Systems and methods for refurbishing chips

Abstract

Aspects of the present disclosure relate to methods and systems for regenerating a sensor chip surface, including techniques for reusing a single sensor chip in multiple sampling cycles by regenerating the surface of the sensor chip between successive sampling cycles. A method is provided for reusing an integrated device for processing a sample, wherein the sample is divided into multiple aliquots, the method including: loading a first aliquot of the multiple aliquots into at least some of a plurality of chambers of the integrated device; sampling an analyte in the first aliquot while the analyte is present in at least some of the plurality of chambers; removing the first aliquot from at least some of the plurality of chambers of the integrated device; and loading a second aliquot of the multiple aliquots into at least some of the plurality of chambers of the integrated device.

Description

[Technical Field]

[0001] This disclosure relates to an integrated device and related instruments capable of performing massively parallel analysis of samples by simultaneously supplying short light pulses to tens of thousands of reaction chambers and receiving fluorescence signals from the reaction chambers for sample analysis. Such instruments may be useful for point-of-care genetic sequencing and personalized medicine. [Background technology]

[0002] Photodetectors are used to detect light in a variety of applications. Integrated photodetectors have been developed that generate electrical signals indicating the intensity of incident light. Integrated photodetectors for imaging applications include pixel arrays that detect the intensity of light received from the entire scene. Examples of integrated photodetectors include charge-coupled device (CCD) image sensors and complementary metal-oxide-semiconductor (CMOS) image sensors.

[0003] Instruments capable of ultra-parallel analysis of biological and chemical samples are generally limited to laboratory use due to their large size, lack of portability, the need for skilled technicians to operate them, the required power output, the need for a controlled operating environment, and the cost. When analyzing samples using such instruments, the common paradigm is to extract the sample clinically or in the field, send it to the laboratory, and await the results. The waiting time for results can range from a few hours to several days. [Overview of the project]

[0004] Aspects of the technology disclosed herein relate to methods for determining the identity or sequence of a sample containing polypeptide molecules or nucleic acid molecules. Further aspects relate to methods and devices for next-generation sequencing of a sample, including single-molecule sequencing. Aspects of the disclosure relate to methods and systems for sequencing polypeptide or nucleic acid molecules using luminescent molecules, such as luminescent labels (e.g., fluorophores).

[0005] A system based on this disclosure enables single-molecule analysis. An exemplary system includes an integrated device (also called a chip) and an apparatus configured to interface with the integrated device. The integrated device may include an array of pixels, where each pixel includes a sample well and at least one photodetector. The sample wells of the integrated device may be configured to accept a sample formed on or through the surface of the integrated device and placed on the surface of the integrated device. In general, the sample wells may be formed as an array of sample wells. Multiple sample wells may have suitable sizes and shapes such that at least a portion of the sample wells accept a single sample (e.g., a sample containing a single type of biomolecule). In some embodiments, the number of samples in the sample wells of the integrated device may be distributed such that some sample wells contain one sample, while other sample wells contain zero, two, or more samples.

[0006] Some embodiments of this disclosure provide a method for reusing an integrated device for processing a sample divided into multiple aliquots. In various embodiments, the sample comprises an analyte, and the analyte comprises a biomolecule containing one or more luminescently labeled molecules, and the method comprises: loading a first aliquot of the multiple aliquots into at least a portion of a plurality of chambers of an integrated device; sampling the analyte of the first aliquot while the analyte is present in at least a portion of the plurality of chambers, comprising determining the luminescence lifetime, luminescence intensity, wavelength, pulse duration, and / or pulse duration of a signal emitted by one or more luminescently labeled molecules; removing the first aliquot from at least a portion of the plurality of chambers of the integrated device; and loading a second aliquot of the multiple aliquots into at least a portion of the plurality of chambers of the integrated device. In some embodiments, the term aliquot may refer to several portions of the same sample, while in other embodiments, the first and second aliquots may include several portions of the first and second sample originating from different sources.

[0007] Some embodiments provide a method for reusing an integrated device for processing a sample, wherein the sample comprises an analyte, and the analyte comprises a biomolecule containing one or more luminescently labeled molecules, and the method comprises loading at least a portion of the sample into a plurality of chambers of the integrated device; sampling the analyte of at least a portion of the sample while the analyte is present in the plurality of chambers, comprising collecting signals emitted by one or more luminescently labeled molecules indicating emission lifetime, emission intensity, wavelength, pulse duration, and / or duration between pulses; and removing the at least a portion of the sample from the plurality of chambers, comprising breaking the covalent bonds between the respective coating molecules bound to the surface of the plurality of chambers and the coupling portion, wherein at least a portion of the sample analyte is bound to the coupling portion.

[0008] Some embodiments provide a method for determining whether a sample is present in one or more chambers of an integrated device, comprising the steps of: loading at least a portion of the sample into one or more chambers of the integrated device; removing at least a portion of the sample from one or more chambers of the integrated device; delivering excitation light to one or more chambers of the integrated device; collecting signals emitted from the plurality of chambers in response to the excitation light in a photodetector region of the integrated device; and determining, based on the signals, whether at least a portion of at least a portion of the sample is present in one or more chambers of the integrated device.

[0009] Some embodiments provide a method for determining whether to continue processing a sample using an integrated device, wherein the sample is divided into a plurality of aliquots, and the method includes a sampling step of sampling an analyte from a first aliquot of a plurality of aliquots while the analyte is present in one or more chambers of the integrated device, the sampling step of exciting the analyte from the first aliquot using excitation light delivered from at least one light source, and collecting a signal emitted from the first aliquot analyte in a photodetector region of the integrated device when excited by light delivered from at least one light source; a step of determining whether to load a second aliquot of the plurality of aliquots into one or more chambers of the integrated device based on the signal emitted from the first aliquot analyte; and a step of loading the second aliquot of the plurality of aliquots into one or more chambers of the integrated device.

[0010] Some embodiments provide a reusable device for processing a sample, wherein the sample is divided into a plurality of aliquots, and the device comprises a plurality of reaction chambers for receiving a first aliquot of the plurality of aliquots, wherein the surfaces of the plurality of reaction chambers are coated with coating molecules configured to bind to coupling portions bound to the analyte of the first aliquot; a photodetector region configured to receive signals emitted from the plurality of chambers in response to excitation light delivered to the plurality of chambers; and at least one processor configured to determine, based on the signals received by the photodetector region, whether the plurality of chambers contain at least a portion of the analyte of the first aliquot.

[0011] Some embodiments provide a method comprising the steps of loading a first sample into a plurality of chambers of a reusable chip, wherein the reusable chip has a plurality of waveguides for directing excitation light received from an apparatus comprising at least one light source to the plurality of chambers, and a plurality of photodetector regions for receiving light emitted from the first sample; delivering excitation light from at least one light source of the apparatus to the plurality of chambers of the reusable chip; sequencing of the analyte of the first sample; removing the first sample from the plurality of chambers of the reusable chip; and loading a second sample into the plurality of chambers of the reusable chip.

[0012] Some embodiments provide a reusable chip for use with an apparatus comprising at least one light source, the reusable chip comprising a plurality of chambers for receiving a first sample, a plurality of waveguides for directing excitation light from at least one light source to the plurality of chambers, and a plurality of photodetector regions for receiving light emitted from the first sample, wherein the reusable chip is configured to allow the first sample to be removed from the plurality of chambers and, after the first sample has been removed, to allow a second sample to be received into the plurality of chambers.

[0013] Some embodiments provide devices and methods for reusing chips through chip surface regeneration, but the devices and methods are suitable for large-scale parallel analysis of biological or chemical samples. In some embodiments, such devices and methods are even more suitable for automated large-scale parallel analysis. [Brief explanation of the drawing]

[0014] [Figure 1A] This is a block diagram of an integrated device and equipment according to some embodiments. [Figure 1B] This is a schematic diagram of an integrated device according to some embodiments. [Figure 1C] This is a schematic diagram of a pixel in an integrated device according to one embodiment. [Figure 1D] It is a circuit diagram of the pixel of FIG. 1C according to some embodiments. [Figure 1E] It is a top view of the pixel of FIG. 1C according to some embodiments. [Figure 1F] It is a plan view of the pixels of FIGS. 1C and 1D according to some embodiments. [Figure 1G] It is a schematic diagram of an alternative pixel of an integrated device according to some embodiments. [Figure 1H] It is a circuit diagram of the pixel of FIG. 1G according to some embodiments. [Figure 1I] It is a plan view of the pixels of FIGS. 1C and 1D according to some embodiments. [Figure 2A] It is an exemplary diagram of an analyte immobilized in a chamber of an integrated device according to some embodiments. [Figure 2B] It is an exemplary diagram of a process for reusing the integrated device of FIG. 1 to process multiple samples according to some embodiments. [Figure 3A] It is an exemplary diagram of a process for regenerating the surface of the chamber of the exemplary integrated device of FIG. 1 according to some embodiments. [Figure 3B] It is another exemplary diagram of a process for regenerating the surface of the chamber of the integrated device of FIG. 1A according to some embodiments. [Figure 3C] It is another exemplary diagram of a process for regenerating the surface of the chamber of the integrated device of FIG. 1A according to some embodiments. [Figure 3D] It is another exemplary diagram of a process for regenerating the surface of the chamber of the integrated device of FIG. 1A according to some embodiments. [Figure 4A] It is an exemplary graph showing the presence of an analyte obtained during the processes of chip loading and regeneration according to some embodiments. [Figure 4B] It is an exemplary graph showing the presence of an analyte obtained during the processes of chip loading and regeneration according to some embodiments. [Figure 4C]An exemplary graph showing the presence of an analyte obtained during chip loading and regeneration processes, according to some embodiments. [Figure 4D] An exemplary graph showing the presence of an analyte obtained during chip loading and regeneration processes, according to some embodiments. [Figure 4E] A diagram of the spectra of samples immersed in an exemplary regeneration solution under different conditions, according to some embodiments. [Figure 4F] An exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4G] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4G-1] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4H] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4H-1] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4I] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4I-1] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4J] A further exemplary graph showing the signal obtained from the reaction chamber of an exemplary integrated device between the regeneration step and the loading step, according to some embodiments. [Figure 4J-1]Further exemplary graphs showing signals obtained from the reaction chamber of an exemplary integrated device between the regeneration process and the loading process, according to some embodiments. [Figure 5A] This flowchart shows an exemplary process for determining whether a sample is present in the chamber of the integration device shown in Figure 1A, according to some embodiments. [Figure 5B] This flowchart shows an exemplary process for determining whether to continue processing a sample using the integrated device shown in Figure 1A, according to some embodiments. [Figure 6A] This is a block diagram of an analytical instrument equipped with a compact mode-locked laser module, according to one embodiment. [Figure 6B] This is a diagram of an exemplary compact mode-locked laser module incorporated into an analytical instrument, according to one embodiment. [Figure 6C] This figure shows an example of a parallel reaction chamber that can be optically excited by a pulsed laser through one or more waveguides, according to some embodiments. [Figure 6D] This figure shows further details of an integrated device reaction chamber, optical waveguide, and time-binning photodetector according to some embodiments. [Figure 7A] Figures 7A and 7B show the results of an experiment evaluating the sequencing accuracy of a DNA analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure, where Figure 7A is a line graph showing the average read length between signals obtained from three sequential runs (two chip regenerations) on an exemplary integrated device between the regeneration and loading steps. [Figure 7B] The figure shows the results of an experiment evaluating the sequencing accuracy of a DNA analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (line graphs showing the average accuracy, maximum accuracy, and “alignment” for each of these three runs). [Figure 7B-1]The figure shows the results of an experiment evaluating the sequencing accuracy of a DNA analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (line graphs showing the average accuracy, maximum accuracy, and “alignment” for each of these three runs). [Figure 8A-8B] Figure 8A shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (this experiment was repeated on two CMOS chips), and Figure 8B shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (this experiment was repeated on two CMOS chips). [Figure 8C-8D] Figure 8C shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (this experiment was repeated on two CMOS chips), and Figure 8D shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (this experiment was repeated on two CMOS chips). [Figure 8E-8F] Figure 8E shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of this disclosure (this experiment was repeated on two CMOS chips), and Figure 8F shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of this disclosure (this experiment was repeated on two CMOS chips). [Figure 8G-8H]Figure 8G shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (this experiment was repeated on two CMOS chips), and Figure 8H shows the signal reading results of an experiment evaluating sequencing accuracy over three read lengths of a peptide analyte using an exemplary method for regenerating a chip in some embodiments of the present disclosure (this experiment was repeated on two CMOS chips). [Figure 9] This figure shows an exemplary process for calibrating a system including integrated devices using photobleaching, according to some embodiments. [Figure 10-1] This figure shows a sample labeled with a dye attached to its surface, according to one embodiment. [Figure 10-2] This figure shows a sample labeled with a dye attached to the inside of a reaction chamber, according to one embodiment. [Figure 11] This figure shows an exemplary graph illustrating the time-dependent excitation output of the light source according to some embodiments. [Figure 12-1] This figure shows an exemplary graph illustrating the signal measured over time from a reference dye, according to some embodiments. [Figure 12-2] This figure shows exemplary histograms of measured fading time and dye intensity for the collection of a single molecule in a reaction chamber according to some embodiments. [Figure 13] This figure shows a sequencing reaction in a reaction chamber according to one embodiment. [Figure 14] This figure shows an exemplary process for quantifying the load in one or more reaction chambers, according to some embodiments. [Figure 15-1] This figure shows an exemplary trace from a chip loading process representing a single loaded well, according to one embodiment. [Figure 15-2] This figure shows an exemplary trace from the chip loading process representing a double-loaded well, according to one embodiment. [Figure 15-3]This figure shows an exemplary trace from the chip loading process representing a multi-load well, according to one embodiment. [Figure 16] This figure shows the periodic pulsed pattern of a fluorescent molecule reversibly bound to a biomolecule, according to one embodiment. [Figure 17] This figure shows an exemplary heat map of a reaction chamber illustrating load percentages in some embodiments. [Modes for carrying out the invention]

[0015] The features and advantages of this disclosure may become more apparent from the detailed description below, along with the drawings. When describing embodiments with reference to the drawings, directional references (such as "top," "bottom," "summit," "bottom," "left," "right," "horizontal," "vertical," etc.) may be used. Such references are intended solely to assist readers viewing the drawings in their normal orientation. These directional references are not intended to describe a preferred or sole orientation of the feature parts of the embodied device. The device may be embodied using other orientations as well.

