Scanning device and method for detecting chemical and biological analytes

Abstract

An apparatus can include a container, a reference surface, a preload, a scanning actuator, and an emitter. The reference surface can form a structural loop with a detector. The preload can be configured to urge the container to contact an area on the reference surface. The scanning actuator can be configured to slide the container along the reference surface in a scanning dimension. The emitter can be configured to direct a signal from the container to a detector and / or direct energy from an energy source to the container when the container is urged to contact the reference surface by the preload.

Description

[0001] Cross-references to related applications

[0002] This application is a divisional application of the invention patent application No. 201880055806.7 entitled "Scanning apparatus and method for detecting chemical and biological analytes", filed on August 15, 2018.

[0003] This application claims priority to U.S. Provisional Application No. 62 / 545,606, filed August 15, 2017, entitled “Scanning Apparatus and Method for Detecting Chemical and Biological Analytes,” the disclosure of which is incorporated herein by reference in its entirety. Background of the Invention

[0004] This disclosure generally relates to the detection of chemical and biological analytes, and has specific applicability to nucleic acid sequencing.

[0005] Determining nucleic acid sequence information is crucial in biological and medical research. Sequence information is used to identify associations between genes and diseases and phenotypes, identify potential drug targets, and understand the mechanisms of disease development and progression. Sequence information is also an important component of personalized medicine, enabling the optimization of disease diagnosis, treatment, or prevention for specific individuals.

[0006] Due to the high cost of operating and maintaining complex instruments in current commercial products, many scientists and medical practitioners are striving to develop modern sequencing technologies. These platforms favor centralized laboratories where expensive "factory-scale" instruments are operated by trained specialists, and batch sampling is used to achieve economies of scale. This centralized system offers little flexibility in terms of performance specifications—users are forced into an ecosystem unnecessarily limited in terms of scope and diversity of use. When it comes to clinical applications, the centralized model is costly for physicians and their patients in terms of the time and money required to transport patient samples from local clinics to remote sequencing laboratories. Further delays can occur as centralized sequencing laboratories wait to receive a sufficient number of samples for economical batch processing. Other application markets such as forensics, veterinary diagnostics, food safety, agricultural analysis, and environmental analysis face similar limitations.

[0007] Therefore, there is a need for a sequencing platform more suitable for use in local laboratories to support decentralized research and clinical care systems. This invention addresses this need and also provides related advantages. Summary of the Invention

[0008] This disclosure provides a detection apparatus that may include: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the inner surface contacts the inner cavity; (b) a reference surface that forms a structural ring with a detector; (c) a preload configured to push the outer surface of the container into contact with a region on the reference surface; (d) a scanning actuator configured to slide the container along the reference surface in a scanning dimension; and (e) a transmitter configured to direct a signal from the inner surface or the inner cavity to the detector when the outer surface of the container is pushed into contact with the reference surface by the preload.

[0009] A method for scanning a container is also provided. The method may include (a) translating a container along a reference surface of a detection device, wherein the container includes an inner cavity and a wall, wherein the inner cavity contains an analyte, wherein during the translation the reference surface contacts at least a portion of the container, and wherein the reference surface forms a structural ring with a detector; and (b) using the detector to detect the analyte at different locations along the container, wherein during the detection the container is pushed against the reference surface by the preload, thereby scanning the container.

[0010] In some embodiments, a method of scanning a container may include: (a) examining a first subset of analytes in the container while applying a preload to a first portion of the container, wherein the preload positions the first subset of analytes to occupy an xy-plane in a detection area, wherein no preload is applied to a second portion of the container; (b) translating the container to position a second subset of analytes in the xy-plane of the detection area; and (c) examining the second subset of analytes in the container while applying the preload to a second portion of the container, wherein the preload positions the second subset of analytes to occupy an xy-plane in the detection area, wherein no preload is applied to the first portion of the container, thereby scanning the container.

[0011] This disclosure provides a reactor apparatus. The reactor apparatus may include: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the inner surface contacts the inner cavity; (b) a reference surface forming a structural ring with an energy source; (c) a preload configured to push the outer surface of the container into contact with a region on the reference surface; (d) a scanning actuator configured to slide the container along the reference surface in a scanning dimension; and (e) an emitter configured to direct energy from the energy source to the inner surface or the inner cavity when the outer surface of the container is pushed into contact with the reference surface by the preload.

[0012] A method for carrying out a reaction in a container is also provided. The method may include (a) translating a container along a reference surface of a reactor apparatus, wherein the container includes an inner cavity and walls, wherein the inner cavity contains reactants, wherein during the translation, the reference surface contacts at least a portion of the container, and wherein the reference surface forms a structural ring with an energy source; and (b) directing energy from the energy source along the container to analytes at different locations, wherein during the directing of energy to the reactants, the container is pushed against the reference surface by the preload, thereby carrying out the reaction in the container.

[0013] A method of carrying out a reaction in a container may include (a) transferring energy from a reactor apparatus to a first subset of reactants in the container while applying a preload to a first portion of the container, wherein the preload positions the first subset of reactants to occupy the xy plane of the reaction zone, wherein no preload is applied to a second portion of the container; (b) translating the container to position a second subset of reactants in the xy plane of the reaction zone; and (c) transferring energy from the reactor apparatus to a second subset of analytes in the container while applying the preload to the second portion of the container, wherein the preload positions the second subset of analytes to occupy the xy plane, wherein no preload is applied to the first portion of the container, thereby carrying out a reaction in the container.

[0014] In a particular embodiment, this disclosure provides a detection apparatus that may include: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the inner surface contacts the inner cavity, and wherein the outer surface has a length l in a scanning dimension x; (b) a reference surface; (c) a preload configured to push the outer surface of the container into contact with a region on the reference surface, optionally the maximum length of the contact region in the scanning dimension x may be 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) an objective lens configured to direct radiation from the container to the detector when the outer surface of the container is pushed into contact with the reference surface by the preload.

[0015] A method for optically scanning a container is also provided. The method may include: (a) providing a container having an inner cavity and walls, wherein the inner cavity contains an optically detectable analyte, and wherein the walls are transparent to the optically detectable analyte; (b) translating the length of the container along a reference surface and detecting the optically detectable analyte at different locations along that length, wherein at any time during the translation, the reference surface contacts only a portion of the container length, wherein during detection, the container is preloaded and pushed against the reference surface, wherein the detection comprises emitting radiation through the walls, then through an objective lens, and then to a detector, thus optically scanning the container.

[0016] This disclosure also provides a detection device comprising: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the wall has a plurality of discrete contacts between the inner surface and the outer surface, wherein the inner surface contacts the inner cavity, and wherein the plurality of discrete contacts occupy a length l in a scanning dimension x; (b) a transmissive surface; (c) a preload configured to push discrete contacts on the outer surface of the container into contact with the transmissive surface, optionally wherein the maximum length of a region of the transmissive surface in the scanning dimension x may be 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 through the transmissive surface. Attached Figure Description

[0017] Figure 1 The dimensions and axes of rotation for the relative orientation of components used to describe the optical system and other devices described herein are shown.

[0018] Figure 2A An exploded outline view of the flow cell and detection device is shown; Figure 2B A contour view of the flow cell in contact with the detection device is shown; Figure 2C A perspective view of the flow cell in contact with the detection device is shown; and Figure 2D An exploded perspective view of the flow cell in contact with the detection device is shown.

[0019] Figure 3A and Figure 3B The front and rear perspective views of the film sprocket mechanism for translating the flow cell relative to the detection device are shown.

[0020] Figure 4A The flow cell box is shown; Figure 4B The film sprocket and guides interacting with the flow cell box are shown; Figure 4C A flow cell is shown; and Figure 4D A perspective view of the film sprocket, guide, flow tank box, flow tank, and motor for the film sprocket is shown.

[0021] Figure 5A and Figure 5B The front and rear perspective views of the spur gear mechanism for translating the flow cell relative to the detection device are shown.

[0022] Figure 6A and Figure 6B The front and rear perspective views of the ball screw mechanism for translating the flow cell relative to the detection device are shown.

[0023] Figure 7A A perspective view of the heating plate and film sprocket scanning mechanism is shown. Figure 7B A perspective view of the objective lens, heating plate, and film sprocket scanning mechanism is shown.

[0024] Figure 8A A perspective view of a jet tank with an attached flow cell is shown; Figure 8B An enlarged view of the attachment point between the flow cell and the jet can is shown; Figure 8C A front view of a jet tank with an attached flow cell is shown; Figure 8D A side view of a jet tank with an attached flow pool is shown; Figure 8E A top view of a jet tank with an attached flow cell is shown; and Figure 8F A perspective view of the jet tank with several jet components emptied is shown.

[0025] Figure 9A A perspective view of the jet tank and flow cell interacting with the detection device is shown; Figure 9B A top view is shown of the jet tank and flow cell interacting with the detection device; and Figure 9C A perspective view of the jet canister detached from the detection device is shown.

[0026] Figure 10A A side view of a jet tank with an attached flow cell and an enlarged view of part c are shown. Figure 10B An enlarged view is shown after the flow cell is released from the jet tank; Figure 10C A top view of the jet canister engaging with components of the detection device is shown; Figure 10D A cross-sectional view of the jet canister (along line m) engaging with components of the detection device is shown; and Figure 10E An enlarged view of the jet canister that engages with the components of the detection device is shown.

[0027] Figure 11 A cross-sectional view of the rigid support aligned with the flow cell and immersion objective is shown. Detailed Implementation

[0028] This disclosure provides apparatus and methods for detecting analytes, such as chemical or biological analytes. Analytes consumed, modified, or generated as part of a target reaction can be detected. Several embodiments of the apparatus and methods are well-suited for detecting repetitive reactions, such as those used to characterize or synthesize polymers. A wide variety of polymers exist in nature and countless polymers can be produced through natural processes or synthetic processes that still utilize a relatively small number of monomers to build blocks. For example, DNA is synthesized in nature from four different nucleotides, just like RNA. Proteins are another ubiquitous polymer, produced from 20 different genetically encoded amino acids. The apparatus and methods of this disclosure can be configured to sequentially detect monomer-built blocks, thereby providing the ability to identify any sequence. In certain embodiments, the apparatus and methods can be configured to detect analytes consumed, generated, or modified during multi-cycle, repetitive reaction processes. For example, intermediate products can be detected in each individual cycle. By a more specific example, nucleic acids can be sequenced by continuously delivering reagents that specifically react or bind to four different types of nucleotide monomers, and components of each reaction (e.g., labeled nucleotides or labeled polymerases) can be detected during or after each cycle. Alternatively, nucleic acids can be synthesized by sequentially delivering one of four different nucleotide monomers or their precursors to a growing polymer in a predetermined sequence, and the product of each cycle (e.g., the blocking moiety released during deprotection) can then be detected. Protein sequencing or synthesis can also be performed cyclically using the apparatus and methods described herein.

