in its entirety, either the commercially available Ion TorrentTM systems from ThermoFisher, Waltham, MA) or as used in nanopore detection, such as those marketed by Oxford NanoporeTM, Oxford UK (eg MinIONTM systems or PromethIONTM) or set forth in US Patent 7,001,792; Soni & Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); or Cockroft, et al. J. Am. Chem. Soc. 130, 818- 820 (2008), each of which is incorporated herein by reference.
[0083] [0083] In a particular modality, the device or the methods established here can be configured for scanning electron microscopy (SEM). Accordingly, an electron beam can be produced by an electron gun and directed to a container by one or more condensing lenses, scanning coils and / or baffle plates. The signal can be detected using an electron detector, such as a scintillator-photomultiplier system (for example, an Everhart-Thornley detector).
[0084] [0084] In particular modalities, a detection device or other device of the present disclosure can provide temperature control of a container that will be detected. Temperature control can be provided by controlling the temperature of an internal chamber that houses the container. Alternatively or additionally, a container that will be detected can be placed in contact with a thermally conductive surface that is controlled in temperature. FIG. 7A shows an exemplary configuration for achieving temperature control of a flow cell via contact with a thermally conductive surface. The rear side of the aluminum body 460 is attached to two thermal elements 450 and 451, which are located to the left and to the right of the conical depression 416. The thermal elements can be polyimide thermo-sheet heaters, Peltier elements, heating elements metal, ceramic heating elements, polymer PTC heating elements or the like. The aluminum body 460 also includes two legs 461 and 462 for attachment to the detection device. As such, the two legs form part of the structural circuit between the reference surface on the aluminum body 460 and the detection device. Optionally, legs 461 and 462 can be made of a material having low thermal conductivity. Thus, the legs can work to attach the aluminum body to a detection device in a way that isolates other components of the detection device from experiencing unwanted temperature fluctuations. The thermal elements 450 and 451 can be activated via wires 452 and 453 to heat or cool the aluminum body 460, so that a flow cell in the cartridge 400 is in contact with the opposite side of the aluminum body 460 and thus controlled in temperature. As shown in FIG. 7B, the conical depression 416 is configured to accept a lens 410 for detecting a flow cell in cartridge 400 through window 418. In the configuration shown, flow cell cartridge 400 is translated via film sprocket 420 under control rotary motor 425.
[0085] [0085] A detection apparatus or other apparatus of the present disclosure may include a fluidic system for delivering reagents to a container that will be detected. Accordingly, one or more reservoirs can be connected fluidly to a container inlet valve. The apparatus may also include a pressure supply for carrying reagents from the reservoirs to the container. The apparatus may include a waste reservoir that is fluidly connected to the container to remove spent reagents. Taking as an example a mode in which the container is a flow cell, the reagents can be distributed via the pump to the flow cell through the inlet, and then the reagents can flow through the outlet of the flow cell into a waste reservoir. . Reservoirs may include reagents for any of a variety of analytical procedures including, but not limited to, nucleic acid sequencing, nucleic acid genotyping, nucleic acid expression analysis, protein sequencing, protein binding analysis (for example, ELISA ), small molecule receptor binding, protein phosphorylation analysis, nucleic acid synthesis or protein synthesis. Alternatively or in addition, the reservoirs may include reagents for a preparative process. Examples of preparative processes include, but are not limited to, nucleic acid synthesis, peptide synthesis, oligonucleotide gene assembly, photolithography, nanofabrication or microfabrication (for example, via laser engraving), laser ablation or the like.
[0086] [0086] A fluidic system can include at least one collector and / or at least one valve to direct reagents from the reservoirs to a container where detection occurs. Collectors are particularly useful in sequencing instruments due to the relatively large number of different reagents that are distributed during a sequencing protocol. Exemplary protocols and useful reagents are set out in more detail below and in references that are incorporated here by reference. The fluid flow from the reservoirs can be selected via valves, such as a solenoid valve (for example, those manufactured by Takasago Electric, Japan), ball valve, diaphragm valve or rotary valve.
[0087] [0087] One or more fluidic components used in a detection apparatus or other apparatus of the present disclosure can be housed in a fluidic carrier that is separable from the detection components. An exemplary fluidic carrier 600 is shown in FIG. 8A. The fluidic conveyor 600 includes a housing 601 having sufficient internal volume to accommodate reagent reservoirs 603, waste reservoirs 602 and a piston shaft 604 for an external pump. Any of a variety of fluidic components can be housed in a fluidic box including, but not limited to, one or more reservoirs, fluid lines, valves or pumps. The fluidic box conveyor includes locks 610 and 611 that are configured to hook with a detection device. See, for example, switch hook 701 in FIG. 9. Flow cell 430 is retained within cartridge 400 and cartridge 400 is retained in fluid box 600 via hook 616 and guides 616 and 617. As shown in the expanded cutout of FIG. 8B and in the side view FIG. 8D, hook 615 includes a tooth 614 that inserts into track 401 to hold cartridge 400 in place. Guides 616 and 617 complete a three-point attachment by engaging the bottom edge of cartridge 400. Preload 620, although shown in the retracted position in FIG.8D, can be extended to push against the rear side of cartridge 400, thereby working with hook 615 and guides 616 and 617 to hold the cartridge in place by compressive forces.