[0016] I. Introduction Aspects of this disclosure relate to methods and systems for regenerating the surface of a sensor chip, where the sensor chip forms all or part of an integrated device. In particular, according to some embodiments, a technique is provided for reusing a single sensor chip in multiple sampling cycles by regenerating the surface of the sensor chip between consecutive sampling cycles.

[0017] The integrated device may include a sensor chip, each having multiple reaction chambers for receiving samples. Sample data can be obtained by exciting a portion of the sample placed in the multiple reaction chambers using excitation light, and collecting the signal emitted from the sample in response to the excitation using a photodetector. To perform sample collection, a portion of the sample present in the reaction chamber is immobilized. For example, the surface of each reaction chamber may be coated with a molecule, such as biotin, while the sample portion may be functionalized with a biotin-binding protein, such as streptavidin, to immobilize a portion of the sample within the reaction chamber. Generally, the sensor chip is placed after sample collection has been performed.

[0018] In many applications, it is desirable to detect multiple analytes present in a single sample. However, the sampling device is limited by the number of pixels and therefore the number of reaction chambers available to accept the sample's analytes. Often, the number of reaction chambers on the sampling device is less than the number of analytes to be sampled. In addition, while samples can be diluted to obtain titration that maximizes the number of reaction chambers accepting a single analyte, many reaction chambers still do not yield useful results because they either do not contain the analyte or contain two or more analytes, making it impossible to obtain the desired single analyte measurement result. Therefore, it is generally impossible for a single sampling device to process samples containing multiple analytes.

[0019] One technique for sampling multiple analytes present in a single sample is to divide the sample into aliquots and process each aliquot individually as a different experiment. For example, each aliquot of a sample may be processed individually by each integration device. However, such a technique is expensive because it requires the use of multiple integration devices.

[0020] The inventors have recognized that it is advantageous to reuse a single integration device for performing multiple experiments on a single sample and / or multiple samples before discarding the integration device. In this way, a sample containing multiple analyte species can be divided into multiple aliquots and processed in succession of experiments using a single integration device. According to the prior art, a sample can be processed by (1) coating the tip surface with a molecule, such as biotin; (2) loading a first aliquot of the sample containing an analyte bound to a coupling portion for binding to the coating, such as streptavidin, and allowing the labeled analyte to bind to the tip surface; (3) rinsing off unbound species; and (4) performing a measurement (e.g., by exciting the analyte bound to the surface of the reaction chamber and collecting the signal emitted from the excited analyte). Diluting the sample to titration (maximizing the number of reaction chambers that accept only a single type of analyte) as described herein still results in several reaction chambers that do not contain the analyte. Therefore, by reloading the integrated device with a second aliquot of the sample, it is possible to measure the analyte in the reaction chamber that remains unoccupied. However, if the reaction chamber is occupied by the initial loading of the first aliquot, the measurement of the analyte in the second aliquot may be hindered or interfered with.

[0021] Accordingly, the inventors have developed a technique for regenerating the surface of a sensor chip that has been used to date. In particular, the technique described herein relates to a method for unloading analytes that have been previously bound to the surface of the sensor chip via biotin-streptavidin binding or other interactions. For example, an example technique for unloading a sensor chip includes (a) destroying the bound anchorages (e.g., streptavidin and biotin); (b) peeling off the molecular (e.g., biotin) coating from the surface of the sensor chip; (c) enzymatically digesting the sample analyte in an occupied reaction chamber; and (d) melting the binding between the sample analyte and the coupling portion (e.g., streptavidin, biotin). After the regeneration of the sensor chip surface, the integrated device can be reloaded with a second aliquot of the sample. Such a technique allows for the processing of an entire sample containing multiple aliquots, even though a multitude of analytes are present in the sample.

[0022] The inventors have also recognized that when integrated devices are reused in multiple experiments, the components of the integrated device may degrade if the sensor chip is used over a long period of time. Therefore, some aspects of the techniques described herein relate to techniques for increasing the service life of integrated devices.

[0023] The techniques described herein for regenerating the surface of sensor chips in integrated devices may, in some embodiments, be used in combination with DNA / RNA and / or protein sequencing applications.

[0024] It should be understood that the integrated devices described herein may incorporate some or all of the technologies described herein, either individually or in combination. Furthermore, in addition to those described herein, other integrated devices may employ the methods and structures described herein.

[0025] Surface functionalization or coating In certain embodiments, the techniques described herein can be used to coat (or function or confine) a target molecule in a desired region of a sample well (also referred to herein as a reaction chamber). In some embodiments, the sample well occupies a volume or space defined by an opening formed on the surface of the integrated device (extending through a first layer of the integrated device into a second layer and reaching the bottom surface distal to the opening). In some embodiments, the exposed surfaces of the first and second layers located between the opening and the bottom surface of the sample well may be referred to as sidewalls (further defining the volume or space occupied by the sample well).

[0026] In several embodiments, when one or more analytes (e.g., DNA analytes or peptide analytes) are immobilized on a bottom surface, it may be desirable to coat the bottom surface to allow the linkage of one or more molecules or complexes. In some embodiments, the bottom surface is coated with a coating molecule. In some embodiments, the coating molecule is an avidin protein. Avidin proteins are biotin-binding proteins and generally have biotin-binding sites in each of their four subunits. Examples of avidin proteins include avidin, streptavidin, traptavidin, tamavidin, bradavidin, xenavidin, and their homologs and variants. In some embodiments, the avidin protein is streptavidin. Since each of the four binding sites has the ability to independently bind to a biotin molecule, the polyvalent nature of avidin proteins can allow for various binding configurations.

[0027] In certain embodiments, the coating molecule is one of biotin, streptavidin, and a biotin-streptavidin complex. In some embodiments, the coating molecule is a biotin analog or a streptavidin analog. In some embodiments, the coating molecule is bisbiotin. In some embodiments, the coating molecule is involved in interactions with a coupling moiety (e.g., a streptavidin moiety), such as bisbiotin-streptavidin binding.

[0028] In some embodiments, the coating molecule is a silane containing an alkyl chain. In some embodiments, the coating molecule is a silane containing an optionally substituted alkyl chain. In some embodiments, the coating molecule is a silane containing a poly(ethylene glycol) chain. In some embodiments, the coating molecule is a silane containing a coupling moiety. For example, the coupling moiety may include a chemical moiety, such as an amine group, a carboxyl group, a hydroxyl group, a sulfhydryl group, a metal, a chelating agent, etc. Alternatively, the coupling moiety may include specific binding elements such as biotin, avidin, streptavidin, neutraavidin, lectins, SNAP-TAG (registered trademark (self-labeled protein tag)), associative or binding peptides or proteins, antibodies or antibody fragments, nucleic acids or nucleic acid analogs, etc. The coupling moiety may include a methyl ether group. Additionally or alternatively, an intermediate binder and / or copolymer may be used to couple to additional groups (in some cases, including both chemical functional groups and specific binding elements) used to couple or bind to the molecule of interest. For example, a coupling moiety, such as biotin, may be layered on a substrate surface and selectively activated within a given area. An intermediate binder, such as streptavidin, may then couple to the biotin coupling moiety. The analyte of interest (which may itself be biotinylated in the exemplary workflow) then couples to streptavidin.

[0029] The chemical structure of biotin is reproduced below:

[0030] [ka] Streptavidin contains four biotin-binding sites. Even after streptavidin has been coated onto the bottom surface of the sample well, three additional binding sites remain for conjugation with biotin or biotin-conjugate polymers.

[0031] Examples of coating molecules include, but are not limited to, trans-cyclooctene (TCO) moieties, tetrazine moieties, azide moieties, alkyne moieties, aldehyde moieties, methyl ether moieties, isocyanate moieties, N-hydroxysuccinimide moieties, thiol moieties, alkene moieties, dibenzocyclooctyl moieties, bicyclononine moieties, and thiamine pyrophosphate moieties. Examples of coating molecules containing covalent coupling moieties include, but are not limited to, azide-silanes and azide-organosilanes, such as azide-PEG-silanes (e.g., azide-PEG3-silane, azide-PEG5-silane), and azide-alkylsilanes (e.g., azide-C 11 Examples include -silanes. In some embodiments, the coupling portion is a non-covalent coupling portion.

[0032] Examples of coupling moieties include, but are not limited to, biotin moieties, avidin proteins, streptavidin proteins, lectin proteins, SNAP tags, methyl ethers, and biotin-streptavidin complexes (e.g., bis-biotin-streptavidin complexes). Exemplary biotin moieties include monobiotin (free biotin) and bisbiotin. In some embodiments, the coupling moiety includes avidin proteins.

[0033] In some embodiments, the method further includes the step of coating the bottom surface of the sample well with a plurality of coating molecules, such as a first coating molecule and a second coating molecule. Examples of the second coating molecule include a biotin moiety, avidin protein, streptavidin protein, an azide moiety, an alkyne moiety, a ketone moiety, and a hydroxylamine moiety. In some embodiments, the second coating molecule is a biotin moiety or a streptavidin protein. In some embodiments, the first and second coating molecules are the same. In some embodiments, the first and second coating molecules are different. In some embodiments, both the first and second coating molecules are biotin. In some embodiments, both the first and second coating molecules are streptavidin. In some embodiments, the second coating molecule is an azide moiety. In some embodiments, the analyte is immobilized on a first coating agent containing a biotin moiety, while the second coupling moiety containing an azide moiety is not conjugated.

[0034] As used herein, in some embodiments, “surface” refers to the surface of a substrate or solid support comprising one or more sample wells. In some embodiments, a solid support refers to a layer, or other structure having a surface capable of supporting a laminated material, such as a functionalized peptide or DNA polymerase as described herein, such as a receptive surface. In some embodiments, the receptive surface of the substrate may optionally have one or more properties, including nanoscale or microscale recessed properties, such as an array of sample wells. In some embodiments, an array is a planar arrangement of elements, such as a sensor or sample wells. An array may be one-dimensional or two-dimensional. A one-dimensional array is an array having one column or one row of elements in a first dimension and multiple columns or rows in a second dimension. The number of columns or rows in the first and second dimensions may or may not be the same. In some embodiments, the array may be, for example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 It may contain individual sample wells.

[0035] II. Overview of Integrated Devices The techniques described herein may be used to regenerate the surface of a sensor chip in an integrated device. In one exemplary embodiment, the integrated device may enable the supply of excitation light from one or more excitation sources located within the apparatus to a reaction chamber containing a sample within the integrated device. The excitation light may be directed, at least partially, toward one or more pixels comprising the reaction chamber by an element, such as a waveguide in the integrated device, in order to illuminate an illumination area within the reaction chamber. The sample (e.g., a fluorescent label) placed in a reaction chamber or reaction component connected to the sample may emit synchrotron radiation in response to irradiation by the excitation light when located within the illumination area of ​​the reaction chamber. In some embodiments, one or more excitation sources are located within the apparatus, into which the integrated device is inserted and operates in cooperation, but all are part of the overall system.