[0029] Regarding scanning detection, various aspects of the invention are illustrated by example. It will be understood that the apparatus and methods described herein can be used for precise spatially resolved manipulation of reagents or substrates in a container, regardless of whether a reagent or substrate is detected. For example, light energy can be transferred to the container to induce a photoreaction at a spatially resolved location within the container, or to create photoreactive materials in a spatially resolved manner.

[0030] This disclosure provides apparatus and methods for observing a container by translational motion of the container relative to a detector. It also provides apparatus and methods for addressing a container by translational motion of the container relative to an energy source, for example, by transferring localized energy. When detecting an analyte, this scanning motion enables the detector to collect signals from successive segments of the container. The total detection field of the collective combination of signals is greater than the static detection field of the detector. Taking a container with an array of analyte markers attached to its inner surface as an example, translation of the container relative to an optical detector can provide an image of the array that is larger than the detector's field of view. Similarly, scanning-based energy transfer can allow sequential reactions to occur within the container.

[0031] A difficulty that plagues many scanning detectors lies in the connection between the mechanism for translating the container relative to the detector and the mechanism for adjusting the rotational registration of the container relative to the detector. Thus, the scanning detector is subjected to a superposition of tolerances, including not only translational tolerances but also rotational tolerances. Relatively small amounts of roll rotation or pitch rotation (i.e., respectively...) Figure 1 As shown, rotations around the x-axis and y-axis can significantly and adversely affect high-resolution imaging of analyte arrays. This adverse effect is exacerbated in optical scanning applications because even small pitch deviations (i.e., rotations around the y-axis) will manifest as increased defocusing when the optical detector scans the container along the x-axis. The longer the scan time, the greater the defocusing.

[0032] A common solution to the high tolerance overlap problem in optical scanners is to use a moving platform with high-precision actuators that can be adjusted in multiple translational and rotational directions. High-precision actuators increase the cost and complexity of the scanner, and such equipment typically requires routine maintenance by highly trained technicians. Specific embodiments of the apparatus and methods described herein avoid these problems by decoupling the mechanism for translating the container relative to the detector from the mechanism for rotating and registering the container relative to the detector. Decoupling translation and rotation registration reduces the tolerance overlap of the translation mechanisms in the detection apparatus and other apparatuses of this disclosure.

[0033] Another advantage of using the container translation device of this disclosure instead of a typical platform is the ability to scan containers much faster. The increased scanning speed depends heavily on the functionality of the container translation device, which is configured to move a mass smaller than that of a typical platform. A smaller mass requires less stabilization time compared to a larger mass moving the same distance. For example, as the required detection resolution increases, the time spent waiting for the container to stabilize before acquiring an image becomes increasingly important, as the container's movement must decay to a sufficiently small average displacement experienced by the features of the observed object to prevent significant distortion in the image. Take a typical nucleic acid sequencing setup as an example: DNA exists in array sites spaced only a few micrometers apart and observed at low micrometer resolution. A typical platform used for moving the array for sequencing requires hundreds of milliseconds of stabilization time to decay to a displacement of less than a few micrometers. By using the device of this disclosure to bypass a typical platform, stabilization times are allowed to be on the order of tens of milliseconds. For nucleic acid sequencing protocols or other repetitive scanning operations, a few milliseconds can add up to hours. For example, for a sequencing protocol acquiring 200 images per cycle and performing 150 cycles per run, a stabilization time saving of 50,000 milliseconds per image is achieved, totaling approximately 4 hours of savings. Similar speed improvements can be achieved for other scanning applications, such as photochemistry, photolithography, microfabrication or nanofabrication (e.g., by laser etching), laser ablation, etc.

[0034] While the apparatus and methods described herein offer advantages in reducing settling time, it should be understood that their use is not necessarily limited to processes that include a settling step. Therefore, the apparatus and methods described herein in the context of so-called “static intensity-modulated” scanning can be applied to continuous scanning operations, such as time-delay integration (TDI) scanning. For example, the apparatus and methods derived herein can be modified for TDI line scan operations, such as those derived by reference in U.S. Patent No. 7,329,860, which is incorporated herein by reference.

[0035] As further elaborated herein, rotational registration of the container relative to the detector can be achieved by physically contacting the container with a reference surface, which is rotationally fixed relative to the detector. In a particular embodiment, as illustrated below, the container can be preloaded and compressed against the reference surface. Translation, respectively, can be achieved by a scanning actuator (e.g., a gear) that interacts directly with another surface of the container (e.g., a guide rail complementary to a gear). The preload and scanning actuator do not need to interact to achieve the movement and registration of the container. For example, when translating the container, no preload needs to be applied to the container. However, for some applications of the apparatus and methods described herein, an interaction between the preload and the scanning actuator may occur. Therefore, a preload can be applied to the container during translation.

[0036] In some embodiments, the container to be tested may be a component of a box. The box can provide a convenient mechanism for transferring the container to the detector. For example, the detector may be kept inside the analyzer to protect it from environmental factors such as moisture, dust, or light. The box may be introduced into the analyzer through a door or opening, bringing the container into contact with the detector. In some embodiments, the analyzer removes the container from the box and translates it through the detector in a manner that does not necessarily involve movement of the box. Alternatively, the container may remain in contact with the box, allowing both the box and the container to be moved for translation or scanning. In another alternative, the box may be a component of the analyzer, and the container may be introduced into the instrument by placing it inside the box.

[0037] Alternatively and / or additionally, the container may be a part of a canister, which also includes a reservoir and a jetting component that delivers reagents to the container during a detection reaction, such as a nucleic acid sequencing reaction. In some embodiments, the canister includes sufficient jetting components to act as a “wet” component, and the analytical instrument housing the detector acts as a “dry” component. The advantage of having separate dry and wet components is that the canister and container can be dedicated to a specific sample or reaction, and that once the reaction is complete, the canister and container can be removed from the analytical instrument and replaced with new canisters and containers dedicated to a second sample or reaction. Because the sample, reagents, and reaction products for each of the two reactions are physically separated from the analytical instrument, cross-contamination that could cause detection artifacts between reactions is avoided.

[0038] The physical separation of components offers another advantage: unnecessary downtime for the analytical instrument can be avoided should the jet components encounter mechanical difficulties. Specifically, unlike many commercially available analytical instruments with permanently integrated fluid dynamics, failures in a jet system can be easily overcome by simply removing the faulty jet canister and replacing it with another, resulting in minimal downtime for the analytical instrument. In some embodiments, the canister is disposable, for example, made from relatively inexpensive components. The canister can be configured to seal reagents within, thereby preventing unnecessary environmental contamination and unnecessary exposure of laboratory personnel and equipment to reagents. Alternatively, the jet canister can be emptied, refilled, and reused if required by a specific application.

[0039] In some embodiments, the jet canister of this disclosure includes not only a reagent reservoir but also one or more waste reservoirs. Unconsumed reagents and / or unwanted reaction products can be collected in the waste reservoir. The advantages of retaining the fluids before and after the reaction in the canister include convenient handling of individual jet components before and after the reaction, minimal user contact with chemical reagents, a compact base surface for the device, and avoidance of unnecessary fluid container expansion.

[0040] Exemplary jet containers, reaction vessels, and jet components, which can be modified according to the teachings herein for use in combination with the detection components of this disclosure, are described in commonly owned U.S. Patent Application Serial No. 15 / 922,661, which claims the benefit of U.S. Provisional Application No. 62 / 481,289, each of which is incorporated herein by reference. Other jet components, particularly suitable for cyclic reactions (e.g., nucleic acid sequencing reactions), are described in U.S. Patent Application Publications Nos. 2009 / 0026082A1; 2009 / 0127589A1; 2010 / 0111768A1; 2010 / 0137143A1; or 2010 / 0282617A1; or each of Patent Nos. 7,329,860; 8,951,781, or 9,193,996, each of which is incorporated herein by reference.

[0041] Details of one or more embodiments are illustrated in the following drawings and description. The drawings and description are provided by way of example for illustrative purposes and are not necessarily intended to limit the scope of the invention. The invention is readily adaptable to modifications in terms of methods and materials, as well as changes in manufacturing methods and apparatus. Such modifications will become apparent to those skilled in the art upon consideration of the following drawings and description.

[0042] This disclosure provides a detection apparatus. The apparatus may include: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the inner surface contacts the inner cavity; (b) a reference surface forming a structural ring with a detector; (c) a preload configured to push the outer surface of the container into contact with a region on the reference surface; (d) a scanning actuator configured to slide the container along the reference surface in a scanning dimension; and (e) a transmitter configured to direct a signal from the inner surface or the inner cavity to the detector when the outer surface of the container is pushed into contact with the reference surface by the preload.

[0043] In a particular embodiment, the detection device may include: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the inner surface contacts the inner cavity, and wherein the outer surface has a length l in the scanning dimension x; (b) a reference surface; (c) a preload configured to push the outer surface of the container into contact with a region on the reference surface, optionally the maximum length of the contact region in the scanning dimension x may be 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) an objective lens configured to direct radiation from the container to the detector when the outer surface of the container is pushed into contact with the reference surface by the preload.

[0044] This disclosure also provides a method for scanning a container. The method may include (a) translating a container along a reference surface of a detection device, wherein the container includes an inner cavity and a wall, wherein the inner cavity contains an analyte, wherein during the translation the reference surface contacts at least a portion of the container, and wherein the reference surface forms a structural ring with a detector; and (b) using the detector to detect the analyte at different locations along the container, wherein during the detection the container is pushed against the reference surface by the preload, thereby scanning the container.

[0045] In some embodiments, a method of scanning a container may include: (a) examining a first subset of analytes in the container while applying a preload to a first portion of the container, wherein the preload positions the first subset of analytes to occupy an xy-plane in a detection area, wherein no preload is applied to a second portion of the container; (b) translating the container to position a second subset of analytes in the xy-plane of the detection area; and (c) examining the second subset of analytes in the container while applying the preload to a second portion of the container, wherein the preload positions the second subset of analytes to occupy an xy-plane in the detection area, wherein no preload is applied to the first portion of the container, thereby scanning the container.

[0046] A method for optically scanning a container is also provided. The method may include: (a) providing a container having an inner cavity and walls, wherein the inner cavity contains an optically detectable analyte, and wherein the walls are transparent to the optically detectable analyte; (b) translating the length of the container along a reference surface and detecting the optically detectable analyte at different locations along that length, wherein at any time during the translation, the reference surface contacts only a portion of the container length, wherein during detection, the container is preloaded and pushed against the reference surface, wherein the detection comprises emitting radiation through the walls, then through an objective lens, and then to a detector, thus optically scanning the container.

[0047] Figure 2 illustrates an exemplary arrangement for scanning the container relative to the detector. Figure 2A and Figure 2BAs shown in the outline diagram, the container is a flow cell 101 aligned with the objective lens 110 via a rigid body 100. The back side of the rigid body 100 has a tapered recess 116 that complements the shape of the objective lens 110. Therefore, the objective lens 110 can be moved closer to the flow cell to obtain the desired focusing or resolution. Any of various recess shapes can be used as needed to accommodate the shape of various objectives or other optical components. The front side of the rigid body 100 has a reference surface 117 that will contact the plane of the flow cell 101. The flow cell 101 is held in contact with the reference surface 117 by a preload that applies a positive pressure to the side of the flow cell 101 opposite to the reference surface 117. The preload is formed by a pressure foot 102, which contacts the flow cell 101 under the force of a spring 103.