[0088] [0088] Fluid case 600 includes openings, as shown in FIG. 8D and FIG. 8F. For purposes of showing fluid connections to flow cell 430, FIG. 8F shows a perspective view of box 600 that has been emptied of several other fluidic components. Aperture 605 is configured to accept the piston of an external pump. The piston can be driven by a detection device to allow control of fluid flow through the flow cell 430 during an analytical procedure (for example, a nucleic acid sequencing procedure), but the piston does not need to directly contact any fluids in the housing 600 or flow cell 430. Therefore, the detection apparatus may constitute a “dry” component that does not make direct contact with fluids, whereas housing 600 and flow cell 430 constitute “wet” components. Fluid box 600 includes two elongated openings 621 and 622 that are configured to accommodate tubes 661 and 662, respectively. The elongated shape allows the tubes to move along the x dimension when the flow cell is translated during scanning. Thus, the tubes can remain engaged with the flow cell and fluid reservoirs during a sweeping operation.
[0089] [0089] The flow cell 430 can be moved independently of the box 600 via movement of the cartridge, as previously established in this document, for example, in connection with FIG. 4. As such, housing 600 remains stationary while flow cell 430 is moved. Alternatively, a flow cell can be attached to a box, so that the box and the flow cell are translated as a unit. In a further alternative, one or more detection components of a detection apparatus can be moved while the flow cell and / or fluidic box are stationary.
[0090] [0090] The interactions between the fluidic box 600 and the components of a detection device are shown in FIG. 9. The perspective view in FIG. 9A and the top view in FIG. 9B, show housing 600 engaged in a way that presses flow cell cartridge 400 between housing 600 and aluminum body 460. When engaged, flow cell cartridge 400 contacts sprocket 420, so that motor 425 can trigger the translation of the flow cell in it. The translation will cause the flow cell to move through the 721 lens, which in turn is configured to direct fluorescence excitation from fluorometer 720 to the flow cell and direct fluorescence emission from the flow cell to fluorometer 720.
[0091] [0091] The mechanism of engaging the box 600 and the flow cell cartridge 400 with a detection device or other device of the present disclosure may be similar to inserting an 8-track cassette in an audio player. Flow cell 430 and cartridge 400 are connected to box 600, so that a user does not need to directly manipulate flow cell 430, instead sending it to the detection apparatus manipulating box 600, much like a user does not. you need to manipulate the tape inside the 8-track cassette. Likewise, individual fluidic components do not need to be handled individually, but can properly engage with actuators on the detection device when box 600 is properly placed on the detection device.
[0092] [0092] Fluid box 600 is disengaged from the detection apparatus in FIG. 9C, which illustrates mechanical elements that can be used by the detection apparatus to control the function of the fluid box 600. The detection apparatus may include a sensor or switch that responds to the presence of the fluid box and acts on functional interactions. In the example of FIG. 9, the switch hook 701 is moved when the housing 600 is properly engaged. This shift can activate one or more functions. For example, the bottom side of fluid box 600 may include one or more openings that are positioned to accept one or more valve actuators 711 on the platform
[0093] [0093] The structural circuit between flow cell 430 and fluorometer 720 includes reference surface 417, aluminum body 460, legs 461 and 462, a plate or base to which legs 461 and 462 are connected and fluorometer 720, which it is also attached to the plate or base.
[0094] [0094] FIG. 10 shows a mechanism that can be used to engage a flow cell with a detection device. FIG. 10A shows a side view and expanded details of the fluid cartridge 600 and the flow cell cartridge 400 when not engaged with a detection apparatus. When fluid box 600 is not engaged, flow cell cartridge 400 is in contact with hook 615 and guides 616 and 617. FIG. 10B shows an expanded detail of the configuration that results when housing 600 is engaged with the detection apparatus. Specifically, the flow cell cartridge 400 is moved towards the wall of the housing 600, disengaging from the hook 615 and the guides 616 and 617.
[0095] [0095] A mechanism for changing the position of the flow cell cartridge 400 is shown in FIG. 10E, which is a detailed view of the interface between the box 600, the flow cell cartridge 400 and the aluminum body 460. FIG. 10E is a detail of FIG. 10D, which is a section along line m in FIG. 10C. When box 600 is properly engaged in the detection apparatus, hook 615 and guides 616 and 617 are inserted into the grooves 471, 472 and 473 in the aluminum body 460. The grooves 471, 472 and 473 are deep enough for the Compression of the housing towards the aluminum body 460 causes the front side of the flow cell cartridge 400 to engage the sprocket 420 and the front side of the flow cell 430 to contact the reference surface 417. The compression also results in the rear side of the flow cell cartridge 400 contacting the compression foot 102. In this way, the flow cell 430 is pressed against the reference surface 417 for alignment with objective 410, which looks at flow cell 430 through the window 418. Flow cell 430 can be transferred via interaction of gearwheel 420 with drilling track 401.
[0096] [0096] Although interactions between a fluidic box and a detection device have been exemplified here using mechanical contacts, it will be understood that other mechanical switching mechanisms can be used. Electronic switches can also be used including, for example, those that are activated by electronic sensors (for example, Bluetooth), magnetic sensors, radio frequency sensors (for example, RFID), pressure sensors, optical sensors (for example, bars) or the like.
[0097] [0097] The fluidic box and the components set out above are exemplary. Other fluidic housings and fluidic components that can be used with a detection apparatus of the present disclosure are set out in the commonly owned US Patent Application 15 / 922,661, which claims the benefit of US Provisional Application 62 / 481,289 and US Patent Application 2017 / 0191125 A1, each of which is incorporated by reference. Furthermore, a similar fluidic box can be used with another device of the present disclosure, such as a reactor device, and the other device can be configured as set out above to interface with a box.