[0036] The concept of reusing integrated devices is described in relation to exemplary system embodiments in which the device (in which the integrated devices are inserted and operate in cooperation) includes at least one laser light source, and the integrated devices include waveguides for directing light, as well as pixels having reaction chambers and detection regions, but the novelty is not limited in that way. Other device and integrated device designs are envisioned, including designs in which the light source is not located within the device, and / or designs in which additional optical components rather than integrated devices are located within the device (e.g., “off-chip”).

[0037] The emitted light from one or more reaction chambers (e.g., at least two reaction chambers in some embodiments) can be detected by one or more photodetectors in the pixels of the integrated device. As described herein, the integrated device may be configured to have a plurality of pixels (e.g., an array of pixels), and thus may have a plurality of reaction chambers and a plurality of corresponding photodetectors. The characteristics of the detected emitted light can provide indication for identifying a label associated with the emitted light. Such characteristics may include any preferred type of characteristics, including the arrival time of photons detected by the photodetector, the amount of photons accumulated by the photodetector over time, and / or the distribution of photons across two or more photodetectors. In some embodiments, the photodetector may have a structure that enables the detection of one or more characteristics associated with the emitted light, such as timing characteristics (e.g., emission lifetime, pulse duration, inter-pulse duration), wavelength, and / or intensity. In one example, one or more photodetectors can detect the distribution of photon arrival times after an excitation light pulse has propagated through an integrated device, and the distribution of arrival times can provide indications for timing characteristics of emitted light (e.g., proxies of emission lifetime, pulse duration, and / or inter-pulse duration).Such information can be found, for example, in U.S. Patent Application No. 16 / 686,028 titled "METHODS AND COMPOSITIONS FOR PROTEIN SEQUENCING" (filed November 15, 2019, agent reference number R0708.70042US02), PCT Application No. PCT / US19 / 61831 titled "METHODS AND COMPOSITIONS FOR PROTEIN SEQUENCING" (filed November 15, 2019, agent reference number R0708.70042WO00), U.S. Patent Application No. 17 / 190,331 titled "INTEGRATED SENSOR FOR MULTI-DIMENSIONAL SIGNAL ANALYSIS" (filed March 2, 2021, agent reference number R0708.70090US01), and "LABELED NUCLEOTIDE COMBINATIONS AND METHODS FOR NUCLEIC ACID The technology may be used in techniques for detecting and / or identifying molecules in a sample, including the techniques described in U.S. Patent Application No. 15 / 600,979 entitled “SEQUENCING” (filed May 22, 2017, attorney reference number R0708.70018US02) and U.S. Patent Application No. 15 / 161,125 entitled “METHODS FOR NUCLEIC ACID SEQUENCING” (filed May 20, 2016, attorney reference number R0708.70020US00), each of these applications being incorporated herein by reference in whole. In some embodiments, one or more photodetectors provide indication of the probability (e.g., emission intensity) of emitted light emitted by a fluorescent label. In some embodiments, one or more photodetectors may be sized and positioned to capture the spatial distribution (e.g., wavelength) of the emitted light. Output signals from one or more photodetectors can be used to distinguish a particular fluorescent label from among several labels, and in this case, the multiple labels can be used to identify a sample or its structure as described herein.

[0038] As used herein, “integrated device” is a device having the ability to interface with a base device. In some embodiments, the integrated device may include one or more sample wells and / or sensors. In some embodiments, the integrated device may have the ability to interface with a base device that emits or detects light. In such embodiments, the integrated device may include one or more sample wells (each comprising a waveguide). The “integrated device” may be referred to herein as a microchip (or “chip”), such as a CMOS chip. An exemplary integrated device is a CMOS chip comprising an array of pixels, where each pixel comprises a sample well and a photodetector.

[0039] In some embodiments, the disclosure provides an integrated device comprising a substrate including sample wells having a metal oxide surface and a silica surface. In some embodiments, the integrated device further comprises a coating layer on the metal oxide surface formed with an amphiphilic reagent comprising hydrophilic head groups and hydrophobic tail groups. In some embodiments, the integrated device further comprises a functionalizing agent bonded to the silica surface, the functionalizing agent comprising a coupling portion. In some embodiments, the substrate comprises an array of sample wells, each having a metal oxide surface and a silica surface. In some embodiments, the array is a microfabricated microarray. In some embodiments, the substrate comprises 128,000 sample wells. In some embodiments, the sample wells include an upper opening formed on the surface of the substrate and a bottom surface distal to the surface of the substrate. In some embodiments, the bottom surface is composed of a silica surface.

[0040] In some embodiments, the sample well includes an immobilization region, which may be an independent region such as the surface of the substrate (to which the analyte of interest is bound), or the bottom surface of the sample well having a polypeptide or nucleic acid coupled to such surface. In some embodiments, the sample well comprises a cavity or well having a defined shape and volume (fabricated within the substrate or device). The sample well may be fabricated using techniques described in the Art (for example, as disclosed in U.S. Patent Application No. 16 / 555,902, which is incorporated in its entirety in this specification).

[0041] In some embodiments, the sample well is formed by a bottom surface containing a non-metallic layer and a side wall surface containing a metallic layer. In some embodiments, the non-metallic layer includes a transparent layer (e.g., glass, silica). In some embodiments, the metallic layer includes a metal oxide surface (e.g., titanium dioxide). In some embodiments, the metallic layer includes a surface deactivation coating (e.g., a phosphorus-containing layer, e.g., an organic phosphonic acid layer). The bottom surface may include a non-metallic layer containing a functional portion. In some embodiments, the sample well comprises an upper surface containing an aluminum film, a titanium nitride (chinite) film, or both. The upper surface may further include a selective surface chemical coating, e.g., coatings known in the art. In some embodiments, the integrated device is configured to interface with a next-generation sequencing system, e.g., a benchtop next-generation sequencing (NGS) system.

[0042] The type of integration device described herein may comprise one or more sample wells configured to receive a molecule of interest therein. In some embodiments, the sample well receives the molecule of interest which may be placed on the surface of the sample well, for example, the bottom surface. In some embodiments, the sample well is formed within the integration device, and the bottom surface of the sample well is distal to the surface of the integration device (in which the sample well is formed). In some embodiments, the bottom surface in which the molecule of interest is placed may be at a distance from a waveguide configured to excite the molecule of interest with a desired level of excitation energy. In some embodiments, the sample well may be positioned with respect to the waveguide such that the evanescent field of an optical mode propagating along the waveguide overlaps with the molecule of interest.

[0043] For example, a schematic overview of an exemplary system 1-100 is shown in Figure 1A. The system comprises both an instrument 1-104 and an integrated device 1-102 that interfaces with it. In some embodiments, the instrument 1-104 may include one or more excitation sources 1-106 integrated as part of the instrument 1-104. In some embodiments, the excitation sources may be external to both the instrument 1-104 and the integrated device 1-102, and the instrument 1-104 may be configured to receive excitation light from the excitation sources and to direct the excitation light to the integrated device. The integrated device may interface with the instrument using any suitable socket to receive the integrated device and to hold the integrated device in an optically precise alignment with the excitation source. The excitation source 1-106 may be configured to provide excitation light to the integrated device 1-102. As schematically shown in Figure 1A, the integrated device 1-102 has a plurality of pixels 1-112, in which case at least a portion of the pixels can perform independent analysis of the sample of interest. Since a pixel receives excitation light from a light source 1-106 separate from that pixel, such a pixel 1-112 can be referred to as a “passive source pixel.” In this case, the excitation light from this source excites some or all of the pixels 1-112. The excitation source 1-106 can be any suitable light source. An example of a suitable excitation source is described in U.S. Patent Application No. 14 / 821,688, filed on 7 August 2015, entitled “INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES” (Agent Reference Number R0708.70004US02), which is incorporated herein by reference in its entirety. In some embodiments, the excitation source 1-106 includes multiple excitation sources that are combined to irradiate the integrated device 1-102 with excitation light. The multiple excitation sources can be configured to produce multiple excitation energies or wavelengths.

[0044] Referring to Figure 1B, pixel 1-112 has a reaction chamber 1-108 configured to receive at least one sample of interest, and a photodetector 1-110 that detects emitted light emitted from the reaction chamber in response to illumination of the sample and at least a portion of the reaction chamber 1-108 by excitation light provided by an excitation source 1-106. In some embodiments, the reaction chamber 1-108 may hold the sample in close proximity to the surface of the integrated device 1-102, which may facilitate the irradiation of the sample with excitation light and the detection of emitted light from the sample or reaction component (e.g., an emission label). As shown in the illustrated embodiment of Figure 1B, the reaction chamber 1-108 and the photodetector 1-110 have a one-to-one correspondence. In some embodiments, as described herein, each pixel may comprise multiple reaction chambers per photodetector.

[0045] Optical elements that couple excitation light from the excitation light source 1-106 to the integrated device 1-102 and guide the excitation light to the reaction chamber 1-108 may be located in either the integrated device 1-102 or the instrument 1-104, or both. The optical elements from the source to the chamber may include one or more lattice couplers located in the integrated device 1-102, which couple the excitation light to the integrated device and waveguides, thereby irradiating the excitation light from instrument 1-104 to the reaction chamber in pixel 1-112. One or more optical splitter elements may be located between the lattice couplers and the waveguides. The optical splitters couple the excitation light from the lattice couplers and can deliver the excitation light to at least one of the waveguides. In some embodiments, the optical splitters may have a structure that allows the excitation light to be delivered substantially uniformly across all waveguides, so that each waveguide receives substantially similar amounts of excitation light. Such embodiments can improve the performance of the integrated device by improving the uniformity of the excitation light received by the reaction chamber of the integrated device.

[0046] The reaction chamber 1-108, a portion of the optical system from the excitation source to the chamber, and the optical system from the reaction chamber to the photodetector are located in integrated device 1-102. The excitation source 1-106 and a portion of the source-to-chamber component are located in instrument 1-104. In some embodiments, a single component may play a role in both coupling the excitation light to the reaction chamber 1-108 and delivering the emitted light from the reaction chamber 1-108 to the photodetector 1-110. Examples of suitable components included in the integrated device for coupling the excitation light to the reaction chamber and / or directing the emitted light to the photodetector are described in "INTEGRATED" filed on August 7, 2015. This is described in U.S. Patent Application No. 14 / 821,688, entitled “DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES” (Agent Reference Number R0708.70004US02), and in U.S. Patent Application No. 14 / 543,865, entitled “INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALZING MOLECULES” (Agent Reference Number R0708.70005US00), filed on 17 November 2014, both of which are incorporated by reference in their entirety.

[0047] Each pixel 1-112 is associated with its own individual reaction chamber 1-108 and at least one photodetector 1-110. Multiple pixels of the integrated device 1-102 can be configured to have any preferred shape, size, and / or dimensions. The integrated device 1-102 can have any preferred number of pixels. The number of pixels in the integrated device 1-102 can be in the range of approximately 100,000 pixels to 64,000,000 pixels, or any value within that range. In some embodiments, the pixels can be arranged in an array of 1024 pixels × 2048 pixels. The integrated device 1-102 can interface with the instrument 1-104 in any preferred manner. In some embodiments, instrument 1-104 may have an interface that detachably couples to integrated device 1-102, thereby allowing a user to attach integrated device 1-102 to instrument 1-104 for use with integrated device 1-102 to analyze at least one sample of interest in a suspension, and to detach integrated device 1-102 from instrument 1-104 to allow attachment of another integrated device. The interface of instrument 1-104 may position integrated device 1-102 to couple with the circuitry of instrument 1-104 and allow it to transmit readout signals from one or more photodetectors to instrument 1-104. Integrated device 1-102 and instrument 1-104 may include a multi-channel high-speed communication link to handle data associated with large pixel arrays (e.g., more than 10,000 pixels).

[0048] Device 1-104 may include a user interface to control the operation of at least one of Device 1-104 and / or Integrated Device 1-102. The user interface may be configured to allow the user to input information into the device, such as commands and / or settings used to control the functions of the device. In some embodiments, the user interface may include buttons, switches, dials, and a microphone for voice commands. The user interface may allow the user to receive feedback on the performance of the device and / or integrated device, such as information obtained by proper alignment and / or readout signals from a photodetector on the integrated device. In some embodiments, the user interface may provide feedback using a speaker to provide audible feedback. In some embodiments, the user interface may include indicator lights and / or a display screen to provide the user with visual feedback.

[0049] In some embodiments, device 1-104 may include a computer interface configured to connect to a computing device. The computer interface may be a USB interface, a FireWire interface, or any other suitable computer interface. The computing device may be any general-purpose computer, such as a laptop or desktop computer. In some embodiments, the computing device may be a server accessible over a wireless network via a suitable computer interface (e.g., a cloud-based server). The computer interface can facilitate the communication of information between device 1-104 and the computing device. Input information for controlling and / or configuring device 1-104 can be provided to and transmitted to the computing device via the computer interface. Output information generated by device 1-104 can be received by the computing device via the computer interface. The output information may include feedback on the performance of device 1-104, the performance of integrated device 1-102, and / or data generated from the readout signals of photodetector 1-110.