[0048] Typically, the reference surface 117 and the pressure foot 102 form a low-friction contact with the flow cell 101. This allows the flow cell to slide down the reference surface 117 and over the pressure foot 102 under preloaded compressive force. This compression aligns the flow cell 101 with the objective lens 110 via a rigid body throughout the scanning of the flow cell 101 via the objective lens 110. The reference surface and the objective lens are components of a structural ring. The structural ring contains structural elements that position the container (e.g., the flow cell) relative to the detector (e.g., via the objective lens). Because the reference surface is pre-aligned with the objective lens, compressing the flow cell to the reference surface prevents unnecessary tilting and rolling of the flow cell relative to the objective lens. The components in the structural ring in Figure 2 include the reference surface 117, which is connected to the rigid body 100, which is connected to the base 114. The base 114 may be connected to a plate or other structural element that is physically connected to components of the optical system, such as those illustrated in Figure 9.

[0049] In the example shown in Figure 2, the reference surface 117 is polished aluminum, which provides rigidity for aligning the flow cell 101 with the objective lens 110, and a low-friction surface for sliding the glass surface of the flow cell 101. Any of a variety of materials that provide rigidity and low friction for the reference surface can be used, including, for example, acetal resins (e.g., available from DuPont, Wilmington, DE). The pressure foot 102 provides a low-friction surface for sliding translation of the glass surface of the flow cell 101 and also provides compressibility to form compliant contact with the flow cell 101 under the force of the spring 103. Any of a variety of materials that provide low friction to the pressure foot can be used, including, for example, those described above with respect to reference surface 117. Optionally, the low-friction material used in the device herein may also be compressible, examples of which include, but are not limited to, polytetrafluoroethylene (PTFE). Perfluoroalkoxyalkanes (PFA), fluorinated ethylene propylene (FEP), polysiloxane foam, nitrile rubber, Buna-N, Perbunan, acrylonitrile butadiene rubber, or nitrile butadiene rubber (NBR). Alternatively or additionally, ball bearings, rollers, and / or lubricating fluids can be used to achieve low friction. Typically, a lubricating fluid is used on the side of the flow cell not between the analyte and the detector, or a fluid that does not interfere with detection is used. In some embodiments, no lubricating fluid is present at the interface between the reference surface and the outer surface of the container wall. For example, lubricating fluid can be avoided to prevent interference when fluid enters the area between the detector and the container.

[0050] In certain embodiments, the container (or box containing the container) is positioned in the xy plane without contacting a reference surface. For example, the container (or box) can be pushed against a fluid bearing or magnetic bearing by a preload, such that the combination of forces provided by the preload and the bearing results in the desired positioning. The fluid bearing can be a gas bearing, whereby gas pressure provides the force for positioning the container (or box). Another useful type of fluid bearing can be a liquid bearing, whereby liquid pressure provides the force for positioning the container (or box). The liquid can be selected to match the refractive index of the system's optical components (e.g., the container walls) to minimize aberrations when detecting optical signals or transmitting radiation.

[0051] like Figure 2C and Figure 2D As shown, the reference surface 117 has a plane forming a flat ring on the front side of the rigid body 100. This ring is raised compared to the front side of the rigid body 100. Raising the reference surface helps prevent unwanted contact between the flow cell 101 and the rigid body 100, which could otherwise generate frictional forces that hinder translation. Raising the reference surface 117 also isolates the area to be detected from the flow cell and prevents unwanted warping that could occur if the flow cell contacts other areas of the rigid body 101. In the example of FIG. 2, the area of ​​the reference surface is smaller than the surface of the flow cell, and therefore only contacts a portion of the flow cell surface. However, in alternative embodiments, the reference surface can be substantially the same size as or larger than the flow cell surface, so that the reference surface can contact almost all surfaces of the flow cell (optionally, except for areas of the flow cell surface juxtaposed with detection windows, objectives, or other emitters).

[0052] In the illustrated example, a reference surface 117 surrounds a circular window 118, which is an aperture through the rigid body 100. Alternatively, the circular window 118 may comprise a material capable of emitting a signal to be detected. For example, the window may be made of quartz, glass, or plastic, which facilitates the emission of the signal to be detected. In some configurations, the window may contain an immersion liquid that contacts the flow cell surface to facilitate detection, as described below. Figure 11As explained in more detail, the circular window 118 is aligned with the front lens 115 of the objective lens 110, allowing the objective lens 110 to observe the flow cell 101 through the window 118. The pressure foot 102 has a flattened annular shape and provides a base surface on the flow cell 101, which is complementary to the base surface of the flattened ring 117 on the opposite side of the flow cell. In this example, the contact area between the preload (via the foot 102) and the container (flow cell 101) is the same as the contact area between the reference surface 117 and the container. Alternatively, the contact area between the preload and the container may be smaller than the contact area between the reference surface and the container. In fact, the contact area between the preload and the container is no larger than the contact area between the reference surface and the container.

[0053] Typically, the complementarity between the base surfaces of the preload and the reference surface can be configured such that the pressure foot 102 has a contact area on the flow cell 101 that excludes the surface area of ​​the flow cell opposite the circular window 118, and also excludes the surface area of ​​the flow cell opposite the rigid body area surrounding the reference surface 117. The complementarity between the base surfaces of the pressure foot 102 and the reference surface 117 helps maintain the flatness of the flow cell surface portion as observed through the window 118. This complementarity can be beneficial for detecting analytes on the inner surface of the flow cell, especially at high magnification and high resolution. Complementarity also facilitates radiography, whereby radiation can be transmitted back and forth through the path defined by the hollow space in the spring 103, the pressure foot 102, and the window 118. The circular shape of the reference surface and the preload is exemplary. Other shapes can be used, including but not limited to squares, rectangles, polyhedra, ellipses, triangles, etc. Moreover, the shapes do not necessarily have to be continuous. Instead, the contact surfaces of the reference surface and / or the preload can be discontinuous regions, such as regions formed by two parallel tracks or by interrupting the aforementioned shapes. Particularly useful applications are nucleic acid microarray detection and nucleic acid sequencing. The shape and orientation of the preload and reference surface can be used to transfer energy to the container or in devices that detect non-optical signals.

[0054] As shown in Figure 2, a flow cell is a particularly useful container for use in the detection apparatus or other devices of this disclosure. Any of a variety of flow cells can be used, including, for example, those comprising at least one channel and an opening at either end of the channel. The opening can be connected to a jet component to allow reagents to flow through the channel. Flow cells are generally configured to allow detection of analytes within the channel, such as in the cavity of the channel or on the inner surface of the wall forming the channel. In some embodiments, a flow cell may include multiple channels, each having an opening at its end. For example, the flow cell shown in Figure 2 has three channels 120, 121, and 122, each with an opening at both ends. The multiple channels can interact with the jet system via a manifold.

[0055] In certain embodiments, the flow cell will include a solid support to which one or more target analytes or reagents are attached. A particularly useful solid support is one with a site array. Arrays offer the advantage of facilitating multiplex detection. For example, different reagents or analytes (e.g., cells, nucleic acids, proteins, candidate small molecule therapeutics, etc.) can be attached to the array by linking each different analyte to specific sites on the array. Exemplary array substrates available include, but are not limited to, BeadChip, which is available from Illumina, Inc. (San Diego, CA). TM Arrays, or those described in U.S. Patent Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO00 / 63437, each of which is incorporated herein by reference. Other examples of commercially available array substrates that may be used include, for example, the Affymetrix GeneChip. TM Array. According to some embodiments, a dot array substrate may also be used. An exemplary dot array is CodeLink, available from Amersham Biosciences. TM Arrays. Another useful array method is to use inkjet printing, such as SurePrint, which is available from Agilent Technologies. TM An array manufactured using technology.

[0056] Other useful array substrates include those used in nucleic acid sequencing applications. For example, arrays (often called clusters) used to generate attached amplicon for genomic fragments can be particularly useful. Examples of substrates that can be modified for use herein include those described in Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO 91 / 06678; WO 04 / 018497 or WO07 / 123744; U.S. Patent Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or 7,405,281; or U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0057] The sites in the array can be spaced less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. In a particular embodiment, each site in the array can have a size greater than approximately 100 nm. 2 250nm 2 500nm 2 1μm 2 2.5μm 2 5μm 210μm 2 100μm 2 or 500μm 2 The area. Alternatively or additionally, the sites in the array may each have an area less than about 1 mm. 2 500μm 2 100μm 2 25μm 2 10μm 2 5μm 2 1μm 2 500nm 2 or 100nm 2 The area. In practice, the size of the sites can range between the upper and lower limits selected from the examples above. The array can have sites of any density, including at least approximately 10 sites / cm². 2 100 sites / cm 2 500 sites / cm 2 1000 sites / cm 2 5,000 sites / cm 2 10,000 sites / cm 2 50,000 sites / cm 2 100,000 sites / cm 2 1,000,000 sites / cm 2 5,000,000 sites / cm 2 Or higher. Embodiments of the apparatus or methods described herein can be used to image an array at a resolution sufficient to distinguish sites of the aforementioned density or site spacing.

[0058] Several embodiments utilize optical detection of analytes in a flow cell. Therefore, the flow cell may include one or more channels, each channel having at least one transparent window. In certain embodiments, the window is permeable to radiation within a specific spectral range, including but not limited to x-rays, ultraviolet (UV), visible light (VIS), infrared (IR), microwaves, and / or radio wave radiation. In some cases, the analyte adheres to the inner surface of the window. Alternatively or additionally, one or more windows may provide a view of an internal substrate to which the analyte is adhered. Exemplary flow cells and physical features of flow cells useful in the methods or apparatus set forth herein are described, for example, in U.S. Patent Application Publication No. 2010 / 0111768A1, WO 05 / 065814, or U.S. Patent Application Publication No. 2012 / 0270305A1, each of which is incorporated herein by reference in its entirety.

[0059] Rectangular flow cell 101 with elongated channels is illustrated in several examples presented herein. In these examples, the maximum length of the contact area between flow cell 101 and reference surface 117 in the scan dimension x is shorter than the length of the flow cell lane in the scan dimension x. More specifically, the diameter of ring 117 is shorter than the length of lanes 120, 121, or 122. Alternatively or additionally, the maximum width w of the contact area between flow cell 101 and reference surface 117 in dimension y is shorter than the width of the flow cell lane in dimension y. Specifically, the diameter of ring 117 may be shorter than the width of any of lanes 120, 121, or 122.