[0098] [0098] Optionally, a detection device or other device of the present disclosure can also include a computer processing unit (CPU) that is configured to operate one or more of the system components established herein. The same or different CPU can interact with the system to acquire, store and process signals (for example, signals detected in a method established here). In particular embodiments, a CPU can be used to determine, from the signals, the identity of the nucleotide that is present at a particular location in a model nucleic acid. In some cases, the CPU will identify a sequence of nucleotides for the model from the signals that are detected.
[0099] [0099] A useful CPU can include, for example, one or more of a personal computer system, server, server computer system, thin client, thick client, portable device or laptop, multiprocessor system, microprocessor based system, decoder, programmable consumer electronics, networked PC, minicomputer system, mainframe computer system, smart phone, or distributed cloud computing environment that includes any of the above systems or devices. The CPU can include one or more processors or processing units, a memory architecture that can include RAM and non-volatile memory. The memory architecture may further include removable / non-removable, volatile / non-volatile computer system storage media. In addition, the memory architecture may include one or more readers to read and write to a non-removable, non-volatile magnetic medium, such as a hard disk, a magnetic disk drive to read and write to a non-volatile removable magnetic disk, and / or an optical disc drive to read or write to a removable non-volatile optical disc, such as a CD-ROM or DVD-ROM. The CPU can also include a variety of readable media from the computer system. These media can be any available media that is accessible by a cloud computing environment, such as volatile and non-volatile media, and removable and non-removable media.
[00100] [00100] The memory architecture can include at least one program product having at least one program module implemented as executable instructions that are configured to control one or more components of a device established herein or to execute one or more portions of a method established here. For example, executable instructions can include an operating system, one or more application programs, other program modules, and program data. Generally, program modules can include routines, programs, objects, components, logic, data structures and so on, which perform particular tasks, such as processing signals detected in a method established here.
[00101] [00101] The components of a CPU can be coupled by an internal bus that can be implemented as one or more of any of several types of bus structures, including a memory bus or memory controller, a peripheral bus, a graphics port and a local processor or bus using any of a variety of bus architectures. For example, and not by way of limitation, these architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA bus (EISA), Video Electronics Standards Association (VESA) local bus and Peripheral Component Interconnects bus (PCI).
[00102] [00102] A CPU can optionally communicate with one or more external devices, such as a keyboard, a pointing device (for example, a mouse), a monitor, such as a graphical user interface (GUI) or other device that facilitates the user interaction with the nucleic acid detection system. Likewise, the CPU can communicate with other devices (for example, via a network card, modem, etc.). This communication can occur via I / O interfaces. Furthermore, a system CPU here can communicate with one or more networks, such as a local area network (LAN), a general wide area network (WAN) and / or a public network (for example, the Internet) via a suitable network adapter.
[00103] [00103] FIG. 11 shows a sectional profile view of an exemplary optical arrangement using immersion optics. The arrangement includes a lens 710 which includes a housing 720 and several lenses 711, 712 and 715. The number, position and shape of the lenses are exemplary and can vary according to the desired prescription. Also included are rigid body 700, flow cell 701 and flow cell cartridge 702. Flow cell cartridge 702 includes inlet 741 and outlet 742 for moving fluid reagents in and out of the flow cell. The underside of the rigid body 700 has a reference surface 717 which is sealed by the flow cell 710 when a preload is applied, for example, as established using the configurations set out above. Opposite this seal, rigid body 700 includes a conical depression 716 that is formed to accept the tip of objective 710. The space 716 between rigid body 700, objective 710 and the seal can be filled with immersion fluid, such as like an oil or aqueous solvent that is combined in index with the objective. As such, the immersion fluid will come in direct contact with the proximal lens 715 of the objective 710 and the surface of the flow cell 701. The fluid can be held in space 716 by seals 731 and 732, which are optionally flexible. Fluid can be added and / or removed from space 716 via line 733. Immersion optics can provide several advantages over optics that image through air including, for example, the ability to obtain a numerical aperture (NA) greater than 0.95, ability to imagine in greater depths in a container and relieve tolerances in the thickness and uniformity of container walls through which the objective resolves objects.
[00104] [00104] The present disclosure provides methods that are particularly useful for performing cyclical reactions. Each cycle can include distributing reagents for the reaction to a flow cell or other container where, optionally, the reaction or reaction products will be observed. Each cycle can also include scanning the container using apparatus or methods set forth herein. The methods are exemplified here in the context of a nucleic acid sequencing reaction. However, those skilled in the art will understand from the teaching described here how to modify the methods and apparatus for other cyclic reactions, such as nucleic acid synthesis reactions, peptide sequencing reactions, peptide synthesis reactions, synthesis reactions small combinatorial or similar molecules. However, the method does not have to be cyclical and can instead be performed in a non-repetitive configuration, for example, to observe a single reaction or a single phenomenon.
[00105] [00105] Particularly useful sequencing reactions are Sequencing By BindingTM (SBBTM) reactions, as described in US Commonly Owned US Patent Publication 2017/0022553 A1; US Patent Application 62 / 447,319 of which US Patent Application 2018/0044727 A1 claims priority; 62 / 440,624 of which US Patent Application 2018/0187245 A1 claims priority; or 62 / 450,397 of which US Patent Application 2018/0208983 A1 claims priority, each of which is incorporated herein by reference. Generally, methods for determining the sequence of a template nucleic acid molecule can be based on the formation of a ternary complex (between polymerase, initiated nucleic acid and cognate nucleotide) under specified conditions. The method may include an examination step followed by a nucleotide incorporation step.