[0050] In some embodiments, device 1-104 may include a processing device configured to analyze data received from one or more photodetectors of integrated device 1-102 and transmit control signals to excitation source 1-106, or to analyze data received from one or more photodetectors of integrated device 1-102 or to transmit control signals to excitation source 1-106. In some embodiments, the processing device may include a general-purpose processor, a specially adapted processor (e.g., a central processing unit (CPU) such as one or more microprocessors or microcontroller cores, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a custom integrated circuit, a digital signal processor (DSP), or a combination thereof). In some embodiments, processing of data from one or more photodetectors may be performed by both the processing device of device 1-104 and an external computing device. In other embodiments, the external computing device may be omitted, and processing of data from one or more photodetectors may be performed solely by the processing device of integrated device 1-102.

[0051] Figure 1B shows a schematic cross-sectional view of an integrated device 1-102 showing a row of pixels 1-112. The integrated device 1-102 may include a coupling region 1-201, a routing region 1-202, and a pixel region 1-203. The pixel region 1-203 may include a plurality of pixels 1-112 having a plurality of reaction chambers 1-108 located on a surface away from the coupling region 1-201 where excitation light (indicated as dashed arrows) couples to the integrated device 1-102. The plurality of reaction chambers 1-108 may be formed by penetrating one or more metal layers 1-106. A single pixel 1-112, indicated by a dotted rectangle, is a region of the integrated device 1-102 including a reaction chamber 1-108 and a photodetector region having one or more photodetectors 1-110. In the illustrated embodiment, a pixel includes a single reaction chamber 1-108. In some embodiments, each pixel may have two or more reaction chambers.

[0052] Figure 1B shows the excitation path (shown as a dashed line) by coupling the excitation light beam to coupling region 1-201 and reaction chamber 1-108. The row of reaction chamber 1-108 shown in Figure 1B can be positioned to optically couple with waveguide 1-220. The excitation light can irradiate a sample positioned within the reaction chamber. The sample or reaction component (e.g., a fluorescent label) can reach an excited state in response to being irradiated with the excitation light. When the sample or reaction component is in an excited state, it can emit emitted light that can be detected by one or more photodetectors associated with the reaction chamber. Figure 1B schematically shows the optical axis of the emitted light (shown as a solid line) from reaction chamber 1-108 to one or more photodetectors 1-110 of pixel 1-112. One or more photodetectors 1-110 of pixel 1-112 can be configured and positioned to detect emitted light from reaction chamber 1-108. A suitable example of a photodetector is described in U.S. Patent Application No. 14 / 821,656, “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” filed on 7 August 2015 (Agent Reference Number R0708.70002US02), which is incorporated herein by reference in its entirety. With respect to individual pixels 1-112, the reaction chambers 1-108 and individual photodetectors 1-110 may be aligned along a common axis (along the y-direction shown in Figure 1A). In this way, the photodetector(s) may overlap with the reaction chamber within pixel 1-112.

[0053] The directionality of the light emitted from reaction chamber 1-108 may vary depending on the position of the sample within reaction chamber 1-108 relative to one or more metal layers 1-106, since one or more metal layers 1-106 may act to reflect the emitted light. Thus, the distance between one or more metal layers 1-106 and a fluorescent marker located within reaction chamber 1-108 may affect the efficiency of one or more photodetectors 1-110, which are located in the same pixel as the reaction chamber, for detecting the light emitted by the fluorescent marker. The distance between one or more metal layers 1-106 and the bottom surface of reaction chamber 1-108, which is close to the location where the sample can be positioned during operation, may be in the range of 100 nm to 500 nm, or any value within that range. In some embodiments, the distance between the metal layers 1-106 and the bottom surface of reaction chamber 1-108 is about 300 nm.

[0054] The distance between the sample and one or more photodetectors can also affect the efficiency of detecting emitted light. Reducing the distance the light must travel between the sample and the photodetectors can improve the detection efficiency of emitted light. In addition, reducing the distance between the sample and the photodetectors can reduce the footprint of the pixels on the integrated device, thereby allowing a greater number of pixels to be included in the integrated device. The distance between the bottom of the reaction chambers 1-108 and one or more photodetectors can be in the range of 1 micrometer to 15 micrometers, or any value within that range. It should be understood that in some embodiments, the emitted light may be provided through means other than the excitation light source and reaction chamber. Therefore, some embodiments may not include the reaction chambers 1-108.

[0055] One or more photonic structures 1-230 may be positioned between the reaction chamber 1-108 and the photodetector 1-110 and may be configured to reduce or prevent excitation light from reaching the photodetector 1-110, otherwise the excitation light may contribute to signal noise when detecting emitted light. As shown in Figure 1B, one or more photonic structures 1-230 may be positioned between the waveguide 1-220 and the photodetector 1-110. One or more photonic structures 1-230 may include one or more photoremoval photonic structures, including spectral filters, polarizing filters, and spatial filters. One or more photonic structures 1-230 may be positioned to align with the individual reaction chambers 1-108 and their individual one or more photodetectors 1-110 along a common axis. A metal layer 1-240, which may function as a circuit of the integrated device 1-102, may also function as a spatial filter or a polarizing filter according to some embodiments. In such embodiments, one or more metal layers 1-240 may be arranged to prevent some or all of the excitation light from reaching the photodetector 1-110.

[0056] The coupling region 1-201 may include one or more optical components configured to couple excitation light from an external excitation source, for example, one or more excitation sources 1-116 shown in Figure 1A. The coupling region 1-201 may include a grating coupler 1-216 positioned to receive part or all of the excitation light beam. Examples of suitable grazing couplers are described in U.S. Patent Application No. 15 / 844,403, “OPTICAL COUPLER AND WAVEGUIDE SYSTEM,” filed December 15, 2017 (Agent No. R0708.70021US01), and U.S. Patent Application No. 16 / 861,399, “SLICED GRATING COUPLER WITH INCREASED BEAM ALIGNMENT SENSITIVITY,” filed April 29, 2020 (Agent No. R0708.70071US01), each of which is incorporated herein by reference in its entirety. The grazing coupler 1-216 may couple excitation light to waveguide 1-220, which can be configured to propagate excitation light to the vicinity of one or more reaction chambers 1-108. Alternatively, the coupling region 1-201 may comprise other known structures for coupling light to the waveguide.

[0057] The excitation sources 1-116 can be positioned and aligned with respect to the integrated device using components located away from the integrated device. Such components may include optical components, including lenses, mirrors, prisms, windows, apertures, attenuators, and / or optical fibers. Additional mechanical components may be included in the apparatus to enable control of one or more alignment components. Such mechanical components may include actuators, stepper motors, and / or knobs. An example of a suitable excitation source and alignment mechanism is described in U.S. Patent Application No. 15 / 161,088, “PULSED LASER AND SYSTEM,” filed May 20, 2016 (Agent Reference Number R0708.70010US02), which is incorporated herein by reference in its entirety. Another example of a beam steering module is described in U.S. Patent Application No. 15 / 842,720, “Compact Beam Shaping and Steering Assembly,” filed on 14 December 2017 (Agent Reference Number R0708.70024US01), which is incorporated herein by reference.

[0058] The sample to be analyzed may be introduced into reaction chambers 1-108 of pixels 1-112. The sample may be any other suitable sample, such as a biological sample or a chemical sample. The sample may contain multiple molecules, and the reaction chamber may be configured to isolate a single molecule. In some examples, the dimensions of the reaction chamber may act to confine a single molecule within the reaction chamber, allowing the measurement to be performed on a single molecule. Excitation light may be irradiated into reaction chambers 1-108 to excite the sample, or at least one fluorescent marker attached to or otherwise associated with the sample, while within the irradiation area of ​​reaction chambers 1-108.

[0059] During operation, parallel analysis of samples in the reaction chamber is performed by exciting some or all of the samples in the wells using excitation light and detecting the signal from the sample emission with a photodetector. The light emitted from the sample is detected by the corresponding photodetector and can be converted into at least one electrical signal. Information on various properties of the emitted light (e.g., wavelength, emission lifetime, intensity, pulse duration, and / or any other suitable properties) can be collected and used for subsequent analysis as described herein. The electrical signal can be transmitted along conductive wires (e.g., metal layers 1-240) in the circuitry of the integrated device, and the conductive wires can be connected to an instrument that interfaces with the integrated device. The electrical signal can then be processed and / or analyzed. Processing or analysis of the electrical signal can be performed on the instrument or on a suitable computing device located away from the instrument.

[0060] Figure 1C shows a cross-sectional view of pixels 1-112 of an integrated device 1-102 according to some embodiments. Figure 1D shows a schematic of pixels 1-112. Figure 1E shows an exemplary array of pixels 1-112 and processing circuit 1-114 that may be included in an integrated device 1-102 according to some embodiments.

[0061] In Figures 1C and 1D, pixels 1-112 include a photodetection region which may be a pinned photodiode (PPD), two charge storage regions which may be storage diodes (SD0 and SD1), and a readout region which may be a floating diffusion (FD) region. Also, as shown in the figure, pixels 1-112 include a drain region D and transfer gates ST0, TX0, TX1, and REJ.

[0062] In some embodiments, the photodetector region PPD, charge storage regions SD0, SD1, and readout region FD are formed on the integrated circuit substrate by doping a portion of the substrate. For example, the substrate may be doped to a low concentration, while the photodetector region PPD, charge storage regions SD0, SD1, and readout region FD may be doped to a higher concentration. In this example, the substrate may be p-type doped to a low concentration, while the photodetector region PPD, charge storage regions SD0, SD1, and readout region FD may be n-type doped. Alternatively, since the embodiments described herein are not so limited, the substrate may be n-type doped to a low concentration, while the photodetector region PPD, charge storage regions SD0, SD1, and readout region FD may be p-type doped.

[0063] In some embodiments, the photodetector region PPD may be configured to generate charge carriers (e.g., photoelectrons) when incident photons are received within the photodetector region PPD. In some embodiments, charge storage regions SD0 and SD1 may be electrically coupled to and / or to the photodetector region PPD. For example, pixels 1-112 may include one or more transfer channels that electrically couple the charge storage regions SD0 and SD1 to and / or to the photodetector region PPD. In some embodiments, the transfer channels may be formed by doping portions of the integrated circuit substrate located between the regions. For example, these portions may be doped with the same conductivity type as the regions (e.g., the n-type doped PPD and the n-type doped channel located between SD0). Referring to Figure 1D, for example, the channel of the transistor coupled between the photodetector region PPD and the charge storage region SD0 is a transfer channel that electrically couples the photodetector region PPD to the charge storage region SD0. Similarly, the channel of the transistor coupled between charge storage regions SD0 and SD1 is a transfer channel that electrically couples charge storage regions SD0 and SD1, and the channel of the transistor coupled between charge storage region SD1 and readout region FD is a transfer channel that electrically couples charge storage region SD1 and readout region FD. The channel of the transistor coupled between photodetector region PPD and drain region D is a transfer channel that couples photodetector region PPD and drain region D.

[0064] In some embodiments, transfer gates ST0, TX0, TX1, and REJ may be configured to control the transfer of charge carriers from the photodetector region PPD to the storage regions SD0, SD1, between the charge storage regions SD0, SD1, and / or between the charge storage regions SD0, SD1 and the readout region FD. For example, transfer gates ST0, TX0, TX1, and REJ may be electrically coupled to a transfer channel to electrically couple the regions of pixels 1-112 and bias the transfer channel in order to transfer charge carriers between regions when an appropriate control signal is applied to the transfer gate. According to various embodiments, the transfer gates may be electrically coupled (e.g., physically) to the transfer channel and / or positioned sufficiently close to the transfer channel and / or isolated by a sufficiently thin insulator to capacitively couple to the transfer channel. In some embodiments, the transfer gates described herein may be formed using a conductive material such as a metal. Alternatively or additionally, in some embodiments, the transfer gates described herein may be formed using a semiconductor material such as polysilicon. In some embodiments, the material used to form the transfer gate described herein may be at least partially opaque.