[0060] Similarly, the maximum diameter or length of window 118 in the scan dimension x can be shorter than the length of the flow pool lane in the scan dimension x. Alternatively or additionally, the maximum diameter or width of window 118 in the y dimension can be shorter than the width of any of lanes 120, 121, or 122. In this configuration, the entire width of the lane can be observed by translation along the y direction. In some embodiments, the area of ​​window 118 and the width of the lane can be configured such that translation in the y dimension is not necessary for observing the entire width of the lane. For example, the maximum width w of the region of window 118 in dimension y is equal to or longer than the width of the flow pool lane in dimension y.

[0061] In certain embodiments, a container, such as a flow cell, may move along an arcuate path during all or part of the scanning operation. (Observation) Figure 1 In the flow cell orientation, the arcuate path may be generated due to rotation about a yaw axis. The arcuate path can be circular, spiral, or other paths required for scanning the container. Optionally, the length or area of ​​the contact region between the container and the reference surface can be smaller than the length or area of ​​the arcuate path. As a more specific example, the diameter of the annular reference surface can be shorter than the length of the arcuate path, or shorter than the length of the lane in the flow cell moving along the arcuate path. Similarly, the maximum diameter or area of ​​the window through which the reference surface is detected can be smaller than the length or area of ​​the arcuate path; or the window can be smaller than the flow cell lane scanned along the arcuate path.

[0062] The flow cell need not be rectangular. Alternative shapes that can be used include, but are not limited to, disks, squares, polygons, or irregular shapes. The lanes of the flow cell can follow linear paths, curved paths, winding paths, etc. Other types of containers can also be used. For example, the apparatus or method of this disclosure can be used to detect the holes in a porous strip or porous plate. The bottom surface of the hole can be pushed toward a reference surface by applying a preload to the top of the container (e.g., by contacting the upper side of the porous plate or porous strip with a pressure foot). Optionally, the hole can have a flat bottom that contacts the reference surface. As a further option, the hole will be larger than the detector's field of view. For example, the shape of the hole can be circular, and its diameter l in the scan dimension x can be longer than the length of the reference surface in the scan dimension x.

[0063] Another exemplary container type is a cylindrical or tubular container, such as a capillary tube. The tube can be fixed to a reference surface under a preload force, as exemplified herein for a flat container. In an exemplary configuration, the length of the tube can be parallel to the scan axis, such that scanning the tube along x will cause the reference surface to move relative to the length of the tube. For a tube configured in this orientation, it is also useful to rotate the tube along a roll axis. This rotation will cause relative movement of the reference surface about a portion of the circumference of the tube. The combination of translation along x and rotation along the roll axis can bring a larger surface area of ​​the tube into contact with the reference surface. For example, the tube and the reference surface can move relative to each other along a helical or spiral path. The reference surface can be flat, as exemplified herein for a flow cell with a flat outer wall. Alternatively, the reference surface can have a curved shape (e.g., a U-shaped or saddle-shaped cross-section) that accommodates and orients the cylindrical or tubular container in contact with it.

[0064] Typically, container walls are made of a rigid material that is not easily bent under operating conditions. In alternative embodiments, the container is made of a flexible material, for example, formed into a sheet, strip, band, or tape that can pass along a reference surface and be detected under a preloaded push. For example, various analytes, such as nucleic acid arrays, can be attached to the surface of the flexible material and detected upon contact with the reference surface. Exemplary flexible materials with attached analytes are described, for example, in U.S. Patent No. 9,073,033 and U.S. Patent Application Publication No. 2016 / 0076025A1, each of which is incorporated herein by reference.

[0065] When using containers with flexible walls, it may be advantageous to pull the wall material onto a reference surface, such as stretching or straightening a portion of the wall material observed by the detector. For example, the reference surface could be a raised edge surrounding a detection window, and the flexible material could be pulled onto the edge to apply tension to the window. This pulling can be achieved, for example, by applying suction to the flexible material using a vacuum suction cup around the raised edge. Suction can be applied as an alternative to or supplement to other preload mechanisms described herein.

[0066] As will be apparent from the examples described herein, containers can be open (e.g., the holes of a well plate, the surface of a chip, or the surface of a sheet) or closed (e.g., the lanes of a flow cell). It will be understood that the holes of a well plate can optionally be covered to form a closed container, and similarly, sheets, strips, tapes, or ribbons can have multiple layers, creating internal cavities between the layers. Alternatively, a container can have one or more fluid-containing open structures, such as grooves, holes, or other concave structures. A container can also have protruding or projecting structures, such as pillars or ridges, and optionally, each protrusion can be individually attached to one or more analytes to be detected or manipulated.

[0067] The preload illustrated in Figure 2 generates a thrust on the side of the container (e.g., a flow cell) opposite the side of the container that contacts the reference surface. The thrust can originate from a spring, clamp, positive air pressure, positive hydraulic pressure, electrostatic repulsion, electrostatic attraction, magnetic attraction, or magnetic repulsion. Alternatively, the preload can be configured to generate a pull force on the container. For example, magnetic or ferromagnetic material inside or on the container can be attracted to the reference surface, or charges inside or on the container can be attracted to the reference surface. In this example, the reference surface or the area surrounding the reference surface can contain magnetic or ferromagnetic material that acts as the preload. In another embodiment, the pull force can be generated by a vacuum chuck configured to apply suction to the area of ​​the container that contacts the reference surface. In another embodiment, a magnetic clamping force can be used to clamp the container between magnetic or ferromagnetic material on or around the reference surface, which attracts magnets or ferromagnets on the opposite side of the container.

[0068] The detection apparatus or other apparatus disclosed herein may include a scanning actuator configured to slide a container along a reference surface. The container may slide along the reference surface and along a preloaded surface. Typically, the scanning actuator is configured to move the container while it contacts the reference surface under the push of the preload. However, the container may also be translated without applying a preload simultaneously. The container may also be moved through a space defined by bearings (e.g., fluid bearings or magnetic bearings) that do not physically contact the container. For example, the container may be positioned against a bearing by the opposing force of the preload. Particularly useful actuators employ one or more gears that interact with perforations or threads on a flow cell or a housing containing the flow cell. Several examples are illustrated below.

[0069] In some embodiments, the scanning actuator may use a film sprocket mechanism. The container to be translated, or the box that holds the container, may contain perforated tracks that engage with sprockets in the detection device to achieve translation. As shown in the exemplary configuration of FIG3, the flow cell 101 is housed in a box 125, which includes two perforated tracks 130 and 140. Perforated track 130 is located near the top edge of box 125 and extends parallel to the longest dimension l of the flow cell. Perforated track 140 is located near the opposite edge of box 125 and also extends parallel to l. Sprockets 150 and 160 are configured to engage with perforated tracks 130 and 140, respectively, when pushed against reference surface 117 by the force of preloaded spring 103. By rotating the engaged sprockets 150 and 160, the flow cell 101 can be translated in a scanning dimension x parallel to l.

[0070] Figure 4A A housing 400 is shown with an insert 403 for a flow cell 430. The insert includes recesses 404 and 405 positioned to facilitate adjustment or removal of the flow cell 430. The housing 400 has a single perforated track 401 near its top edge 402. Figure 4B As shown, the perforation is complementary to the teeth on the sprocket 420, and the perforated track 401 is inserted into the face of the housing 400, thus providing a track for engagement with the guide 410. The guide 410 is inserted into the perforated track 401 to prevent the housing 400 from rotating along the yaw axis during translation under the action of the sprocket 420, thereby preventing unnecessary yaw rotation of the flow cell 430 relative to the detector. Figure 4C As shown, the flow tank 430 includes a base plate 431 sized to pressure fit with the insert 403, and also includes a top plate 440. A channel 443 is formed between plates 431 and 440 due to the presence of a partition or gasket. The top plate 440 also includes holes 441 and 442 that serve as the inlet and outlet of the channel 443. Figure 4D The image shows a perspective view of a box 400 with an assembled flow cell 430, a sprocket 420 with a motor 425, and a guide 410.

[0071] Another useful mechanism for scanning actuation is a spur gear that meshes with teeth on the edge of the flow cell or the edge of the box that holds the flow cell in place. Figure 5A A cartridge 200 is shown, which pressure-fits with a flow tank 101 and has a serrated bottom edge 240 and a smooth top edge 241. When the cartridge 200 is pushed into contact with a reference surface on the rigid body 100 by a preloaded spring 103, the serrated bottom edge 240 engages with a spur gear 230. Rotation of the spur gear 230 translates the cartridge 200 and the flow tank 101. When the cartridge 200 is positioned such that the flow tank 101 contacts the reference surface on the rigid body 100, wheel guides 210 and 220 engage with the smooth edge 241 of the cartridge 200. The wheel guides prevent the cartridge 200 and the flow tank 101 from rotating about a yaw axis.

[0072] Scanning actuation can also employ ball screws, which engage with threaded snaps on the flow cell or the housing that holds the flow cell. Figure 6A A housing 300 is shown, which pressure-fits with a flow tank 101 and has a threaded snap 311 at the top and two guide snaps 312 and 313 at the bottom. When the housing 300 is pushed into contact with a reference surface on the rigid body 100 by a preloaded spring 103, the threaded snap 311 engages a screw 310. By rotating the screw 310 against the threads of the snap 311, the housing 300 and the flow tank 101 are translated. When the housing 300 is positioned so that the flow tank 101 contacts the rigid body 117, the guide snaps 312 and 313 engage with a guide rail 320. The guide snaps 312 and 313 serve to prevent the housing 300 and the flow tank 101 from rotating about a yaw axis.

[0073] Scanning actuation can utilize mechanical contact between a motor and a container (or container housing), as described above. Alternatively or additionally, the interaction between the motor and the container (or container housing) can be mediated by magnetic attraction. For example, the container or housing can have a magnetic or ferromagnetic material that interacts with the magnetic or ferromagnetic components of the actuator.

[0074] Whether mechanical contact or other interactions are used to mediate actuation, linear motors can be used to drive scanning motion. Exemplary linear motors that can be used include synchronous linear motors, induction linear motors, unipolar linear motors, and piezoelectric linear motors.

[0075] The apparatus disclosed herein may also include a y-actuator configured to change the relative translational position of the detector and the container along the y-axis. Taking the apparatus shown in FIG2 as an example, the y-actuator may be operated, for example, by changing the relative translational position of the objective lens 110 and the reference surface 117. Alternatively or additionally, the y-actuator may be operated by changing the relative translational position of the flow cell 101 and the reference surface 117. Translation along the y-axis allows addressing of different lanes of the flow cell. When the width of a lane is greater than the field of view of the objective lens, y-translation can be used to detect multiple stripes of the lane (i.e., a first stripe may be detected by scanning along the x-axis, and a second stripe may be addressed by first scanning along the y-axis and then by a second scan along the x-axis). The y-actuator may be configured similarly to the x-actuator configuration illustrated herein. For example, the y-actuator may be configured to translate the flow cell as it is preloaded onto the reference surface. Other stepper motors or translation actuators may also be used for x or y translation.