[00106] [00106] The examination phase can be carried out in a flow cell (or other container), the flow cell containing at least one template nucleic acid molecule initiated with an initiator distributing reagents to the flow cell to form a first reaction mixture. The reaction mixture can include the initiated template nucleic acid, a polymerase and at least one type of nucleotide. The interaction of the polymerase and a nucleotide with the initiated template nucleic acid molecule (s) can be observed under conditions where the nucleotide is not added covalently to the primer (s); and the next base in each model nucleic acid can be identified using the observed interaction of the polymerase and nucleotide with the initiated model nucleic acid molecule (s). The interaction between the initiated model, polymerase and nucleotide can be detected in a variety of schemes. For example, nucleotides can contain a detectable marker. Each nucleotide can have a distinguishable marker in relation to other nucleotides. Alternatively, some or all of the different types of nucleotides can have the same marker and the types of nucleotides can be distinguished based on separate distributions of different types of nucleotides to the flow cell. In some embodiments, the polymerase can be labeled. Associated polymerases that are associated with different types of nucleotides may have unique markers that distinguish the type of nucleotide to which they are associated. Alternatively, the polymerases can have similar markers and the different types of nucleotides can be distinguished based on separate distributions of different types of nucleotides to the flow cell. Detection can be carried out by scanning the flow cell using an apparatus or method set forth herein.
[00107] [00107] During the examination phase, discrimination between correct and incorrect nucleotides can be facilitated by stabilization of the ternary complex. A variety of conditions and reagents can be useful. For example, the primer may contain a reversible blocking fraction that prevents covalent nucleotide fixation; and / or cofactors that are needed for extension, such as divalent metal ions, may be absent; and / or divalent inhibitor cations that inhibit polymerase based primer extension may be present; and / or the polymerase that is present in the examination phase may have a chemical modification and / or mutation that inhibits the extension of the primer; and / or the nucleotides may have chemical modifications that inhibit incorporation, such as 5 'modifications that remove or alter the native triphosphate fraction. The examination phase may include scanning the flow cell using the apparatus and methods set forth herein.
[00108] [00108] The extension phase can then be performed by creating conditions in the flow cell where a nucleotide can be added to the primer in each model nucleic acid molecule. In some embodiments, this involves removing reagents used in the examination phase and replacing them with reagents that facilitate extension. For example, examination reagents can be replaced by a polymerase and nucleotide (s) that are (are) capable of extension. Alternatively, one or more reagents can be added to the examination phase reaction to create extension conditions. For example, divalent catalytic cations can be added to an examination mixture that was deficient in cations and / or polymerase inhibitors can be removed or deactivated and / or competent extension nucleotides can be added and / or an unlocking reagent can be added to make the primer extension (s) competent and / or extension-competent polymerase can be added.
[00109] [00109] It will be understood that any of a variety of nucleic acid sequencing reactions can be performed using an apparatus and method of the present disclosure. Other exemplary sequencing methods are set out below.
[00110] [00110] Sequencing synthesis techniques (SBS) can be used. SBS generally involves the enzymatic extension of a nascent primer by iteratively adding nucleotides against a template strand to which the primer is hybridized. Briefly, SBS can be initiated by contacting target nucleic acids, attached to sites in a container, with one or more labeled nucleotides, DNA polymerase, etc. Those sites where a primer is extended using the target nucleic acid as a template will incorporate a labeled nucleotide that can be detected. Detection may include scanning using an apparatus or method set forth herein. Optionally, the labeled nucleotides can further include a reversible termination property that terminates the additional primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminating fraction can be added to a primer so that subsequent extension cannot occur until an unlocking agent is delivered to remove the fraction. Thus, for modalities that use reversible termination, an unlocking reagent can be delivered to the container (before or after detection occurs). Washes can be performed between the various stages of distribution. The cycle can be performed n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, reagents and detection components that can be readily adapted for use with a detection apparatus produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456: 53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; US patent 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281 and US Patent Application 2008/0108082 A1, each of which is incorporated herein by reference. Also useful are SBS methods that are commercially available from Illumina, Inc. (San Diego, CA).
[00111] [00111] Some modalities of SBS include detection of a released proton by incorporating a nucleotide in an extension product. For example, sequencing based on detecting protons released may use reagents and an electrical detector that are commercially available from ThermoFisher (Waltham, MA) or described in US Patent Publication 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1, each of which is incorporated herein by reference.
[00112] [00112] Other sequencing procedures can be used, such as pyro sequencing. Pyrosquencing detects the release of inorganic pyrophosphate (PPi) when nucleotides are incorporated into a nascent primer hybridized to a template nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al. Science 281 (5375), 363 (1998); US Patents
[00113] [00113] Link sequencing reactions are also useful including, for example, those described in Shendure et al. Science 309: 1728-1732 (2005); US patent 5,599,675; or US Patent 5,750,341, each of which is incorporated herein by reference. Some modalities may include hybridization sequencing procedures as described, for example, in Bains et al., Journal of Theoretical Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251 (4995), 767-773 (1995); or WO 1989/10977, each of which is incorporated herein by reference. In both ligation sequencing and hybridization sequencing procedures, primers that are hybridized to nucleic acid models undergo repeated cycles of oligonucleotide ligation extension. Typically, oligonucleotides are fluorescently labeled and can be detected to determine the model sequence, for example, using an apparatus or scanning method set forth herein.