[0065] In some embodiments, when a control signal is received by the transfer gate, the transfer gate may electrically couple the control signal to the transfer channel, biasing the transfer channel and thereby increasing its conductivity. In some embodiments, the transfer channel may be doped with a dopant concentration of the same conductivity type but lower than that of the region of pixels 1-112 electrically coupled by the transfer channel, thereby creating an intrinsic electric potential barrier between the regions. The intrinsic electric potential barrier may exist between the regions even when no external electric field is applied to the transfer gate or transfer channel. For example, the dopant concentration of the transfer channel between the photodetector region PPD and the charge storage region SD0 may create an intrinsic electric potential barrier between the photodetector region PPD and the charge storage region SD0. In some embodiments, a control signal may be applied to the transfer gate configured to reduce the intrinsic electric potential barrier between the regions electrically coupled by the transfer channel, thereby increasing the conductivity of the transfer channel and causing the transfer of charge carriers between the regions. For example, in the case of an n-type doped transfer channel, the control signal may have a voltage that is at least the threshold voltage of the transfer channel greater than the voltage in one of the regions (e.g., the source terminal of the transfer channel), the threshold voltage depending on the size of the transfer channel, the substrate voltage of the integrated device 1-102 adjacent to the transfer channel, and other such parameters. Similarly, in the case of a p-type doped transfer channel, the control signal may have a voltage that is at least the threshold voltage lower than the voltage in one of the regions. In some embodiments, the control circuit of the integrated device 1-102 may be configured to generate and provide such a control signal to the transfer gate, as further described herein.

[0066] In Figure 1C, pixels 1-112 are shown in a configuration in which the photodetector region PPD, charge storage regions SD0, SD1, and readout region FD are configured to receive incident photons in a direction that is spaced apart from the transfer gates REJ, ST0, TX0, and TX1 (e.g., front illumination). However, it should be understood that in some embodiments, the photodetector region PPD may be configured to receive incident photons in a direction that is spaced apart from the transfer gates REJ, ST0, TX0, and TX1 (e.g., back illumination). In some embodiments, such a configuration can improve the electrical properties of the transfer gates because the influence of the optical properties of the transfer gates on the incident photons is reduced.

[0067] In Figure 1D, pixels 1-112 further include a reset (RST) transfer gate configured to be coupled to a read region FD and to a high voltage VDDP, and a row selection (RS) transfer gate coupled between the read region FD and the bit line. When the integrated device 1-102 is coupled to a power supply (e.g., at least a DC power supply), the transfer gate RST may be coupled to the high voltage VDDP, which is supplied by the power supply and / or regulated by the voltage regulator of the integrated device 1-102.

[0068] In some embodiments, the transfer gate RST may be configured to reset the voltage of the read region FD. For example, when a reset signal is applied to the transfer gate RST, the transfer gate RST may bias the transfer channel to electrically couple the read region FD to a high voltage VDDP, thereby increasing the conductivity of the transfer channel and transferring charge carriers from the read region FD to the high voltage VDDP. In some embodiments, the reset transfer gate RST may be further configured to reset the voltages of the charge storage regions SD0 and / or SD1. For example, when a reset signal is applied to the reset transfer gate RST and a control signal is applied to the transfer gate TX1, the transfer gate TX1 may transfer charge carriers in the charge storage region SD1 to the read region FD, and the transfer gate RST may transfer the charge carriers to the high voltage VDDP. Similarly, when a reset signal is applied to the reset transfer gate RST and control signals are applied to the transfer gates TX1 and TX0, transfer gate TX0 may transfer charge carriers in charge storage area SD0 to SD1, transfer gate TX1 may transfer charge carriers in charge storage area SD1 to read area FD, and transfer gate RST may transfer charge carriers to high voltage VDDP. In some embodiments, the integrated device 1-102 may be configured to reset the read area FD and charge storage areas SD0 and SD1 before collecting and reading charge carriers. For example, the integrated device 1-102 may be configured to reset the read area FD, then the charge storage area SD1, and then the charge storage area SD0 before collecting and reading charge carriers.

[0069] In some embodiments, bit lines may be coupled to processing circuits on and / or external circuits on the integrated device 1-102 configured to receive voltage levels indicating charge carriers read into the read area FD. In some embodiments, the processing circuit 1-114 may include an analog-to-digital converter (ADC). In some embodiments, the integrated device 1-102 may be configured to reset the voltage of the read area FD for each pixel before reading out charge carriers. For example, the integrated device 1-102 may be configured to reset the voltage of the read area FD, sample the voltage, transfer charge carriers to the read area FD, and sample the voltage again. In this example, the second sampled voltage may indicate the number of charge carriers transferred to the read area FD when compared to the first sampled voltage. In some embodiments, the integrated device 1-102 may be configured to sequentially read charge carriers from each pixel 1-112 to bit lines, such as row by row and / or column by column. It should be understood that several arrays of pixels 1-112 may have multiple bit lines electrically coupled to different pixels and / or groups of pixels 1-112. In some embodiments, pixels from multiple columns may be read simultaneously to individual processing circuits. For example, the first pixels of each column (e.g., pixels (1,1) and (1,2)) may be read simultaneously to individual processing circuits, and then the second pixels of each column (e.g., pixels (2,1) and (2,2)) may be read simultaneously to individual processing circuits. It should be understood that in some embodiments, processing circuits may be provided for each row of the array, either as a substitute for each column or in addition to each column. In some embodiments, the integrated device 1-102 may include multiple units of processing circuits (each electrically coupled to a bit line, for example).

[0070] It should be understood that, depending on the various embodiments, the transfer gates described herein may include one or more semiconductor materials and / or metals, and may include the gates of field-effect transistors (FETs), the bases of bipolar junction transistors (BJTs), and so on. It should also be understood that the control signals described herein, applied to various transfer gates, may change shape and / or voltage depending on the potential of the semiconductor region and regions electrically coupled to the semiconductor region (e.g., adjacent regions).

[0071] In some embodiments, the pixels described herein may include more than two charge storage regions. For example, pixels 2-112 described herein in relation to Figures 1G-1I include three charge storage regions.

[0072] Figure 1E is a plan view of alternative pixels 1-112' according to some embodiments. In some embodiments, pixels 1-112' may be configured in the manner described for pixels 1-112. In Figure 1E, the drain region D of pixel 1-112' is located on the same side as the charge storage regions SD0, SD1 and the readout region FD of the photodetector region PPD. Also, as shown in Figure 1E, the photodetector region PPD may include a mask having a triangular opening, the triangular opening having a base on the side of the photodetector region adjacent to the charge storage regions SD0, SD1 and the drain region D, and a corresponding apex on the side of the photodetector region PPD opposite to the drain region D and the charge storage regions SD0, SD1.

[0073] In some embodiments, the photodetector region PPD may be configured to induce an intrinsic electric field in the direction from the photodetector region PPD toward the charge storage regions SD0, SD1, and drain region D. For example, the photodetector region PPD may be formed by doping the substrate of integrated device 1-102 through an aperture, resulting in a higher dopant concentration in the region of the substrate exposed through the aperture than in the region covered by the mask during doping. In this example, the greater the amount of dopant (e.g., n-type dopant) at the base of the triangular aperture, the lower the potential at the base of the photodetector region PPD adjacent to the drain region D and charge storage region SD0 may be compared to the potential at the apex of the photodetector region PPD on the opposite side. The intrinsic electric field in the photodetector region PPD may exist even when there is no external electric field applied to pixels 1-112. The inventors have recognized that the intrinsic electric field of the photodetector region PPD increases the rate of charge transfer from the photodetector region PPD to the drain region D and / or storage regions SD0, SD1, thereby increasing the efficiency of charge carrier discharge and / or collection during the operation of pixels 1-112. In the example in Figure 1E, the intrinsic electric field can be directed along the dotted arrow between the drain region and the charge storage region SD0. For example, the intrinsic electric field can cause charge carriers to flow along the dotted arrow, and an external electric field induced by a control signal applied to the transfer gate REJ or ST0 can cause charge carriers to flow into the drain region D or the charge storage region SD0, respectively.

[0074] Figure 1F is a schematic top view of pixels 1-112' according to some embodiments. As shown in Figure 1F, the contact may be positioned above the portion of pixels 1-112'. In some embodiments, the contact may be configured to prevent incident photons from reaching portions of pixels 1-112' other than the photodetector region PPD, and / or from reaching the photodetector region of adjacent pixels at oblique angles of incidence. For example, the contact may be elongated in a direction parallel to the optical axis in which the photodetector PPD is configured to receive incident photons. In some embodiments, the contact may be formed using an opaque material such as tungsten. The inventors have recognized that the contacts described herein prevent many or all incident photons from reaching the charge storage regions SD0, SD1 along optical paths other than the optical axis, thereby preventing incident photons from generating noise charge carriers in the charge storage regions SD0, SD1.

[0075] In Figure 1F, a pair of contacts are positioned on either side of the photodetector region PPD, with the first contact of the pair positioned closer to the apex of the triangular opening of the mask, and the second contact of the pair positioned closer to the base of the triangular opening of the mask. The second contact may be configured to prevent incident photons from reaching the charge storage regions SD0, SD1. A third contact is positioned at the end of pixels 1-112 opposite to the end where the photodetector region PPD is positioned. The first and third contacts are positioned between pixels 1-112 and individual adjacent pixels, and the second contact is positioned between the photodetector region PPD and the transfer gates ST0, REJ. It should be understood that in some embodiments, the pair of contacts on either side of the photodetector region PPD may be replaced by at least one contact wall that at least partially surrounds the photodetector region PPD, such as a single cylindrical contact wall.

[0076] Figure 1G is a cross-sectional view of an alternative exemplary pixel 2-112 that may be included in an integrated device 1-102 according to some embodiments. In some embodiments, pixel 2-112 may be configured in the manner described with respect to pixel 1-112 in relation to Figures 1A to 1F. For example, as shown in Figure 1G, region FD, drain region D, and transfer gates may each be configured in the manner described with respect to pixel 1-112. Pixel 2-112 further includes a charge storage region SD2 electrically coupled between a charge storage region SD1 and a read region FD. For example, multiple transfer channels may electrically couple charge storage region SD1 to charge storage region SD2 and charge storage region SD2 to read region FD. In Figure 1G, transfer gate TX0 is configured to control the transfer of charge carriers from charge storage region SD1 to charge storage region SD2, and transfer gate TX1 is configured to control the transfer of charge carriers from charge storage region SD2 to read region FD.

[0077] Figure 1H is a schematic diagram of pixel 2-112 according to one embodiment. As shown in Figure 1H, the transfer channel electrically coupling charge storage region SD1 to charge storage region SD2 is a transistor channel having a transfer gate TX0, and the transfer channel electrically coupling charge storage region SD2 to readout region FD is a transistor channel having a transfer gate TX1. Other transistors in pixel 2-112 shown in Figure 1H, such as a transistor having a reset gate RST and a transistor having a row selection transfer gate RS, may be configured in the manner described with respect to pixel 1-112. For example, an array of pixel 2-112 may be arranged in a configuration that includes processing circuits as described herein with respect to pixel 1-112.

[0078] Figure 1I is a top view of a pixel 2-112' that may be included in an integrated device 1-102 according to some embodiments. In some embodiments, the pixel 2-112' may be configured in the manner described herein with respect to the pixel 1-112'. For example, the photodetector region PPD of the pixel 2-112' may be configured to induce an intrinsic electric field in the direction from the photodetector region PPD toward the charge storage region SD0 and the drain region D.

[0079] In some embodiments, the integration device includes a sample preparation module capable of performing methods for preparing biological samples for, for example, detection and / or analysis. In some embodiments, the methods for preparing biological samples described herein may be used to identify the characteristics or features of a sample, including the identity or sequence (e.g., nucleotide sequence or amino acid sequence) of one or more target molecules within the biological sample. In some embodiments, the biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, or plant sample. In some embodiments, the biological sample is a blood sample, saliva sample, sputum sample, fecal sample, urine sample, buccal swab sample, amniotic fluid sample, semen sample, synovial membrane sample, spinal cord sample, or pleural fluid sample. In some embodiments, the biological sample is derived from a human, non-human primate, rodent, dog, cat, horse, or any other mammal. In some embodiments, the biological sample is derived from a bacterial cell culture (e.g., a pathogenic Escherichia coli (E. coli) cell culture). In some embodiments, one or more target molecules are nucleic acids. In some embodiments, one or more target molecules are proteins.

[0080] In some embodiments, a method for preparing a biological sample may include one or more sample transformation steps, such as dissolution of the sample, purification of the sample, fragmentation of the sample, purification of the fragmented sample, library preparation (e.g., nucleic acid library preparation), purification of the library preparation, enrichment of the sample (e.g., using affinity SCODA), and / or detection / analysis of a target molecule.