[0076] In certain embodiments, the apparatus of this disclosure may include a rotary actuator configured to change the relative translational position of the detector and the container along an arcuate path. Figure 1 Taking the exemplary flow cell with the shown orientation as an example, a rotary actuator can rotate the flow cell along a yaw axis. Rotation along the yaw axis is particularly useful for scanning lanes or features along curved paths. Additional or alternative rotary actuators can rotate the container along a rolling axis. Rotation along the yaw axis can be particularly useful when the container is a tube or cylinder oriented with its length along the x-axis.

[0077] Several embodiments of this disclosure are illustrated with reference to objectives having several lenses for focusing and converging radiation from an object (e.g., a container such as a flow cell). It will be understood that any of a variety of optical elements can be used as objectives in the apparatus or methods of this disclosure, including, for example, lenses, mirrors, optical fibers, fiber bundles, lens arrays, or other optical elements that focus radiation from an object being observed, whether or not such optical element is also capable of focusing radiation. Objectives or other optical components used in the apparatus or methods produced herein can be configured to emit any radiation across a wide range of spectra, including but not limited to X-rays, ultraviolet (UV), visible light (VIS), infrared (IR), microwaves, and / or radio waves.

[0078] The objective lens used in the apparatus described herein can be positioned to guide radiation from the inner surface or cavity of the container through the container wall to the detector when the outer surface of the container contacts the reference surface. In a particular embodiment, the objective lens and other optional components of the optical system can be configured for epiluminescence detection (i.e., epiluminescence), thereby guiding excitation radiation from the radiation source through the objective lens, then through the container wall, to the inner surface or cavity of the container; and thereby guiding emission from the inner surface or cavity of the container back through the wall and through the objective lens (i.e., both excitation and emission pass through the objective lens). Alternatively, the objective lens and other optional components of the optical system can be configured for transmissive fluorescence, thereby guiding excitation radiation from the radiation source through a first wall of the container to the inner surface or cavity of the container; and thereby guiding emission from the inner surface or cavity of the container through another wall of the container and through the objective lens (i.e., emission passes through the objective lens, excitation does not pass through the objective lens). Other useful configurations for fluorescence detection include those that excite the container by total internal reflection fluorescence (TIRF) or by waveguide. In any of the various configurations, the radiation source can form a structural ring with the reference surface, such that the container in contact with the reference under preload will be correctly oriented relative to the radiation source.

[0079] The objectives shown in Figures 2, 3, 5, and 6 are exemplary and have four lenses. Any number or type of lenses can be included to suit a specific application. Particularly useful objectives have a numerical aperture of at least 0.1 and at most 0.9. Numerical apertures greater than 0.95 can be achieved using immersion objectives, which are further elaborated below. Objectives or other emitters can be configured to operate in conjunction with a detection system capable of resolving features (e.g., nucleic acid sites) spaced less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm on a surface. Detection systems including objectives or other emitters can be configured to resolve surface areas less than about 1 mm². 2 500μm 2 100μm 2 25μm 2 10μm 2 5μm 2 1μm 2 500nm 2 or 100nm 2 Its characteristics.

[0080] The optical system used in the apparatus or method described herein may have a minimum thickness of 0.1 mm. 2 0.5mm 2 1mm 2 2mm 2 3mm 2 4mm 2Or a wider field of view. Alternatively and / or additionally, the field of view can be configured to a maximum of 4mm. 2 3mm 2 2mm 2 1mm 2 0.5mm 2 0.1mm 2 Or smaller.

[0081] The objective lens or other suitable component of the detection system used in the apparatus described herein can be configured to focus on the analyte inside or on the container. For example, the apparatus may also include a focusing actuator configured to change the relative position of the objective lens and the reference surface in the focusing dimension. Under preload, physically aligning the container with the reference surface effectively fixes the container's position in the z-axis, thereby facilitating accurate and stable focusing throughout the scanning operation.

[0082] The apparatus described herein can employ components used in optical subsystems or nucleic acid sequencing systems. Several such detection apparatuses are configured for optical detection, such as the detection of fluorescence signals. Examples of detection apparatuses and components thereof that can be used herein for detecting containers are described, for example, in U.S. Patent Application Publication No. 2010 / 0111768A1 or U.S. Patent Nos. 7,329,860; 8,951,781 or 9,193,996, each of which is incorporated herein by reference. Other detection apparatuses include commercially available detection apparatuses for nucleic acid sequencing, such as those developed by Illumina. TM ,Inc. (e.g., HiSeq) TM MiSeq TM NextSeq TM or NovaSeq TM Systems), Life Technologies TM (e.g., AABI PRISM) TM or SOLiD TM (system), Pacific Biosciences (e.g., using SMRT) TM Technological systems, such as Sequel TM or RS II TM (system) or Qiagen (e.g., Genereader) TMThe detection device provided by the system. Other useful detectors are described in U.S. Patent Nos. 5,888,737; 6,175,002; 5,695,934; 6,140,489; or 5,863,722; or U.S. Patent Publications 2007 / 007991A1, 2009 / 0247414A1 or 2010 / 0111768; or WO2007 / 123744, each of which is incorporated herein by reference in its entirety. In certain embodiments, the platform of a known sequencing system can be replaced by the scanning device described herein.

[0083] Typically, an objective lens is an optical element in a detection device that is closest to (i.e., most proximate to) the container being tested (e.g., a 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 optical fibers, may be provided by the container or a housing to which the container is attached. For example, the objective lens of the detection device may be configured to direct excitation, emission, or other signals to optical components present on the container or housing. Thus, optical components close to the sample can be provided by the detection device or, alternatively, by the container holding the sample.

[0084] In the methods or apparatus described herein, the detection device used to observe the container does not need to be capable of optical detection. For example, the detector could be an electronic detector for detecting protons or pyrophosphates (see, for example, U.S. Patent Application Publication Nos. 2009 / 0026082A1; 2009 / 0127589A1; 2010 / 0137143A1; or 2010 / 0282617A1, each of which is incorporated herein by reference in its entirety, or an Ion Torrent commercially available from Thermo Fisher, Waltham, MA). TM (System) or electronic detectors for detecting nanopores, such as those from Oxford Nanopore TM Those commercialized by Oxford UK (e.g., MinION) TM or Promethion TM The system) or those set forth in U.S. Patent No. 7,001,792; Soni and Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanod. 2, 459-481 (2007); or Cockroft et al., J. Am. Chem. Soc. 130, 818-820 (2008), each of which is incorporated herein by reference.

[0085] In one particular embodiment, the apparatus or method described herein can be configured for scanning electron microscopy (SEM). Thus, an electron beam can be generated by an electron gun and guided to a container by one or more condenser lenses, scanning coils, and / or deflection plates. The signal can be detected using an electron detector such as a scintillator-photomultiplier tube system (e.g., an Everhart-Thornley detector).

[0086] In certain embodiments, the detection apparatus or other devices of this disclosure can provide temperature control of the container to be tested. Temperature control can be provided by controlling the temperature of the internal chamber containing the container. Alternatively or additionally, the container to be tested can be placed in contact with a temperature-controlled, thermally conductive surface. Figure 7A An exemplary configuration for achieving temperature control of a flow cell through contact with a thermally conductive surface is shown. The back of an aluminum body 460 is attached to two heating elements 450 and 451 located on the left and right sides of a conical recess 416. The heating elements can be polyimide heating plates, Peltier elements, metal heating elements, ceramic heating elements, polymer PTC heating elements, etc. The aluminum body 460 also includes two legs 461 and 462 for attachment to a detection device. Thus, the two legs form part of a structural ring between a reference surface on the aluminum body 460 and the detection device. Optionally, legs 461 and 462 can be made of a material with low thermal conductivity. Therefore, the legs can serve to attach the aluminum body to the detection device in such a way that other components of the detection device are protected from unwanted temperature fluctuations. The heating elements 450 and 451 can be activated by wires 452 and 453 to heat or cool the aluminum body 460, causing the flow cell in the cartridge 400 to contact the opposite sides of the aluminum body 460 and thus be temperature-controlled. Figure 7B As shown, the tapered recess 416 is configured to receive the objective lens 410 to inspect the flow cell in the chamber 400 through the window 418. In the illustrated configuration, the flow cell chamber 400 is translated via a film sprocket 420 under the control of a rotary motor 425.

[0087] The detection apparatus or other apparatus disclosed herein may include a jet system for delivering reagents to a container to be tested. Thus, one or more reservoirs may be fluidly connected to an inlet valve of the container. The apparatus may also include a pressure source for driving reagents from the reservoir to the container. The apparatus may include a waste reservoir fluidly connected to the container to remove used reagents. Taking an embodiment where the container is a flow cell as an example, reagents may be delivered to the flow cell via an inlet via a pump, and then the reagents may flow through the flow cell outlet to the waste reservoir. The reservoir 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 (e.g., ELISA), small molecule receptor binding, protein phosphorylation analysis, nucleic acid synthesis, or protein synthesis. Alternatively or additionally, the reservoir may include reagents for preparation processes. Exemplary preparation processes include, but are not limited to, nucleic acid synthesis, peptide synthesis, oligonucleotide assembly into genes, photolithography, nanofabrication or microfabrication (e.g., via laser etching), laser ablation, etc.

[0088] The jet system may include at least one manifold and / or at least one valve for directing reagents from a reservoir to a container for detection. Manifolds are particularly useful in sequencing instruments because relatively large quantities of different reagents are delivered in sequencing protocols. Exemplary schemes and useful reagents are further described in detail below and in references incorporated herein by reference. The fluid flow rate of the reservoir can be selected by valves such as solenoid valves (e.g., those manufactured by Takasago Electric, Japan), ball valves, diaphragm valves, or rotary valves.

[0089] In the detection apparatus or other devices disclosed herein, one or more jet components can be housed in a jet canister detachable from the detection component. Figure 8A An exemplary jet container 600 is shown. The jet container 600 includes a housing 601 with sufficient internal volume to accommodate a reagent reservoir 603, a waste reservoir 602, and a piston shaft 604 for an external pump. Any of various jet components can be accommodated in the jet container, including, but not limited to, one or more reservoirs, fluid lines, valves, or pumps. The jet container includes latches 610 and 611 configured to engage with hooks in a detection device. See, for example, hook key 701 in Figure 9. A flow cell 430 is secured within a housing 400, which is secured to the jet container 600 by hooks 616 and guides 616 and 617. Figure 8B Enlarged sectional view and Figure 8D As shown in the side view, hook 615 includes teeth 614 that insert into track 401 to secure box 400 in place. Guides 616 and 617 complete the three-point attachment by engaging the bottom edge of box 400. Preload member 620, although in Figure 8DThe box is shown in the retracted position, but can be extended and pushed toward the back of the box 400, thereby working with the hook 615 and the guides 616 and 617 to secure the box in place by compressive force.