[00114] [00114] Some modalities may use methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and gamma-phosphate-labeled or zero-mode waveguides (ZMW). Techniques and reagents for sequencing via FRET and or ZMW detection that can be modified for use in an apparatus or method established herein are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008); or US Patents 7,315,019; 8,252,911 or 8,530,164, the disclosures of which are hereby incorporated by reference.
[00115] [00115] The steps for the above sequencing methods can be performed cyclically. For example, the steps for examining and extending an SBBTM method can be repeated, so that in each cycle a single correct nearby nucleotide is examined (ie, the correct nearby nucleotide being a nucleotide that correctly binds to the nucleotide in a nucleic acid of template is located immediately 5 'from the base in the template which is hybridized to the 3' end of the hybridized primer) and subsequently a single correct nucleotide nearby is added to the primer. Any number of cycles of a sequencing method set forth herein can be performed including, for example, at least 1, 2, 5, 10, 20, 25, 30, 40, 50, 75, 100, 150 or more. Alternatively or additionally, no more than 150, 100, 75, 50, 40, 30, 25, 20, 10, 5, 2 or 1 cycles are performed.
[00116] [00116] Nucleic acid model (s) to be sequenced can be added to a container using any of a variety of known methods. In some embodiments, a single nucleic acid molecule will be sequenced. The nucleic acid molecule can be delivered to a container and, optionally, can be attached to a surface in the container. In some embodiments, the molecule is subjected to single molecule sequencing. Alternatively, multiple copies of the nucleic acid can be made and the resulting set can be sequenced. For example, nucleic acid can be amplified on a surface (for example, on the inner wall of a flow cell) using techniques set out in more detail below.
[00117] [00117] In multiplex modalities, a variety of different nucleic acid molecules (i.e., a population having a variety of different sequences) is sequenced. The molecules can optionally be attached to a surface in a container. Nucleic acids can be fixed at unique sites on the surface and unique nucleic acid molecules that are spatially distinguishable from each other can be sequenced in parallel. Alternatively, the nucleic acids can be amplified on the surface to produce a plurality of sets attached to the surface. Sets can be spatially distinguishable and sequenced in parallel.
[00118] [00118] A method set out in this document can use any of a variety of amplification techniques in a container. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), bridge amplification or random primer amplification (RPA). In particular embodiments, one or more primers used for amplification can be attached to a surface in a container. In such embodiments, the extension of the primers fixed to the surface along the model nucleic acids will result in copies of the models being fixed to the surface. Methods that result in one or more sites on a solid support, where each site is attached to multiple copies of a particular nucleic acid model, can be called "clustering" methods.
[00119] [00119] In PCR modalities, one or both of the primers used for amplification can be attached to a surface. Formats using two fixed primer species are often referred to as bridge amplification, because double-stranded amplicons form a bridge-like structure between the two fixed primers that flank the model sequence that has been copied. Exemplary reagents and conditions that can be used for bridge amplification are described, for example, in US Patent 5,641,658 or 7,115,400; US Patent Publication 2002/0055100 A1, 2004/0096853 A1, 2004/0002090 A1, 2007/0128624 A1 or 2008/0009420 A1, each of which is incorporated herein by reference. PCR amplification can also be performed with one of the amplification primers attached to the surface and the second primer in solution. An exemplary format that uses a combination of a primer fixed to the solid phase and a solution phase initiator is known as a primer walk and can be performed as described in US Patent 9,476,080, which is incorporated herein by reference. Another example is emulsion PCR that can be performed as described, for example, in Dressman et al., Proc. Natl. Acad. Sci. USA 100: 8817-8822 (2003), WO 05/010145, or US Patent Publication 2005/0130173 A1 or 2005/0064460 A1, each of which is incorporated herein by reference.
[00120] [00120] RCA techniques can be used in a method established here. Exemplary reagents that can be used in an RCA reaction and principles by which RCA produces amplicons are described, for example, in Lizardi et al., Nat. Genet. 19: 225-232 (1998) or US Patent Application 2007/0099208 A1, each of which is incorporated herein by reference. The primers used for RCA can be in solution or attached to a surface in a flow cell.
[00121] [00121] MDA techniques can also be used in a method of the present disclosure. Some reagents and conditions useful for MDA are described, for example, in Dean et al., Proc Natl. Acad. Sci. USA 99: 5261-66 (2002); Lage et al., Genome Research 13: 294-307 (2003); Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20: 1691-96 (1992); or US Patent 5,455,166; 5,130,238; or 6,214,587, each of which is incorporated by reference. Primers used for MDA can be in solution or attached to a surface in a container.
[00122] [00122] In particular modalities, a combination of the amplification techniques exemplified above can be used. For example, RCA and MDA can be used in a combination where RCA is used to generate a concatemeric amplicon in solution (for example, using solution phase initiators). The amplicon can then be used as a model for MDA using primers that are attached to a surface in a container. In this example, amplicons produced after the combined RCA and MDA steps will be attached to the container. Amplicons will generally contain concatemeric repeats of a target nucleotide sequence.