[0081] In some embodiments, a sample (e.g., a sample containing cells or tissue) may be prepared, e.g., lysed (e.g., destroyed, decomposed, and / or otherwise digested). In some embodiments, a sample containing cells or tissue is lysed using any known physical or chemical methodology to release a target molecule (e.g., a target nucleic acid or target protein) from the cells or tissue. In some embodiments, a sample may be lysed using any method known to those skilled in the art. For example, a sample may be lysed using electrolytic methods, enzymatic methods, surfactant-based methods, and / or mechanical homogenization. In some embodiments, a sample (e.g., complex tissue, Gram-positive bacteria, or Gram-negative bacteria) may require multiple lysis methods to be carried out in sequence. In some embodiments, a sample may be prepared, e.g., lysed in the presence of a buffer system. This buffer system may be used to create a slurry-like sample, suspend the sample, and / or stabilize the sample during the execution of any known lysis methodology, including the methods described herein. In some embodiments, methods for inducing lysis by mechanical homogenization include, but are not limited to, bead beating, heating (e.g., above 50°C, 60°C, 70°C, 80°C, 90°C, or 95°C, until sufficiently hot to break down the cell walls), syringe / needle / microchannel passage (to induce shearing), sonication, or maceration with a grinder. In some embodiments, sample preparation involves cell disruption (i.e., removal of undesirable cell and tissue elements after lysis). In some embodiments, cell disruption is accompanied by protein and / or nucleic acid precipitation. In some embodiments, after precipitation, the lysis and disrupted sample is centrifuged. In some embodiments, the supernatant is discarded after centrifugation. In some embodiments, proteins or peptides are subjected to immunoprecipitation.

[0082] In some embodiments, a sample (e.g., a sample containing a target nucleic acid or target protein) may be purified in a method according to the disclosure, for example, after dissolution. In some embodiments, the sample may be purified using chromatography (e.g., affinity chromatography that selectively binds to the sample) or electrophoresis. In some embodiments, the sample may be purified in the presence of a precipitating agent. In some embodiments, after the purification step or method, the sample may be washed and / or released from the purification matrix (e.g., an affinity chromatography matrix) using an elution buffer. In some embodiments, the purification step or method may include the use of a reversibly switchable polymer, such as an electroactive polymer. In some embodiments, the sample may be purified by electrophoretically passing the sample through a porous matrix (e.g., cellulose acetate, agarose, acrylamide).

[0083] In some embodiments, a sample (e.g., a sample containing a target nucleic acid or target protein) may be fragmented (i.e., digested) in a method according to the present disclosure. In some embodiments, a nucleic acid sample may be fragmented to produce fragments ranging from small (<1 kilobase) fragments for sequence-specific identification to large (up to 10+ kilobase) fragments for long-read sequencing applications. Fragmentation of nucleic acids or proteins may be achieved in some embodiments using mechanical (e.g., fluid shearing), chemical (e.g., iron (Fe+) cleavage), and / or enzymatic (e.g., tagmentation using restriction enzymes, transposases) methods. In some embodiments, a protein sample may be fragmented to produce peptide fragments of arbitrary length. Protein fragmentation may be achieved in some embodiments using chemical and / or enzymatic (e.g., proteolytic enzymes, e.g., trypsin) methods. In some embodiments, the average fragment length may be controlled by reaction time, temperature, and the concentration of the sample and / or enzyme (e.g., restriction enzymes, transposases). In some embodiments, nucleic acids may be fragmented by tagmentation so that nucleic acid fragmentation and labeling with fluorescent molecules (e.g., fluorophores) occur simultaneously. In some embodiments, the fragmented sample undergoes a purification round (e.g., chromatography or electrophoresis) to remove small and / or undesirable fragments, as well as residual payload, chemicals and / or enzymes (e.g., transposases) used during the fragmentation process.

[0084] In some embodiments, a sample (e.g., a sample containing a target nucleic acid or target protein) may be enriched with respect to the target molecule in a method according to this disclosure. Enrichment is commonly used when the complexity of an unenriched sample exceeds the capabilities of the sequencing platform, or when the abundance of the target molecule present in the sample is low (e.g., it cannot be readily detected by the sequencing platform). Enrichment involves the use of a mechanism for selectively amplifying the target molecule. This enrichment may involve the use of antibodies, aptamers, size-based selection, or electrostatic charge-based selection for selectively amplifying the target molecule(s) (e.g., target protein(s) or target nucleic acid(s)). Enrichment may be commonly used when the intention of sample preparation is to sequence a specific target molecule. When the target molecules are related or homologous, enrichment may be used to perform or carry out proteomic, genomic, or metagenomic analysis or investigation.

[0085] In some embodiments, the sample is enriched for the target molecule using electrophoresis. In some embodiments, the sample is enriched for the target molecule using affinity SCODA. In some embodiments, the sample is enriched for the target molecule using electric field inversion gel electrophoresis (FIGE). In some embodiments, the sample is enriched for the target molecule using pulsed-field gel electrophoresis (PFGE). In some embodiments, the matrix used during the enrichment period (e.g., a porous medium, an electrophoretic polymer gel) contains immobilized affinity agents (also known as "immobilized capture probes") that bind to the target molecule present in the sample. In some embodiments, the matrix used during the enrichment period contains one, two, three, four, five, or more unique immobilized capture probes, each binding to a unique target molecule and / or to the same target molecule with different binding affinities.

[0086] In addition to, or as an alternative to, the amplification of the target molecule, the sample may be enriched by depleting undesirable non-target molecules (e.g., highly abundant proteins (e.g., albumin)) (e.g., for low-abundance target molecules). Depletion of undesirable non-target molecules can be carried out using capture strategies similar to those discussed above. When using a depletion strategy, a capture probe binds to the undesirable non-target molecule, allowing the target molecule to remain in the solution. This strategy also allows for enrichment of the target molecule (i.e., an increase in the relative concentration of the target molecule(s)).

[0087] In some embodiments, the target nucleic acid is enriched before sequencing (e.g., enriched using electrophoresis, e.g., affinity SCODA). In some embodiments, a method for sequencing multiple target nucleic acids (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 50, or more) present in a sample (e.g., a purified sample, cell lysate, a single cell, a population of cells, or a tissue) is presented herein. In some embodiments, the sample is prepared as described herein before sequencing the target nucleic acid or multiple target nucleic acids present in the sample (e.g., lysed, purified, fragmented, and / or enriched with respect to the target nucleic acid). In some embodiments, the target nucleic acid is an enriched target nucleic acid (e.g., enriched using electrophoresis, e.g., affinity SCODA).

[0088] In some embodiments, in methods based on the present disclosure, a target molecule or target molecule(s) may be detected after enrichment and subsequent release to enable analysis of the target molecule(s) and its upstream sample. In some embodiments, the target nucleic acid may be detected using gene sequencing, absorption, fluorescence, electrical conductivity, capacitance, surface plasmon resonance, hybrid capture, antibody, direct labeling of nucleic acid (e.g., end labeling, labeled tagmentation payload), nonspecific labeling with intercalating dyes (e.g., ethidium bromide, SYBR dye), or any other known methodology for nucleic acid detection. In some embodiments, the target protein or peptide fragment may be detected using absorption, fluorescence, mass spectrometry, amino acid sequencing, or any other known methodology for protein or peptide detection.

[0089] In some embodiments, the device further comprises a pump (e.g., a peristaltic pump) configured to transport one or more fluids into, inside or outside any one of the device's modules. In some embodiments, the device further includes a pump (e.g., a peristaltic pump) configured to transport one or more fluids into or through any of the microfluidic channels of the device or cartridges accepted by the device's modules. In some embodiments, the device is configured to transport fluids with a flow resolution of 1000 microliters or less, 100 microliters or less, 50 microliters or less, or 10 microliters or less. In some embodiments, the device is configured to accept two or more cartridges simultaneously. In some embodiments, the device is configured to establish fluid communication between two or more cartridges accepted simultaneously by the device. In some embodiments, the device is configured to accept two or more cartridges sequentially.

[0090] In some embodiments, the peristaltic pump comprises a device including rollers and a fluid device (e.g., a cartridge). In some embodiments, the peristaltic pump comprises a device including rollers and a rotational oscillating mechanism connected to the rollers. In some embodiments, the device includes a sample preparation module, which includes a peristaltic pump (including a fluid cartridge including a base layer having a surface with channels), in which case at least a portion of at least a part of the channels has a substantially triangular cross-section (having one vertex at the base of the channels and the other two vertices on the surface of the base layer). A detection module may be located downstream of the sample preparation module. In some embodiments, the sample preparation region includes two or more fluid cartridges.

[0091] According to some embodiments, a fluid system is provided. According to some embodiments, the fluid system can be used to transfer fluid to a fluid container, for example, using a peristaltic pump. According to some embodiments, the fluid system includes a cartridge or module configured to accept a fluid container using alignment properties. According to certain embodiments, the cartridge or module is configured to fluidly connect to the fluid container via alignment properties. This may allow simultaneous alignment and fluid connection between the device's cartridge or module and the fluid container. According to certain embodiments, such versatility in the connection advantageously reduces the dead volume of the fluid connection between the device's cartridge or module and the fluid container (for example, by reducing the need for extra channel length).

[0092] Two components are fluidically connected if, in some configurations of the embodiment, fluid can pass between them. For example, a first fluid component and a second fluid component can be in fluid communication if they are connected by a channel, microchannel, or pipe. As another example, two components separated by a valve can still be considered fluidically connected, insofar as the valve can be configured to allow fluid flow between the two components. In contrast, two components that are mechanically connected and have no fluid path between them are not considered fluidically connected. Fluidly connected components can be directly fluidically connected (i.e., connected by a fluid path that does not pass through any intermediate components). However, in some cases, fluidly connected components may be connected by a fluid path that passes through one, two, three, four, five, eight, ten, fifteen, twenty, or more intermediate components.

[0093] The device includes a channel, which, in some embodiments, is fluidly connected to an alignment feature of the device. The alignment feature may be a first alignment feature of the device. In some embodiments, the device further includes a second alignment feature, which, according to certain embodiments, is connected to the channel via a fluid connection established, for example, by a fluid path penetrating a fluid container. However, in some embodiments or configurations, the second alignment feature is not connected to the channel. In some embodiments, the device is configured to receive a fluid container. For example, the device is configured to hold a fluid container, according to some embodiments. In some embodiments, the device includes a cartridge. In some embodiments, the device includes a solid substrate (e.g., a solid substrate of the cartridge). The solid substrate may include a recess configured to receive a fluid container.

[0094] A fluid container may have any of a variety of suitable shapes. For example, according to a particular embodiment, a fluid container may have a substantially rectangular cross-section. In some embodiments, a fluid container has a narrow cross-section when compared to the minimum lateral dimension of the fluid container. In some embodiments, a fluid container may be configured to house a substrate (e.g., an integrated device, a chip). According to some embodiments, a fluid container is a flow cell (e.g., a cell including an interior, an inlet, and an outlet, configured to receive fluid through the inlet and transport fluid through the outlet). For example, a fluid container may be a flow cell containing an integrated device. In some embodiments, a fluid container has an interior (e.g., an internal chamber). In some embodiments, the interior of the device has the ability to be at least partially or completely filled with fluid. In some embodiments, the interior of the fluid device has a certain volume. In some embodiments, a fluid container has two or more interiors. For example, a fluid container may include one, two, three, four, five, or more interiors. According to some embodiments, at least some of these interiors are fluidly connected to each other. In some embodiments, these interiors are divided from one another so that fluid cannot flow directly from one interior to another. Incorporating multiple interiors into a fluid container is advantageous because, according to some embodiments, it may be possible to simultaneously fill multiple integrated devices or several parts of integrated devices. Simultaneous filling of multiple integrated devices or several parts of integrated devices may be possible to increase the uniformity of the fluid flow and / or enable the analysis of multiple samples (which may simultaneously interact with other integrated devices or several parts of integrated devices).

[0095] Each interior of a fluid container may include an inlet and an outlet. According to some embodiments, the inlet and / or outlet may be connected to a fluid device (e.g., via the properties of one or more alignments). In some embodiments, a fluid (e.g., a fluid sample containing biomolecules, such as peptides or polynucleotides) flows through one or more interiors of the fluid container. The flow of fluid through the interiors can, in some embodiments, at least partially or completely fill the interior of the fluid container.

[0096] In some embodiments, the device cartridge or module includes a base layer having a surface containing channels. In some embodiments, at least a portion of the channels has a substantially triangular cross-section with one vertex at the base of the channels and two other vertices on the surface of the base layer. In some embodiments, at least a portion of the channels has a surface layer. The surface layer may include an elastomer. The surface layer may be configured to substantially seal the surface openings of the channels.

[0097] As used herein, the term “channel” is known to those skilled in the art and may mean a structure configured to contain and / or transport a fluid. A channel generally includes walls; a base (e.g., a base connected to and / or formed from the walls); and surface openings in one or more portions of the channel that can be opened, covered, and / or sealed. In some embodiments, the sealed surface portion is completely sealed. In some embodiments, the sealed surface portion is substantially sealed. A surface opening can be substantially sealed if more than 50%, 60%, 75%, 90%, or 95% of the surface opening is sealed. In some embodiments, the surface opening is sealed by an elastomer.