[0090] Jet tank 600 includes, for example Figure 8D and 8F The opening is shown. This is to illustrate the fluid connection of the flow cell 430. Figure 8F A perspective view of canister 600, emptied of several other jet components, is shown. Opening 605 is configured to receive a piston from an external pump. The piston can be driven by a detection device to allow control of the fluid flow rate through flow cell 430 during analysis procedures (e.g., nucleic acid sequencing procedures), but the piston does not need to be in direct contact with any fluid in canister 600 or flow cell 430. Thus, the detection device can constitute a “dry” component that does not come into direct contact with the fluid, while canister 600 and flow cell 430 constitute “wet” components. Jet canister 600 includes two elongated openings 621 and 622, configured to receive tubes 661 and 662, respectively. The elongated shape allows the tubes to move along the x-axis as the flow cell translates during scanning. Thus, the tubes can remain engaged with the flow cell and jet reservoir during scanning operations.

[0091] The flow cell 430 can be translated independently of the tank 600 by the movement of the box as previously described herein (e.g., in conjunction with Figure 4). Thus, when the flow cell 430 moves, the tank 600 remains stationary. Alternatively, the flow cell can be attached to the tank, such that the tank and the flow cell can be translated as a single unit. In another alternative, one or more detection components of the detection device can be moved while the flow cell and / or the jet tank are stationary.

[0092] Figure 9 illustrates the interaction between the jet canister 600 and the detection device components. Figure 9A Perspective view and Figure 9B The top view shows the can 600 engaged, with the flow cell box 400 clamped between the can 600 and the aluminum body 460. When engaged, the flow cell box 400 contacts the film sprocket 420, allowing the motor 425 to drive translation of the flow cell within it. This translation moves the flow cell past the objective lens 721, which is configured to guide fluorescence excitation from the fluorometer 720 to the flow cell and fluorescence emission from the flow cell to the fluorometer 720.

[0093] The mechanism by which the canister 600 and the flow pool box 400 engage with the detection device or other device of the present invention can be analogous to inserting an 8-track box into an audio player. The flow pool 430 and box 400 are connected to the canister 600 so that the user does not need to directly handle the flow pool 430, but rather passes it to the detection device by handling the canister 600, just as the user does not need to handle the tape inside the 8-track box. Similarly, when the canister 600 is correctly placed in the detection device, it does not need to be handled individually for each jet component, but can be properly engaged with the actuator in the detection device.

[0094] exist Figure 9C The jet canister 600 detached from the detection device. Figure 9C Mechanical elements that can be used by a detection device to control the function of jet can 600 are shown. The detection device may include a sensor or switch that responds to the presence of the jet can and initiates functional interactions. In the example of Figure 9, the hook key 701 is displaced when the can 600 is properly engaged. This displacement can activate one or more functions. For example, the bottom surface of the jet can 600 may include one or more openings positioned to receive one or more valve actuators 711 on the platform 710. Although the valve actuator is shown in a protruding position for illustrative purposes, it can retract into the platform 710 when the jet can 600 is not present. The valve actuator can be raised in response to the displacement of the hook key 701 and / or in response to the control software of the detection device. Thus, the one or more valve actuators 711 can be used to control the flow rate of fluid entering and leaving the flow pool and / or between reservoirs within the can. In another example, the pump component 702 of the detection device may engage with the jet component of the can 600 via the opening 710, for example, by inserting a piston. The interaction between the pump component 702 and the fluid container 600 can be directly actuated by the displacement of the hook 701 and / or in response to the control software of the detection device.

[0095] The structural ring between the flow cell 430 and the fluorometer 720 includes a reference surface 417, an aluminum body 460, legs 461 and 462, a plate or base to which the legs 461 and 462 are attached, and the fluorometer 720 is also attached to the plate or base.

[0096] Figure 10 shows a mechanism that can be used to engage the flow cell with the detection device. Figure 10A A side view and enlarged details of the jet canister 600 and flow tank 400 are shown when not engaged with the detection device. When the jet canister 600 is not engaged, the flow tank 400 is in contact with the hook 615 and guides 616 and 617. Figure 10B Enlarged details of the configuration produced when the tank 600 engages with the detection device are shown. Specifically, the flow cell box 400 moves toward the wall of the tank 600, disengaging from the hook 615 and the guides 616 and 617.

[0097] exist Figure 10E The diagram shows a mechanism for changing the position of the flow cell box 400. Figure 10E This is a detailed view of the interface between the tank 600, the flow tank box 400, and the aluminum body 460. Figure 10E yes Figure 10D Details Figure 10D It is along Figure 10C A cross-sectional view of line m. When the can 600 is properly engaged with the detection device, hooks 615 and guides 616 and 617 are inserted into recesses 471, 472, and 473 in the aluminum body 460. Recesses 471, 472, and 473 are deep enough that compression of the can towards the aluminum body 460 causes the front of the flow cell box 400 to engage with the sprocket 420, and the front of the flow cell 430 to contact the reference surface 417. Compression also causes the back of the flow cell box 400 to contact the pressure foot 102. In this way, the flow cell 430 is pressed against the reference surface 417 for alignment with the objective lens 410, which observes the flow cell 430 through window 418. The flow cell 430 can be translated by the interaction of the sprocket 420 and the perforated track 401.

[0098] Although mechanical contact has been used to illustrate the interaction between the jet canister and the detection device in this document, it should be understood that other mechanical switching mechanisms can be used. Electronic switches can also be used, including those activated by, for example, electronic sensors (e.g., Bluetooth), magnetic sensors, radio frequency sensors (e.g., RFID), pressure sensors, optical sensors (e.g., barcodes), etc.

[0099] The jet canisters and components described above are exemplary. Other jet canisters and jet components that can be used with the detection apparatus of this disclosure are described in commonly owned U.S. Patent Application Serial No. 15 / 922,661 (which claims the benefit of U.S. Provisional Application No. 62 / 481,289) and U.S. Patent Application Publication No. 2017 / 0191125A1, each of which is incorporated herein by reference. Moreover, similar jet canisters can be used with other devices of this disclosure (e.g., reactor devices), and these other devices can be configured to engage with the canister as described above.

[0100] Optionally, the detection apparatus or other apparatus of this disclosure may also include a computer processing unit (CPU) configured to operate one or more system components described herein. The same or different CPUs may interact with the system to acquire, store, and process signals (e.g., signals detected in the methods described herein). In certain embodiments, the CPU may be used to determine the identity of nucleotides present at a specific location in the template nucleic acid based on the signal. In some cases, the CPU will identify the nucleotide sequence of the template based on the detected signal.

[0101] A useful CPU may include, for example, one or more of the following: personal computer systems, server computer systems, thin clients, fat clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics network PCs, minicomputer systems, mainframe computer systems, smartphones, or distributed cloud computing environments that include any of the above systems or devices. The CPU may include one or more processors or processing units and may include a memory architecture that includes RAM and non-volatile memory. The memory architecture may also include removable / non-removable, volatile / non-volatile computer system storage media. Furthermore, the memory architecture may include one or more readers for reading and writing non-removable non-volatile magnetic media, such as hard disk drives; disk drives for reading and writing removable non-volatile media; and / or optical disk drives for reading or writing removable non-volatile optical discs (e.g., CD-ROMs or DVD-ROMs). The CPU may also include various computer system readable media. Such media can be any available media accessible in a cloud computing environment, such as volatile and non-volatile media, as well as removable and non-removable media.

[0102] The memory architecture may include at least one program product having at least one program module implemented as executable instructions configured to control one or more components of the apparatus described herein or to perform one or more parts of the methods described herein. For example, executable instructions may include an operating system, one or more application programs, other program modules, and program data. Typically, program modules may include routines, programs, objects, components, logic, data structures, etc., that perform specific tasks, such as processing signals detected in the methods described herein.

[0103] CPU components can be coupled via an internal bus, which can be implemented as any one or more of several types of bus architectures, including a memory bus or memory controller, a peripheral bus, a graphics acceleration port, and a processor or local bus using any of a variety of bus architectures. By way of example and not limitation, such architectures include the Industry Standard Architecture (ISA) bus, the Micro Channel Architecture (MCA) bus, the Enhanced ISA (EISA) bus, the Video Electronics Standards Association (VESA) local bus, and the Peripheral Component Interconnect (PCI) bus.

[0104] The CPU can optionally communicate with one or more external devices (e.g., a keyboard, a pointing device (e.g., a mouse), a display (e.g., a graphical user interface (GUI)), or other devices that facilitate user interaction with the nucleic acid detection system). Similarly, the CPU can communicate with other devices (e.g., via a network interface card, modem, etc.). Such communication can be performed through I / O interfaces. Furthermore, the CPU of this system 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 (e.g., the Internet) via a suitable network adapter.

[0105] Figure 11 A cross-sectional view of an exemplary optical apparatus using immersion optics is shown. The apparatus includes an objective lens 710, which comprises a housing 720 and several lenses 711, 712, and 715. The number, position, and shape of the lenses are exemplary and can be varied according to desired specifications. A rigid body 700, a flow cell 701, and a flow cell housing 702 are also included. The flow cell housing 702 includes an inlet 741 and an outlet 742 for transferring fluid reagents into or out of the flow cell. The bottom surface 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, using the configuration described above. In contrast to this seal, the rigid body 700 includes a tapered recess 716 shaped to receive the tip of the objective lens 710. The space 716 between the rigid body 700, the objective lens 710, and the seal can be filled with an immersion liquid, such as an oil or aqueous solvent matching the refractive index of the objective lens. In this way, the immersion liquid will directly contact the surfaces of the proximal lens 715 of the objective lens 710 and the flow cell 701. The fluid can be held in the space 716 by optional flexible seals 731 and 732. Fluid can be added and / or removed from the space 716 via conduit 733. Compared to optics that image through air, immersion optics offer several advantages, such as the ability to achieve a numerical aperture (NA) greater than 0.95, the ability to image containers at greater depths, and reduced tolerances to the thickness and uniformity of the container walls through which the objective lens resolves objects.

[0106] This disclosure provides methods particularly useful for performing cyclic reactions. Each cycle may include delivering reagents for the reaction to a flow cell or other container, where the reaction or reaction products may optionally be observed. Each cycle may also include scanning the container using the apparatus or methods described herein. The method is illustrated herein by way of example in the case of a nucleic acid sequencing reaction. However, those skilled in the art will understand from the teachings herein how the methods and apparatus can be modified for other cyclic reactions, such as nucleic acid synthesis reactions, peptide sequencing reactions, peptide synthesis reactions, combinatorial small molecule synthesis reactions, etc. However, the method need not be cyclic, but may be performed in a non-repeatable configuration, for example, to observe a single reaction or phenomenon.

[0107] Particularly useful sequencing reactions are achieved through Binding TM (SBB TM Sequencing is performed using reactions as described in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1; U.S. Patent Application Publication No. 2018 / 0044727 A1, U.S. Patent Application Serial No. 62 / 447,319, which claims priority thereto; U.S. Patent Application Publication No. 2018 / 0187245 A1, U.S. Patent Application Serial No. 62 / 440,624, which claims priority thereto; or U.S. Patent Application Publication No. 2018 / 0208983 A1, U.S. Patent Application Serial No. 62 / 450,397, which are each incorporated herein by reference. Typically, methods for determining the sequence of a template nucleic acid molecule can be based on the formation of a ternary complex (polymerase, initiating nucleic acid, and homologous nucleotides) under specified conditions. The method may include an examination phase followed by a nucleotide incorporation phase.