[00123] [00123] Nucleic acid templates that are used in a method or composition in this document can be DNA, such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA) or the like. RNA can also be used, such as mRNA, ribosomal RNA, tRNA or the like. Nucleic acid analogs can also be used as models in this document. Thus, a mixture of nucleic acids used herein can be derived from a biological source, synthetic source or amplification product. Primers used here can be DNA, RNA or the like.
[00124] [00124] Exemplary organisms from which nucleic acids can be derived include, for example, those of a mammal, such as rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat , dog, primate, human or non-human primate; a plant, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola or soy; an algae, such as Chlamydomonas reinhardtii; a nematode, such as Caenorhabditis elegans; an insect, such as Drosophila melanogaster, mosquito, fruit fly, bee or spider; a fish, such as zebrafish; a reptile; an amphibian, such as a frog or Xenopus laevis; a dictyostelium discoideum; a fungus, such as pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe; or a plasmodium falciparum. Nucleic acids can also be derived from a prokaryote, such as a bacterium, Escherichia coli, staphylococci or mycoplasma pneumoniae; an archae; a virus, such as Hepatitis C virus or human immunodeficiency virus; or a viroid. Nucleic acids can be derived from a homogeneous culture or population of the above organisms or, alternatively, from a collection of several different organisms, for example, in a community or an ecosystem. Nucleic acids can be isolated using methods known in the art including, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference. Cells, tissues, biological fluids, proteins and other samples can be obtained from these organisms and detected using an apparatus or method established here.
[00125] [00125] A template nucleic acid can be obtained from a preparative method, such as genome isolation, genome fragmentation, gene cloning and / or amplification. The model can be obtained from an amplification technique, such as polymerase chain reaction (PCR),
[00126] [00126] The present disclosure further provides a detection apparatus that includes (a) a container having a lumen and a wall, in which the wall has an internal surface and an external surface, in which the wall has a plurality of discrete contacts between the inner surface and the outer surface, where the inner surface contacts the lumen and where the plurality of discrete contacts occupies a length ℓ in a scan dimension x; (b) a transmissive surface; (c) a preload configured to urge discrete contacts on the outer surface of the container to contact the transmissive surface; optionally, the transmissive surface area may have a maximum length in the scan dimension x which is shorter than the length ℓ; (d) a scanning actuator configured to slide the container along the transmissive surface in the scanning dimension x; and (e) a detector configured to acquire signals from the discrete contacts across the transmissive surface.
[00127] [00127] As exemplified in several modalities in this document, optical signals can be retransmitted to a detection device via transmissive surface that is transparent to optical signals. One purpose serves as a useful transmitter of optical signals from a container to a detector. In some embodiments, the transmitter is an array of lenses. The lenses in the array can be configured to collect signals from (or direct energy to)
[00128] [00128] Each lens in a lens array can be aligned with its own optical train to direct radiation to one or more detectors. Alternatively, multiple lenses can be combined in a common optical train to direct radiation to one or more detectors. Optical trains can include any of a variety of optical components including, but not limited to, a collimating lens to collimate signals from the matrix of sites, a color separation element to spectrally separate radiation; and a focusing lens to focus radiation from the sites to a detector. Exemplary configurations for a lens array and an array of sites observed by the lens are provided in US Patent 9,581,550, which is incorporated herein by reference. For example, the matrix sites can be zero-mode waveguides (ZMWs).
[00129] [00129] Other transmitters can be used as appropriate for the energy or signal to be transmitted. For example, a transmissive surface can conduct electrical signals, thermal signals, magnetic signals, pressure signals, audio signals or the like. Temporary electrical contacts, such as spring pins, can be used to transmit electrical signals between the transmissive surface and the container. A transmitter that is present in an apparatus set forth in this document can transmit energy in a variety of ways including, but not limited to, the aforementioned signals.
[00130] [00130] In a specific embodiment, the transmissive surface or the internal surface of the container includes an electronic detector, such as a field effect transistor (FET) or complementary metal oxide semiconductor (CMOS). Particularly useful electronic detectors include, for example, those used for nucleic acid sequencing applications, such as those used for proton detection, as set out in US Patent Application Publication 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1, each of which is incorporated herein by reference. Also useful are electronic detectors used to detect optical signals including, for example, those set out in US Patent Application Publication 2009/0197326 A1; 2015/0293021 A1; 2016/0017416 A1; or 2016/0356715 A1, each of which is incorporated herein by reference.
[00131] [00131] The apparatus and methods of the present disclosure have been exemplified in the context of use for nucleic acid sequencing reactions. The apparatus and methods can also be used for other analytical applications. Generally, analytical applications that are performed in scanning microscopes can be applied to devices and methods of the present disclosure. For example, the methods or apparatus can be configured to scan microarrays that are used to analyze enzyme activity, ligand binding to receptors, binding complementary nucleic acids together, presence of mutations (such as single nucleotide polymorphisms (SNPs) ) in nucleic acids, level of expression for RNA species. Microarrays that are detected via optical markers, such as fluorophores, are particularly applicable. Larger biological samples, such as cells or tissues, can be detected using a method or apparatus in this document. Again, the detection modalities that use optically detected probes or spots are particularly applicable. Other uses include evaluating manufactured products for which quality or other characteristics are assessed via microscopic scanning. Exemplary products include, but are not limited to, computer chips, sensors, electronic components and other devices that are microfabricated or nanofabricated. Tests known in the molecular diagnostic technique can be modified for use in an apparatus or method established here, such as binding assays (for example, enzyme-linked immunosorbent assay (ELISA)), real-time polymerase chain reaction assays and similar.