[0098] As used herein, the term “microchannel” refers to a channel having at least one dimension of size 1000 microns or less. For example, a microchannel may have at least one dimension (e.g., width, height) of size 1000 microns or less (e.g., 100 microns or less, 10 microns or less, 5 microns or less). In some embodiments, a microchannel has at least one dimension of 1 micron or more (e.g., 2 microns or more, 10 microns or more). Combinations of the ranges cited above are also possible (e.g., 1 micron or more and 1000 microns or less, 10 microns or more and 100 microns or less). Other ranges are also possible. In some embodiments, a microchannel has a hydraulic diameter of 1000 microns or less. As used herein, the term “hydraulic diameter” (DH) is known to those skilled in the art and can be determined as DH = 4A / P, where A is the cross-sectional area of ​​the fluid flow through the channel and P is the wetted perimeter of the cross-section (the circumference of the channel cross-section in contact with the fluid).

[0099] In some embodiments, a fluid component can act to move a liquid to and from a device's reservoir and / or channels (e.g., incubation channels). In some embodiments, a device component can act to move a liquid through a channel(s) of a device, for example, to and from a fluid device's reservoir and / or other channels (e.g., incubation channels). In some embodiments, a device component moves a liquid through a peristaltic pump transport mechanism (e.g., a device) configured to interact with an elastomer component (e.g., an elastomer-containing surface) associated with the channels of a cartridge or module for pumping the fluid through the channels.

[0100] In some embodiments, fluid exchange occurs during the loading period of a module or cartridge. For example, in some embodiments, fluid exchange occurs during the loading period of a biological sample into the sample preparation module of an integrated device. Fluid exchange during the sample loading period may involve the addition or removal of a solution (e.g., a buffering solution or a solution for dissolving the sample) into a reservoir containing the sample. In some embodiments, fluid exchange during the sample loading period may involve the fluid transfer of the sample from a first channel, microchannel, or reservoir (e.g., a channel, microchannel, or reservoir used for sample input) to a second channel, microchannel, or reservoir (e.g., for dissolution, fragmentation, digestion, enrichment, etc.). Furthermore, in some embodiments, fluid exchange occurs during the loading period of one or more target molecules into a detection module or device (e.g., a sequencing module or sequencing device as described herein). In some embodiments, fluid exchange occurring during the period in which one or more target molecules are loaded onto the detection module or device involves the addition or removal of a solution involved in detection (e.g., a solution containing a fluorophore or reference molecule).

[0101] In some embodiments, fluid exchange occurs during the detection period of one or more target molecules (e.g., a target protein, polypeptide, or nucleic acid to be sequenced) or during the data acquisition period. For example, in some embodiments, fluid exchange occurs during the detection and / or data acquisition period in a sample well (e.g., a sample well containing one or more target molecules) or reaction chamber. In some embodiments, fluid exchange occurring during the detection and / or data acquisition period involves the addition or removal of a solution used for detection and / or data acquisition (e.g., a buffer containing a fluorophore for detection). In some embodiments, fluid exchange occurring during the detection and / or data acquisition period involves the addition or removal of a solution used to strip, reload, and / or unload the sample well or reaction chamber (a solution used to strip coating molecules from the reaction chamber surface).

[0102] In some embodiments, the device, the method of using it, and the associated fluids are among the many disclosures, including U.S. Patent Application No. 17 / 083,106, filed October 28, 2020, entitled “Peristaltic Pumping of Fluids and Associated Methods, Systems, and Devices”; U.S. Patent Application No. 17 / 083,126, filed October 28, 2020, entitled “Peristaltic Pumping of Fluids for Bioanalytical Applications and Associated Methods, Systems, and Devices”; and “Systems and Methods for Sample Preparation” filed October 28, 2020. As described in U.S. Patent Application No. 17 / 082,223 entitled “PREPARATION)”; U.S. Patent Application No. 17 / 082,226 entitled “METHODS AND DEVICES FOR SEQUENCING” filed on 28 October 2020; U.S. Patent Application No. 17 / 153,490 entitled “COMPOUNDS AND METHODS FOR SELECTIVE C-TERMINAL LABELING” filed on 20 January 2021; and International Patent Application No. PCT / US2021 / 028471 entitled “DEVICES AND METHODS FOR SEQUENCING” filed on 21 April 2021, the entire contents of the above paragraphs are incorporated herein by reference.

[0103] III. Technologies for reusing chips As described herein, the inventors have developed a technique for reusing the sensor chip of an integrated device to process a continuous portion of a sample. The inventors have recognized that simply loading additional aliquots of sample onto the integrated device without doing anything else can lead to inaccurate results because the previously loaded sample can interfere with signal acquisition of the newly loaded sample. Therefore, the technique described herein relates to a method for unloading the previously loaded sample from the reaction chamber of the integrated device and reusing the integrated device after subsequent sample loading.

[0104] Figure 2A is an example diagram of an analyte 159 immobilized within a chamber 158 of an integrated device, based on one embodiment. The analyte 159 may include biomolecules, such as polypeptides or polynucleotides. Figure 2A presents an example of a reaction chamber 158 with the analyte 159 to be sampled (e.g., a peptide chain). The analyte 159 may be bound to the surface 150 of the reaction chamber 158 via a binding between a coating molecule 152 (e.g., biotin) coating the surface 150 of the reaction chamber 158 and a coupling moiety 154 (e.g., streptavidin) bound to the analyte 159. The binding between the coating molecule 152 and the coupling moiety 154 immobilizes the analyte 159 within the reaction chamber 158 while the analyte 159 is being sampled. The analyte 159 may be labeled with a fluorescent dye. Labeling of the analyte may be achieved using recognition molecules 160A, 160B configured to bind to a specific analyte. The reaction chamber 158 may be positioned in close proximity to a photodetector region 161, which includes a complementary metal oxide semiconductor chip 164 and a photonic component 162 for collecting synchrotron radiation from the reaction chamber 158. After sampling, at least a portion of the analyte 159 may be cut by a cutter molecule 156 as described herein.

[0105] Figure 2B shows an example method for reusing the integrated device of Figure 1A to process multiple samples, based on some embodiments. Process 200 is initiated in act 202, in which a first aliquot of the sample can be loaded into the reaction chamber of the integrated device. For example, a sample containing multiple analytes can be divided into multiple aliquots so that it can be processed sequentially using the integrated device. The first aliquot can be loaded into the reaction chamber of the integrated device. For example, the surface of the reaction chamber can be coated with a coating molecule, such as biotin. The analyte of the first aliquot of the sample may be bound to a coupling portion, such as streptavidin. When the first aliquot is loaded into the reaction chamber, the analyte is fixed (or added) to the surface of the reaction chamber by the coupling portion (of the sample) binding to the coating molecule (fixed on the surface of the reaction chamber).

[0106] In operation 204 of Figure 2B, the first aliquot of the analyte can be sampled while the analyte is present in the reaction chamber. The step of sampling the analyte may include, in operation 205A, delivering excitation light to the reaction chamber to excite the analyte and / or a fluorescent label coupled thereto. The excited sample emits a signal (e.g., photons) that can be collected by the photodetector region of the integrated device in operation 205B. The emitted signal may, in some embodiments, be used to identify the analyte.

[0107] In some embodiments, the analyte comprises one or more peptides to be sampled. In some embodiments, the analyte comprises one or more peptides, and an excited fluorescent label is conjugated to an amino acid recognition molecule. In some embodiments, the identification of the peptide analyte can be carried out by contacting the polypeptide with one or more amino acid recognition molecules (associating with one or more types of terminal amino acids). In some embodiments, a cleavage molecule or cutter, as shown in Figure 2A, may be used to isolate a portion of the analyte (e.g., by separating a peptide from a peptide chain), and the next portion of the analyte can be sampled.

[0108] Once sufficient signal has been collected from the analyte, in operation 206, the first aliquot may be removed from the reaction chamber of the integration device. For example, it may be desirable to load an additional sample (i.e., an additional aliquot of the sample) into the integration device in order to collect information about the additional sample. However, as described herein, previously processed analytes present in the reaction chamber may interfere with signal collection of the newly loaded analyte. Therefore, the first aliquot may be removed from the integration device before loading the additional sample into the integration device.

[0109] Exemplary techniques for unloading a sensor chip include (a) breaking the bonded anchorage between the coupling portion and the coating molecule (e.g., between streptavidin and biotin); (b) peeling the molecular (e.g., biotin) coating from the surface of the sensor chip; (c) enzymatically digesting the sample analyte in the occupied reaction chamber; and (d) dissolving the bond between the sample analyte and the coupling portion (e.g., streptavidin, biotin). Such techniques are further described herein.

[0110] In operation 206, after the step of removing the first aliquot from the integration device, a second aliquot of the sample may be loaded into the reaction chamber in operation 208. The analyte of the second aliquot may similarly be bound to a coupling portion configured to bind to a coating molecule that coats (e.g., is bound to) the surface of the reaction chamber. The analyte of the second aliquot may be sampled to collect information about the analyte.

[0111] The above steps of loading and unloading multiple aliquots of a sample onto an integration device can be repeated as needed. The steps allow for processing samples containing a large number of analyte species and processing each aliquot sequentially by dividing the sample into multiple aliquots. The unloading techniques described herein prevent interference (which may result from previously loaded analytes emitting new signals during the sampling period of newly loaded aliquots). Any of the unloading techniques described below can be used in combination (e.g., sequentially) to achieve the researcher's objectives.

[0112] Breaking of the bond between the coating molecule and the coupling site. A first exemplary technique for unloading a portion of a sample from the reaction chamber of an integrated device involves breaking the covalent bond between the coating molecules coating the surface of the reaction chamber and the coupling portion bound to the analyte of the sample. Breaking the bond between the coating molecules and the coupling portion allows the immobilized analyte to be released from the surface of the reaction chamber, enabling the analyte to be flushed out of the integrated device. In some embodiments, the step of breaking the covalent bond between the coating molecules and the coupling portion includes a step of dissociating the coupling portion from the coating molecules.

[0113] Figure 3A shows an example process for regenerating the surface of the chamber of the integrated device of Figure 1A, based on some embodiments. Process 310 begins in operation 312 (a first aliquot of analyte is sampled while it is present in the reaction chamber of the integrated device). For example, excitation light emitted from at least one light source may be delivered to the reaction chamber, and a signal emitted from the reaction chamber (e.g., from the first aliquot of analyte and / or a fluorescent label coupled thereto) may be detected in the photodetector region of the integrated device.

[0114] After the sampling step in operation 314, the first aliquot may be removed from the reaction chamber. For example, the first aliquot may be removed from the integration device to allow loading of a second aliquot of the sample without signal interference from the first aliquot. Operation 314 provides an example of unloading the first aliquot from the integration device, which involves breaking the bond between the coating molecules (e.g., biotin, streptavidin) coating the surface of the reaction chamber and the coupling portion (e.g., streptavidin, biotin) bound to the sample analyte immobilized on the surface of the reaction chamber.

[0115] The inventors have recognized that prior art for disrupting the bond between molecules, such as biotin, and streptavidin is unsuitable. Such art generally requires harsh conditions, including high temperatures (e.g., 90 degrees Celsius) and chemicals that may cause damage to the integrated device. Such art can be time-consuming to implement (e.g., exceeding 60 minutes), which contradicts the need to continuously process consecutive aliquots of a sample on a single integrated device.

[0116] Therefore, the inventors have developed an improved technique for breaking the bond between the coating molecule and the coupling moiety. An example method for breaking the coating molecule-coupling moiety is initiated in operation 315A. In operation 315A, the surface of the reaction chamber can come into contact with (e.g., immerse in) the regeneration solution. The regeneration solution facilitates the breaking of the bond between the coating molecule and the coupling moiety.

[0117] In some embodiments, the regeneration solution comprises an acetate and water. In some embodiments, the acetate is ammonium acetate. Ammonium acetate can improve the solubility of the analyte (e.g., peptides or other biological samples) so that the analyte dissolves and / or is flushed away once it is released from the surface of the reaction chamber. In other embodiments, the acetate is sodium acetate. Existing solutions that break the bond between the binding molecule and the coating molecule, as described herein, contain highly irritating chemicals that may damage the components of the integrated device. Water, in contrast, is compatible with the components of the integrated device and does not pose a risk of causing damage to the integrated device.