[0108] The detection phase can be performed in a flow cell (or other container) containing at least one template nucleic acid molecule, which is initiated with primers by delivering reagents into the flow cell to form a first reaction mixture. The reaction mixture may include the initiated template nucleic acid, a polymerase, and at least one nucleotide type. The interaction between the polymerase and the nucleotide with the initiated template nucleic acid molecule can be observed under conditions where the nucleotide is not covalently added to the primer; and the observed interaction between the polymerase and the nucleotide with the initiated template nucleic acid molecule can be used to identify the next base in each template nucleic acid. The interaction between the initiated template, polymerase, and nucleotide can be detected using various protocols. For example, the nucleotide may contain a detectable label. Each nucleotide may have a label that distinguishes it from other nucleotides. Alternatively, some or all different nucleotide types may have the same label, and the nucleotide types may be distinguished based on the individual delivery of different nucleotide types to the flow cell. In some embodiments, the polymerase may be labeled. Polymerases associated with different nucleotide types may have unique labels that distinguish them from the nucleotide types they are associated with. Alternatively, polymerases may have similar labels, and different nucleotide types may be distinguished based on the individual delivery of different nucleotide types to the flow cell. The detection can be performed by scanning the flow cell using the apparatus or method described herein.

[0109] During the testing phase, ternary complex stabilization can facilitate the differentiation of correct and incorrect nucleotides. Various conditions and reagents can be used. For example, primers may contain reversible blocking portions to prevent covalent attachment of nucleotides and / or may be devoid of cofactors required for extension (e.g., divalent metal ions); and / or may contain inhibitory divalent cations that inhibit polymerase-based primer extension; and / or the polymerase present in the testing phase may have chemical modifications and / or mutations that inhibit primer extension; and / or nucleotides may have chemical modifications that inhibit the patient, such as removal or alteration of the 5' modification of the native triphosphate moiety. The testing phase may include scanning the flow cell using the apparatus and methods described herein.

[0110] The extension phase can then be performed by creating conditions in a flow cell that allow nucleotides to be added to primers on each template nucleic acid molecule. In some embodiments, this involves removing the reagents used in the testing phase and replacing them with reagents that facilitate extension. For example, the testing reagents can be replaced with an extension-enabled polymerase and nucleotides. Alternatively, one or more reagents can be added to the testing phase reaction to generate extension conditions. For example, a catalytic divalent cation can be added to a cation-deficient testing mixture, and / or a polymerase inhibitor can be removed or destroyed, and / or an extension-enabled nucleotide can be added, and / or an unblocking agent can be added to enable the primers to extend, and / or an extension-enabled polymerase can be added.

[0111] It will be understood that the apparatus and methods of this disclosure can be used to perform any of a variety of nucleic acid sequencing reactions. Other exemplary sequencing methods are described below.

[0112] Synthesis sequencing (SBS) technology can be used. SBS typically involves the enzymatic extension of nascent primers by iteratively adding nucleotides to a template strand that hybridizes with the primer. In short, SBS can be initiated by contacting a target nucleic acid attached to a container site with one or more labeled nucleotides, DNA polymerase, etc. Those sites that use the target nucleic acid as a template for primer extension will incorporate detectable labeled nucleotides. Detection can include scanning using the apparatus or methods described herein. Optionally, the labeled nucleotides may also include reversible termination properties, terminating further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible termination moiety can be added to the primer, such that subsequent extension does not occur until a desealing reagent is delivered to remove this moiety. Therefore, in embodiments using reversible termination, a desealing reagent can be delivered to the container (before or after detection). Washing can be performed between the various delivery steps. This cycle can be performed n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. For example, exemplary SBS procedures, reagents, and detection components that can be readily adapted for use with detection devices produced by the methods of this disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; WO 91 / 06678; WO 07 / 123744; U.S. Patent Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019, or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082A1, each of which is incorporated herein by reference. SBS methods commercially available from Illumina, Inc. (San Diego, CA) are also useful.

[0113] Some SBS embodiments include the detection of protons released after nucleotides are incorporated into the extension product. For example, sequencing based on the detection of released protons can use reagents and electrodetectors commercially available from Thermo Fisher (Waltham, MA) or described in U.S. Patent Application Publication Nos. 2009 / 0026082A1; 2009 / 0127589 A1; 2010 / 0137143 A1; or 2010 / 0282617 A1, each of which is incorporated herein by reference.

[0114] Other sequencing procedures, such as pyrosequencing, can be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when nucleotides are incorporated into nascent primers that hybridize with the 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 6,210,891; 6,258,568 and 6,274,320, each incorporated herein by reference). In pyrosequencing, the released PPi can be detected by the conversion of ATP sulfatase to adenosine triphosphate (ATP), and the resulting ATP can be detected by photons generated by luciferase. Therefore, the sequencing reaction can be monitored by a luminescent detection system configured to scan the container using the apparatus and methods described herein.

[0115] Ligation sequencing reactions are also useful, including, for example, those described in Shendure et al., Science 309:1728-1732 (2005); U.S. Patent No. 5,599,675; or U.S. Patent No. 5,750,341, each of which is incorporated herein by reference. Some embodiments may include hybridization sequencing presentation, for example, as described 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 hybridizing with the nucleic acid template undergo repeated extension cycles via oligonucleotide ligation. Typically, oligonucleotides are fluorescently labeled and can be detected to determine the sequence of the template, for example, using the scan loading or methods described herein.

[0116] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interaction or zero-mode waveguide (ZMW) between a polymerase carrying a fluorophore and a γ-phosphate-labeled nucleotide. For example, modifications may be made to the techniques and reagents used in the apparatus or methods described herein for sequencing by FRET and / or ZMW detection, as described in, for example, 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 U.S. Patent Nos. 7,315,019; 8,252,911 or 8,530,164, the disclosures of which are incorporated herein by reference.

[0117] The steps of the sequencing method described above can be repeated. For example, SBB can be repeated. TM The method includes checking and extension steps to check for a single next-correct nucleotide in each cycle (i.e., the next correct nucleotide is the nucleotide that correctly binds to the 5'-positioned nucleotide of the template nucleic acid immediately adjacent to the 3' end of the hybridization primer), followed by adding the single next-correct nucleotide to the primer. Sequencing methods with any number of cycles described herein can be performed, including, for example, at least 1, 2, 5, 10, 20, 25, 30, 40, 50, 75, 100, 150, or more cycles. Alternatively or additionally, no more than 150, 100, 75, 50, 40, 30, 25, 20, 10, 5, 2, or 1 cycle may be performed.

[0118] The nucleic acid template to be sequenced can be added to the container using any of a variety of known methods. In some embodiments, a single nucleic acid molecule is to be sequenced. The nucleic acid molecule can be delivered to the container and can optionally be attached to a surface within the container. In some embodiments, the molecule is sequenced as a single molecule. Alternatively, multiple copies of the nucleic acid can be prepared, and the resulting whole can be sequenced. For example, the nucleic acid can be amplified on a surface (e.g., on the inner wall of a flow cell) using techniques described in further detail below.

[0119] In multiple embodiments, a variety of different nucleic acid molecules (i.e., a population with a variety of different sequences) are sequenced. The molecules may optionally be attached to a surface within the container. Nucleic acids may be attached to unique sites on the surface, and individual nucleic acid molecules spatially distinct from one another can be sequenced in parallel. Alternatively, nucleic acids can be amplified on the surface to generate multiple surface-attached aggregates. These aggregates can be spatially distinct and sequenced in parallel.

[0120] The methods described herein can be used in containers using any of a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), bridging amplification, or random primer amplification (RPA). In certain embodiments, one or more primers for amplification may be attached to the surface of the container. In such embodiments, the surface-attached primers, extending along the template nucleic acid, will result in copies of the template being attached to the surface. Methods for generating one or more sites on a solid support, wherein each site is attached to multiple copies of a specific nucleic acid template, may be referred to as “clustering” methods.

[0121] In PCR embodiments, one or two primers for amplification may be attached to the surface. The use of two attached primers is commonly referred to as bridging amplification because the double-stranded amplicon forms a bridge-like structure between the two attached primers, flanking the replicated template sequence. Exemplary reagents and conditions that can be used for bridging amplification are described, for example, in U.S. Patent Nos. 5,641,658 or 7,115,400; U.S. Patent Publications 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 may also be performed using one of an amplification primer attached to the surface and a second primer in solution. An exemplary form of using a combination of solid-phase and liquid-phase primers is called primer walking, and can be performed as described in U.S. Patent No. 9,476,080, which is incorporated herein by reference. Another example is emulsion PCR, which can be performed, for example, as described in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145 or U.S. Patent Publication Nos. 2005 / 0130173A1 or 2005 / 0064460 A1, each of which is incorporated herein by reference.

[0122] RCA technology can be used in the methods described herein. Exemplary reagents that can be used in RCA reactions and the principles of RCA amplicon generation are described, for example, in Lizardi et al., Nat. Genet. 19:225-232 (1998) or U.S. Patent Application Publication No. 2007 / 0099208 A1, each of which is incorporated herein by reference. Primers used for RCA can be in solution or attached to the surface of a flow cell.

[0123] MDA technology can also be used in the methods of this disclosure. Some reagents and useful conditions for MDA are described, for example, 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 U.S. Patent Nos. 5,455,166; 5,130,238; or 6,214,587, each of which is incorporated herein by reference. Primers used for MDA can be in solution or attached to a surface in a container.

[0124] In certain embodiments, combinations of the amplification techniques illustrated above can be used. For example, RCA and MDA can be used in combination, where RCA is used to generate polyplastic amplicones in solution (e.g., using liquid-phase primers). The amplicon can then be used as a template for the MDA using primers attached to the surface of the container. In this example, the amplicon generated after the RCA and MDA combination step will be attached to the container. The amplicon will typically contain a polyplastic repeat of the target nucleotide sequence.

[0125] The nucleic acid templates used in the methods or compositions described herein can be DNA, such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), etc. RNA, such as mRNA, ribosomal RNA, tRNA, etc., can also be used. Nucleic acid analogs can also be used as templates in this paper. Therefore, the mixtures of nucleic acids used herein can be derived from biological sources, synthetic sources, or amplification products. The primers used herein can be DNA, RNA, or analogs thereof.

[0126] Exemplary organisms from which nucleic acids can be obtained include, for example, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, primates, humans, or non-human primates; plants such as Arabidopsis thaliana, maize, sorghum, oats, wheat, rice, rapeseed, or soybeans; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila melanogaster, mosquitoes, fruit flies, bees, or spiders; fish such as zebrafish; reptiles; amphibians such as frogs or Xenopus laevis; dictyostelium discoideum; and fungi such as Pneumocystis carinii. Nucleic acids can originate from prokaryotes, such as *Carinii*, *Takifugu rubripes*; yeasts, such as *Saccharomyces cerevisiae* or *Schizosaccharomyces pombe*; or *Plasmodium falciparum*. Nucleic acids can also originate from prokaryotes, such as bacteria, such as *Escherichia coli*, *Staphylococci*, or *Mycoplasma pneumoniae*; archaea; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Nucleic acids can originate from homogeneous cultures or populations of the above-mentioned organisms, or alternatively from, for example, a collection of several different organisms in a community or 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 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 the apparatus or methods described herein.