[00132] [00132] The apparatus and the methods established here in the context of detection of reactions can be readily modified for use in preparative methods. In particular embodiments, the present disclosure provides a reactor apparatus. A reactor apparatus may include (a) a container having a lumen and a wall, the wall having an inner surface and an outer surface, the inner surface contacting the lumen; (b) a reference surface that forms a structural circuit with an energy source; (c) a preload configured to urge the outer surface of the container to contact an area on the reference surface; (d) a scan actuator configured to slide the container along the reference surface in a scan dimension; and (e) a transmitter configured to direct energy from the energy source to the inner surface or the lumen, when the outer surface of the container is propelled by the preload to contact the reference surface.
[00133] [00133] A method for carrying out reactions in a vessel is also provided. The method may include (a) translating a container along a reference surface of a reactor apparatus, in which the container comprises a lumen and a wall, in which the lumen comprises reagents, in which the reference surface contacts at least a portion of the container during translation and where the reference surface forms a structural circuit with the energy source; and (b) directing energy from the energy source to the reagents, in which the container is driven to the reference surface by a preload directing the energy to the reagents, thereby carrying out reactions in the container.
[00134] [00134] A method for carrying out reactions may include (a) distributing energy from a reactor apparatus to a first subset of reagents in a container while applying a preload to a first portion of the container, where the preload positions the first subset of reagents to occupy an xy plane of a reaction zone, where the preload is not applied to a second portion of the container; (b) transfer the container to position a second subset of reagents on the xy plane of the reaction zone reaction; and (c) distributing energy from the reactor apparatus to the second subset of the analytes in the container while applying the preload to a second portion of the container, where the preload positions the second subset of the analytes to occupy the xy plane, in that the preload is not applied to the first portion of the container, thereby carrying out reactions in the container.
[00135] [00135] Exemplary energy sources that can be used in the device here include, but are not limited to, radiation sources, such as laser, light emitting diode (LED), lamp, microwave source or x-ray generator; electricity source; ion beam source, such as a duoplasmitron; electron emitter, such as hot filament or hollow cathode; electric current source; or voltage source.
[00136] [00136] Throughout this application, several publications, patents and / or patent applications were referenced. The disclosures of these documents in their entirety are hereby incorporated by reference in this order.
[00137] [00137] The term "comprising" is intended here to be opened, including not only the elements recited, but also encompassing any additional elements.
[00138] [00138] As used here, the term “each”, when used in reference to a collection of items, is intended to identify an individual item in the collection, but does not necessarily refer to all items in the collection. Exceptions can occur if the explicit disclosure or context clearly determines otherwise.
[00139] [00139] A number of modalities have been described. However, it will be understood that several modifications can be made. Therefore, other modalities are within the scope of the following claims.

权利要求:
Claims (39)
[1]
1. Detection apparatus, characterized by the fact that it comprises (a) a container comprising a lumen and a wall, in which the wall comprises an internal surface and an external surface, in which the internal surface contacts the lumen; (b) a reference surface that forms, with a detector, a structural loop in which the reference surface remains rotationally fixed in relation to the detector; (c) a preload configured to urge the outer surface of the vessel to contact an area on the reference surface; (d) a scanning actuator configured to slide the container along the reference surface and along the preload in a scanning dimension, while the reference surface remains rotationally fixed in relation to the detector; and (e) a transmitter configured to direct a signal from the inner surface or the lumen to the detector when the outer surface of the container is driven by the preload to contact the reference surface.
[2]
2. Apparatus according to claim 1, characterized by the fact that the preload is configured to create a pulling force or a pushing force in the container.
[3]
Apparatus according to any of claims 1 to 2, characterized in that the preload has a contact area with the container, which is not greater than the contact area between the reference surface and the container .
[4]
Apparatus according to any of claims 1 to 3, characterized in that the preload comprises a compressible material configured to contact the container or at least one ball bearing configured to contact the container.
[5]
Apparatus according to any one of claims 1 to 4, characterized by the fact that the scan dimension is in an xy plane of a Cartesian coordinate system.
[6]
6. Apparatus according to claim 5, characterized by the fact that it further comprises an actuator z configured to change the relative position of the transmitter and the reference surface in the Z dimension of the Cartesian coordinate system.
[7]
Apparatus according to any of claims 5 to 6, characterized by the fact that the scanning dimension is linear along dimension X of the xy plane.
[8]
8. Apparatus according to claim 7, characterized by the fact that it further comprises an actuator y configured to change the relative translational position of the container and the reference surface along the dimension y.
[9]
Apparatus according to any of claims 7 to 8, characterized in that the outer surface has length ℓ in the scanning dimension x, in which the contact area on the reference surface has a maximum length in the scanning dimension x which is shorter than length ℓ.
[10]
Apparatus according to any one of claims 5 to 9, characterized by the fact that the scan dimension is an arcuate path in the xy plane.
[11]
Apparatus according to any one of claims 5 to 10, characterized in that the transmitter comprises a lens array, wherein each lens in the array is configured to observe a discrete area in the xy plane.
[12]
Apparatus according to claim 11, characterized by the fact that each lens in the lens array is configured to observe a single site in an array of sites that are present in the container.
[13]
13. Apparatus according to any of claims 1 to 12, characterized by the fact that the sweep actuator comprises a gear configured to interact with perforations in the container.
[14]
Apparatus according to any of claims 1 to 13, characterized in that the sweep actuator comprises a magnetic linear drive configured to interact with a magnetic or ferromagnetic component of the container.
[15]
Apparatus according to any one of claims 1 to 14, characterized in that the signal comprises radiation and the transmitter comprises an optical lens.
[16]
16. Apparatus according to claim 15, characterized by the fact that the reference surface surrounds an aperture for the objective.
[17]
17. Apparatus according to claim 16, characterized by the fact that the preload has a contact area with the container that is opposite the reference surface surrounding the opening.
[18]
18. Apparatus according to any of claims 1 to 17, characterized by the fact that it further comprises a radiation source that forms a structural circuit with the reference surface.
[19]
19. Apparatus according to claim 18, characterized by the fact that the radiation source is configured to direct energy through the transmitter and then to the inner surface or the lumen when the outer surface of the container is propelled by the pre- charge to contact the reference surface.
[20]
Apparatus according to any of claims 1 to 19, characterized in that the container comprises a flow cell having an inlet and outlet in fluid communication with the lumen and in which the internal surface of the wall comprises a matrix of different nucleic acid sites.
[21]
21. Method for sweeping a container, characterized by the fact that it comprises (a) moving a container along a reference surface of a detection apparatus, in which the container comprises a lumen and a wall, in which the lumen comprises analytes , where the reference surface contacts at least a portion of the container during translation and where the reference surface forms, with a detector, a structural circuit in which the reference surface remains rotationally fixed in relation to the detector during translation; and (b) detecting the analytes at different locations along the container using the detector, where the vessel is propelled to the reference surface by a preload during detection, thereby sweeping the container.
[22]
22. Method according to claim 21, characterized in that the reference surface contacts only a portion of the length of the container at any time during translation,
[23]
23. Method according to any of claims 21 to 22, characterized in that the signals comprise optical signals produced by at least one of the analytes.
[24]
24. Method, according to claim 23, characterized by the fact that the detection apparatus further comprises a lens that is configured to transmit optical signals from the lumen to the detector.
[25]
25. Method, according to claim 24, characterized by the fact that it also comprises focusing the objective by changing the relative distance between the objective and the reference surface.
[26]
26. Method according to any of claims 24 to 25, characterized in that the objective observes the container through an opening in the reference surface.
[27]
27. Method, according to claim 26, characterized by the fact that the preload has an area of contact with the container that is opposite to an area of the reference surface that surrounds the opening.
[28]
28. Method according to any of claims 21 to 27, characterized in that the translation comprises changing the translational position of the container and the reference surface in a linear direction or in an arcuate direction.
[29]
29. Method according to any one of claims 21 to 28, characterized in that the translation comprises contacting a rotating gear with perforations in the container or in a cartridge that holds the container.
[30]
30. Method according to any one of claims 21 to 29, characterized in that the translation comprises magnetic interactions of a linear actuator with a magnetic or ferromagnetic material in the container or in a cartridge that holds the container.
[31]
31. Method according to any of claims 21 to 30, characterized in that the container is propelled to the reference surface by a preload during translation.
[32]
32. Method according to any of claims 21 to 31, characterized in that the detection comprises detecting analytes that are attached to an internal surface of the wall.
[33]
33. Method according to claim 32, characterized by the fact that the analytes form a matrix of sites on the inner surface of the wall.
[34]
34. Method according to claim 33, characterized in that the detection apparatus further comprises a lens array which is configured to transmit optical signals from the site array to the detector.
[35]
35. Method according to claim 34, characterized by the fact that each lens in the lens array is configured to observe a single site in an array of sites.
[36]
36. Method according to claim 35, characterized in that the detection comprises resolving sites in the matrix that are present at a density of at least 100 sites per cm2.
[37]
37. Method according to claim 36, characterized in that the scanning of the container is carried out in a method of sequencing nucleic acids at the sites.
[38]
38. Reactor apparatus, characterized by the fact that it comprises (a) a container comprising a lumen and a wall, wherein the wall comprises an inner surface and an outer surface, where the inner surface contacts the lumen; (b) a reference surface that forms, with an energy source, a structural loop in which the reference surface remains rotationally fixed in relation to the energy source; (c) a preload configured to urge the outer surface of the vessel to contact an area on the reference surface; (d) a scanning actuator configured to slide the container along the reference surface and along the preload in a scanning dimension, while the reference surface remains rotationally fixed in relation to the power source; and (e) a transmitter configured to direct energy from the energy source to the inner surface or the lumen, when the outer surface of the container is propelled by the preload to contact the reference surface.
[39]
39. Method for carrying out reactions in a vessel, characterized by the fact that it comprises (a) transferring a vessel along a reference surface of a reactor apparatus, in which the vessel comprises a lumen and a wall, in which the lumen comprises reagents, in which the reference surface contacts at least a portion of the container during translation, in which the reference surface forms, with an energy source, a structural circuit in which the reference surface remains rotationally fixed in relation to the source of energy during translation; and (b) directing energy from the energy source to the reagents at different locations along the container, where the container is propelled to the reference surface by a preload while directing the energy to the reagents, thereby carrying out reactions in the container.
Petition 870200029933, of 03/05/2020, p. 67/84 Rolling Shaft Tilt Shaft 1/18
Scan
Yaw Axis
DETAIL C SCALE 2: 1

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SG11202000964UA|2020-02-27|

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