[0127] Template nucleic acids can be obtained from preparation methods such as genome isolation, genome fragmentation, gene cloning, and / or amplification. Templates can be obtained from amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), etc. Exemplary methods for isolating, amplifying, and fragmenting nucleic acids to produce templates for analysis on an array are described in U.S. Patent Nos. 6,355,431 or 9,045,796, each of which is incorporated herein by reference. Amplification can also be performed using methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.

[0128] This disclosure also provides a detection device comprising: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the wall has a plurality of discrete contacts between the inner surface and the outer surface, wherein the inner surface contacts the inner cavity, and wherein the plurality of discrete contacts occupy a length l in a scanning dimension x; (b) a transmissive surface; (c) a preload configured to push discrete contacts on the outer surface of the container into contact with the transmissive surface, optionally wherein the maximum length of a region of the transmissive surface in the scanning dimension x may be 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 through the transmissive surface.

[0129] As illustrated in several embodiments herein, optical signals can be relayed to the detection device through a light-transmitting surface. Objective lenses serve as useful transmitters of optical signals from the container to the detector. In some embodiments, the transmitter is a lens array. Lenses in the array can be configured to collect signals from different regions of the xy-plane (or direct energy to different regions of the xy-plane). Lenses can be arranged to collect signals from consecutive regions of the xy-plane, or alternatively, the observed region can be separated by gaps that are not observed when that region is observed. In some embodiments, the container includes an array of sites configured for observation via the lens array. Each lens can be configured to simultaneously observe one or more sites in the site array. For example, each lens can be configured to observe at least 1, 4, 9, 16, 25, 36, 49, 64, 81, 100, or more sites in the site array. Alternatively or additionally, each lens can be configured to observe at most 100, 81, 64, 49, 36, 25, 16, 9, 4, or 1 site in the site array. Therefore, an embodiment is provided in which each lens is configured to observe a single point.

[0130] Each lens in the lens array can be aligned with its own optical assembly to direct radiation to one or more detectors. Alternatively, multiple lenses can be combined into a common optical assembly to direct radiation to one or more detectors. The optical assembly can include any of a variety of optical components, including but not limited to: a collimating lens for collimating signals from the site array, a dichroic element for spectrally separating radiation, and a focusing lens for focusing radiation from the sites onto the detectors. Exemplary configurations of lens arrays and site arrays observed through lenses are provided in U.S. Patent No. 9,581,550, which is incorporated herein by reference. For example, a site in the array can be a zero-mode waveguide (ZMW).

[0131] Other transmitters may be used depending on the energy or signal to be transmitted. For example, a transmissive surface can conduct electrical, thermal, magnetic, pressure, and audio signals. Temporary electrical contacts, such as pogo pins, can be used to transmit electrical signals between the transmissive surface and the container. The transmitters present in the apparatus described herein can transmit various forms of energy, including but not limited to the signals mentioned above.

[0132] In certain embodiments, the transmissive or inner surface of the container includes an electronic detector, such as a field-effect transistor (FET) or a complementary metal-oxide-semiconductor (CMOS). Particularly useful electronic detectors include, for example, those used in nucleic acid sequencing applications, such as those for proton detection as described in U.S. Patent Application Publications 2009 / 0026082 A1; 2009 / 0127589 A1; 2010 / 0137143 A1; or 2010 / 0282617 A1, each of which is incorporated herein by reference. Electronic detectors for detecting optical signals are also useful, including, for example, those set forth in U.S. Patent Publications 2009 / 0197326 A1; 2015 / 0293021 A1; 2016 / 0017416 A1; or 2016 / 0356715 A1, each of which is incorporated herein by reference.

[0133] The apparatus and methods of this disclosure have already been illustrated in the context of nucleic acid sequencing reactions. These apparatus and methods can also be used for other analytical applications. Typically, analytical applications performed in scanning microscopy can be applied to the apparatus and methods of this disclosure. For example, the methods or apparatus can be configured to scan microarrays for analyzing enzyme activity, ligand-receptor binding, binding of complementary nucleic acids to each other, the presence of mutations in nucleic acids (e.g., single nucleotide polymorphisms (SNPs)), and expression levels of RNA material. Microarrays detected by optical labeling, such as fluorophores, are particularly suitable. Larger biological samples, such as cells or tissues, can be detected using the methods or apparatus described herein. Similarly, detection methods utilizing optically detected probes or dyes are particularly suitable. Other uses include the evaluation of finished products, assessing their quality or other characteristics by scanning with a microscope. Exemplary products include, but are not limited to, computer chips, sensors, electronic components, and other microfabricated or nanofabricated devices. Tests known in the field of molecular diagnostics can be modified for use in the apparatus or methods described herein, such as binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), real-time polymerase chain reaction assays, etc.

[0134] The apparatus and methods described herein in the context of detection reactions can be readily modified for use in preparation methods. In a particular embodiment, this disclosure provides a reactor apparatus. The reactor apparatus may include: (a) a container having an inner cavity and a wall, wherein the wall has an inner surface and an outer surface, wherein the inner surface contacts the inner cavity; (b) a reference surface forming a structural ring with an energy source; (c) a preload configured to push the outer surface of the container into contact with a region on the reference surface; (d) a scanning actuator configured to slide the container along the reference surface in a scanning dimension; and (e) an emitter configured to direct energy from the energy source to the inner surface or the inner cavity when the outer surface of the container is pushed into contact with the reference surface by the preload.

[0135] A method for carrying out a reaction in a container is also provided. The method may include (a) translating a container along a reference surface of a reactor apparatus, wherein the container includes an inner cavity and walls, wherein the inner cavity contains reactants, wherein during the translation, the reference surface contacts at least a portion of the container, and wherein the reference surface forms a structural ring with an energy source; and (b) directing energy from the energy source along the container to analytes at different locations, wherein during the directing of energy to the reactants, the container is pushed against the reference surface by the preload, thereby carrying out the reaction in the container.

[0136] The method of carrying out the reaction may include (a) transferring energy from the reactor apparatus to a first subset of reactants in the container while applying a preload to a first portion of the container, wherein the preload positions the first subset of reactants to occupy the xy plane of the reaction zone, wherein no preload is applied to a second portion of the container; (b) translating the container to position a second subset of reactants in the xy plane of the reaction zone; and (c) transferring energy from the reactor apparatus to a second subset of analytes in the container while applying the preload to the second portion of the container, wherein the preload positions the second subset of analytes to occupy the xy plane, wherein no preload is applied to the first portion of the container, thereby carrying out the reaction in the container.

[0137] Exemplary energy sources that may be used in the apparatus described herein include, but are not limited to, radiation sources such as lasers, light-emitting diodes (LEDs), lamps, microwave sources, or X-ray generators; power supplies; ion beam sources such as dual plasma tubes; electron emitters such as heated filaments or hollow cathodes; current sources; or voltage sources.

[0138] Throughout this application, various publications, patents, and / or patent applications are cited. The disclosures of these documents are incorporated herein by reference in their entirety.

[0139] The term "includes" is intended to be open-ended in this document, including not only the listed elements but also any other elements.

[0140] As used herein, the term "each" is intended to identify an individual item in a collection of items, but not necessarily every single item in the collection. Exceptions may occur if the explicit public information or context otherwise specifies otherwise.

[0141] Many embodiments have been described. However, it will be understood that various modifications can be made. Therefore, other embodiments are within the scope of the appended claims.

Claims

1. A method for optically scanning a container, comprising: (a) A container is provided, the container including an inner cavity and a wall, wherein the inner cavity contains an optically detectable analyte, and wherein the wall is transparent to the optically detectable analyte; (b) Providing a detection apparatus comprising a preload device, a rigid body, a detector, and an objective lens, wherein a reference surface is located on a first surface of the rigid body, the objective lens is located on a second surface of the rigid body, and the objective lens is positioned to emit radiation from the first surface of the rigid body to the detector, the detection apparatus further comprising 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 relative to the detector; and (c) The container is optically scanned in the following manner: (i) Slide the length of the container along the reference surface such that at any time during the sliding, the reference surface is in direct contact only with a portion of the length of the container. (ii) Detecting the optically detectable analyte by the detector, including by acquiring a signal emitted from the optically detectable analyte through the wall of the container and through the objective lens toward the detector, and (iii) During the test, the preload device applies a force to force the container into contact with the reference surface.

2. The method of claim 1, wherein during the detection, the preload device pushes the container toward the reference surface.

3. The method of claim 2, wherein the preload device comprises a ball bearing that contacts the container during the detection.

4. The method of claim 3, wherein the ball bearing is forced toward the container by a spring.

5. The method of claim 1, wherein the first contact area between the preload device and the container and the second contact area between the reference surface and the container are complementary.

6. The method of claim 1, wherein the detection device further comprises a rotating gear, wherein the container further comprises one or more perforations, and wherein the sliding further comprises the rotating gear of the detection device contacting the one or more perforations.

7. The method of claim 1, wherein the container is forced to the reference surface by the preload device during the sliding.

8. The method of claim 1, wherein the objective lens observes the container through an opening in the reference surface.

9. The method of claim 8, wherein the contact area between the preload device and the container is opposite to the area of ​​the reference surface surrounding the opening.

10. The method of claim 1, wherein the optically detectable analyte is attached to the inner surface of the wall in contact with the cavity.

11. The method of claim 10, wherein the optically detectable analyte forms an array of distinguishable sites on the inner surface of the wall.

12. The method of claim 11, wherein the array has a per cm 2 A density of at least 1000 sites, wherein the detection includes acquiring an image of the array at a resolution that distinguishes each site in the array.

13. The method of claim 10, further comprising: Nucleic acid is sequenced at an array of distinguishable sites, based at least on a scan of the container.

14. The method of claim 13, wherein sequencing the nucleic acid comprises delivering polymerase and nucleotides to the container.

15. The method of claim 14, wherein the polymerase and nucleotides are delivered in each of a plurality of repeated cycles, and wherein the container is scanned during each of the plurality of cycles.

16. The method of claim 14, wherein sequencing the nucleic acid comprises labeling sites of the array with fluorescent tags.

17. The method of claim 16, wherein the fluorescent tag is excited by radiation passing through the objective lens, and wherein the signal includes fluorescence emitted from the tag.

18. The method of claim 1, wherein during optical scanning of the container, the position of the reference surface is fixed relative to the position of the detector.

19. The method of claim 1, wherein the reference surface comprises a plane forming a flat ring around a window passing through the rigid body, and wherein a signal acquired during the detection is emitted through the window after being emitted through the wall and before being emitted through the objective lens.

20. The method of claim 1, wherein the objective lens comprises an array of lenses, wherein each of the lenses is configured to collect signals from a location in a different region along the length of the container.

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