[0118] The regeneration solution may further contain compounds (e.g., organic compounds) designed to disrupt the coupling moiety and coating molecules. For example, in some embodiments, the regeneration solution may contain organic compounds for disrupting the biotin-streptavidin complex, such as fluorinated alcohols (or fluoroalcohols), e.g., hexafluoro-2-propanol (i.e., 1,1,1,3,3,3-hexafluoro-2-propanol, hexafluoro-iso-propanol, or "HFIP"), or hexafluoro-2-propanol analogs. Such compounds can break the bond between the coating molecule and the coupling moiety, thereby allowing the release of the analyte from the reaction chamber surface. In some embodiments, the compound may further disrupt the coupling moiety (e.g., streptavidin) complex itself by breaking the bond between the bonding molecule and the analyte.

[0119] In some embodiments, the regeneration solution comprises alcohol, acetate (e.g., ammonium acetate), and water. In some embodiments, the regeneration solution comprises fluorinated alcohol, acetate, and water. In some embodiments, the regeneration solution comprises fluorinated ketone, acetate, and water. In some embodiments, the regeneration solution comprises an alcoholic irritant, acetate, and water. In some embodiments, the regeneration solution further comprises free biotin or its analogues, e.g., monobiotin, iminobiotin, and / or desthiobiotin.

[0120] In some embodiments, free biotin analogs may contain biotin with a polyethylene glycol (PEG) linker or another linker to increase the solubility of the regeneration solution. Exemplary free biotin analog chemical structures are shown below.

[0121] [ka] As described herein, existing techniques for destroying molecules are redundant and contradict the need for continuous processing of samples. In some embodiments, regeneration solutions having compositions such as those described herein have, or are designed to do so, the ability to destroy coating molecules and coupling portions within 60 minutes. In some embodiments, the immersion step is performed for individual periods of 30 minutes or less. Shorter immersion periods allow for processing multiple aliquots of the sample using a steady flow. In some embodiments, the regeneration solutions described herein are suitable for automated chip regeneration methods.

[0122] Existing techniques for molecular disruption, as described herein, must be performed at high temperatures, e.g., 90 degrees Celsius. Regeneration solutions having compositions such as those described herein have the ability to disrupt coating molecules and coupling moieties at room temperature (e.g., at least 20 degrees Celsius and no more than 22 degrees Celsius). Lower operating temperatures reduce the risk of damage to samples, integrated devices, and / or operators that may result from the use of high temperatures.

[0123] In some embodiments, the regeneration solution contains ethylenediaminetetraacetic acid (EDTA). In some embodiments, the regeneration solution contains formamide. In some embodiments, the regeneration solution contains EDTA and formamide. In some embodiments, the regeneration solution contains sodium acetate and formamide. In some embodiments, the regeneration solution contains a fluoroalcohol selected from hexafluoro-2-propanol, nonafluoro-tert-butyl alcohol, 2,2,3,3,4,4,4-heptafluoro-1-butanol, and octafluoro-2-butanone. In some embodiments, the regeneration solution contains hexafluoro-2-propanol. In some embodiments, the regeneration solution contains an analog of hexafluoro-2-propanol, e.g., nonafluoro-tert-butyl alcohol, 2,2,3,3,4,4,4-heptafluoro-1-butanol, octafluoro-2-butanol, or octafluoro-2-butanone. In some embodiments, the regeneration solution comprises one or more of nonafluoro-tert-butyl alcohol, 2,2,3,3,4,4,4-heptafluoro-1-butanol, and octafluoro-2-butanone, acetates (e.g., ammonium acetate), and water.

[0124] In operation 315B, each coupling portion is destroyed from the coating molecule by breaking the bond between the bonding molecule and the coating molecule, for example by applying a regeneration solution to the tip. For example, as described herein, the regeneration solution may contain a compound for destroying the bonding molecule from the coupling portion, such as hexafluoro-2-propanol (e.g., HFIP). HFIP is a non-toxic alcohol with an aromatic odor. HFIP is an irritant because it is corrosive to skin and mucous membranes. In some embodiments, the solution contains an amount of hexafluoro-2-propanol greater than about 1–5 mL, 3–7 mL, 4–8 mL, 5–9 mL, 5–10 mL, 8–10 mL, 9–11 mL, 10–12.5 mL, or more than 12.5 mL.

[0125] In some embodiments, the solution contains an amount of HFIP greater than approximately 1, 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or 12.5 mL. In some embodiments, the solution contains an amount of HFIP of about 1–15 mL, 1–12.5 mL, 1–5 mL, 3–7 mL, 4–8 mL, 6–9 mL, 5–10 mL, 7–11 mL, 8–10 mL, 9–11 mL, 10–12.5 mL, or more than 12.5 mL. In some embodiments, the solution contains an amount of HFIP of about 9 mL. In some embodiments, the solution contains an amount of HFIP of 9 mL.

[0126] The regeneration solution may further contain ammonium acetate (C2H4O2·H3N). In some embodiments, the solution contains 0.1, 0.2, 0.25, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, or 1.0 mL of 5M ammonium acetate. In some embodiments, the solution contains 0.5 mL of 5M ammonium acetate.

[0127] In some embodiments, the solution contains a biotin compound. Adding a biotin compound may enhance competition for the streptavidin binding site on the functionalized sample well surface. Adding a biotin compound may (further or separately) promote the dissociation of biotin-streptavidin interactions on the sample well surface, as mediated by the hexafluoro-2-propanol component of the solution. The solution may contain monobiotin or bisbiotin. In some embodiments, the solution is pegylated biotin, such as biotin-PEG3. PEG linkers may enhance the solubility of biotin in the solution. In some embodiments, the solution contains biotin-PEG3, biotin-PEG4, biotin-PEG5, biotin-PEG6, biotin-PEG7, or biotin-PEG8. In some embodiments, the regeneration solution contains a biotin compound at concentrations between 10 and 500 μM, between 50 and 250 μM, between 100 and 250 μM, between 100 and 500 μM, between 100 and 200 μM, or between 100 and 150 μM. In some embodiments, the regeneration solution contains a biotin compound at concentrations between 100 μM and 200 μM.

[0128] In an exemplary embodiment, the regeneration solution contains 9 mL of HFIP, 0.5 mL of 5 M ammonium acetate, 1 mL of water, and biotin-PEG3 (bisbiotin-PEG4) at a concentration between 100 μM and 200 μM.

[0129] In some embodiments of the regeneration solution, for example, in embodiments used for the identification and / or sequencing of nucleic acid analytes, the regeneration solution contains a DNase enzyme. The DNase enzyme may be provided as part of a nucleoside digestion mix.

[0130] In exemplary embodiments, the DNase enzyme (or nucleoside digestion mix) is applied to the chip separately from the regeneration solution. Therefore, in some embodiments, the chip regeneration method presented herein involves simultaneous or separate application of the regeneration solution and the DNase enzyme. In some embodiments, the DNase enzyme is applied to the chip before the regeneration solution. Therefore, in these embodiments, the nucleic acid (DNA) analyte is at least partially enzymatically digested with DNase before the surface of the sample well comes into contact with the regeneration solution.

[0131] In some embodiments, the step of immersing the surface of the reaction chamber in the regeneration solution further induces covalent bonding between the coupling portion and the analyte to be broken. As a result of this breakdown, the analyte and coupling portion are released from the surface of each reaction chamber in operation 315C. The released analyte and coupling portion may be flushed out of the reaction chamber in operation 315D. As described herein, the regeneration solution may contain components to increase the solubility of the analyte, such as ammonium acetate. Therefore, in some embodiments, the analyte may be dissolved in the regeneration solution additionally or alternatively.

[0132] After removing the first aliquot from the reaction chamber of the integrated device, step 310 can proceed to operation 316. The removal step described herein ensures that there is no interference caused by signals emitted from previously loaded aliquots when sampling the loaded aliquot later. Thus, once the first aliquot has been removed, in operation 316, a second aliquot can be loaded into the reaction chamber of the integrated device. The second aliquot may contain an analyte, but the analyte is bound to each coupling portion. The coating molecules coating the surface of the reaction chamber may remain in the reaction chamber even after the first aliquot has been removed. Thus, step 310 may eliminate the need for a recoating step of loading additional coating molecules into the reaction chamber of the integrated device before loading the second aliquot. The coupling portions of the second aliquot may bind to the remaining coating molecules and fix the analyte of the second aliquot to the surface of the reaction chamber so that the sampling step can be carried out.

[0133] Following operation 316, a second aliquot of the analyte can be sampled. For example, excitation light emitted from at least one light source can be delivered to the reaction chamber, and a signal emitted from the reaction chamber (e.g., from the second aliquot of the analyte and / or a fluorescent label coupled thereto) can be detected in the photodetector region of the integrated device. The above steps of loading and unloading the integrated device via techniques for breaking the bonds between the coating molecules and coupling portions can be repeated multiple times as needed.

[0134] In some embodiments, the method includes a step of disrupting one or more bonds between a coupling moiety and a plurality of coating molecules (e.g., a first coating molecule and a second coating molecule), wherein the first and second coating molecules are functionalized to the well surface. In some embodiments, the first and second coating molecules are different. In some embodiments, the first coating molecule is a streptavidin protein, and the second coating molecule is not a streptavidin protein (e.g., an avidin protein or an azide moiety). In some embodiments, the method includes a step of disrupting bonds between a coating molecule and a plurality of types of coupling moieties (e.g., first and second coupling moieties), wherein the coating molecules are functionalized to the well surface. In some embodiments, the first and second coupling moieties are different. In some embodiments, the first coupling moiety is a biotin moiety, and the second coupling moiety is not a biotin moiety (e.g., a SNAP-tag or an azide moiety). In all such embodiments, the use of the regeneration solution based on the disclosed method can be used to break down the bonds between multiple types of coating molecules and multiple types of coupling moieties that are combined together.

[0135] In some embodiments, the analyte may be at least partially enzymatically digested to break the bond between the coating molecule and the coupling moiety before the reaction chamber surface comes into contact with the regeneration solution in operation 315A. For example, in some embodiments (e.g., in DNA sequencing applications), the sampled analyte is so large that contact between the reaction chamber surface and the regeneration solution (containing compounds designed to break the coupling moiety and coating molecule) may be prevented due to the size of the analyte. Therefore, in some embodiments, at least a portion of the sampled analyte in the reaction chamber may be enzymatically digested to allow the compounds designed to break the coupling moiety and coating molecule to come into contact with the reaction chamber surface. For example, if the analyte contains a DNA strand (e.g., in DNA sequencing), at least a portion of the DNA strand may be enzymatically digested before the reaction chamber surface comes into contact with the regeneration solution (e.g., using endonuclease (e.g., DNase) and / or exonuclease digestion).

[0136] Removal of reaction chamber coating A second exemplary technique for unloading a portion of a sample from the reaction chamber of an integrated device involves detaching coating molecules from the reaction chamber surface. By detaching the coating molecules from the reaction chamber surface, coupling portions bound to the coating molecules and the analytes attached to them are released from the reaction chamber, and the released components can be dissolved and / or flushed from the integrated device.

[0137] For example, Figure 3B shows an example method for regenerating the chamber surface of the integrated device shown in Figure 1A, which, according to some embodiments, involves stripping a coating from the reaction chamber surface. Step 320 begins in operation 322, during which a sample is taken while a first aliquot of analyte is present in the reaction chamber of the integrated device. For example, excitation light emitted from at least one light source may be delivered to the reaction chamber, or a signal emitted from the reaction chamber (e.g., from the first aliquot of analyte and / or a fluorescent label coupled thereto) may be detected in the photodetector region of the integrated device.

[0138] After the sampling step, in operation 324, the first aliquot may be removed from the reaction chamber. For example, the first aliquot may be removed from the assembly device to allow loading of a second aliquot of the sample without signal interference from the first aliquot. Operation 324 provides a second example of unloading the first aliquot from the assembly device, which involves removing a coating containing one or more coating molecules (e.g., biotin, streptavidin) from the surface of the reaction chamber.

[0139] In particular, during operation 325A, one or more coating molecules coating the surface of the reaction chamber are peeled off from the surface of the reaction chamber. In some embodiments, the step of peeling off one or more coating molecules includes the addition of any regeneration solution as disclosed above. In some embodiments, this step includes the addition of a solution of HFIP or 1,1,1,3,3,3-hexafluoro-2-propanol.

[0140] As described herein, the coupling portion binds to the coating molecule in the reaction chamber, fixing the first aliquot of the sample analyte bound to the coupling portion to the surface of the reaction chamber, thereby enabling sampling. As a result of removing the coating, the analyte and coupling portion are also released from the surface of each reaction chamber in operation 325B. The released analyte and coupling portion can be flushed out of the reaction chamber in operation 325C. In some embodiments, the released components can be additionally or alternatively dissolved by immersing the surface of the tip in a solution, such as one of the regeneration solutions described herein. In some embodiments, the disclosed method includes the step of peeling off the first and second coating molecules from the surface of the reaction chamber, except that the first and second coating molecules are different. In such embodiments, the first coating molecule may contain streptavidin protein, and the second coating molecule may contain a portion that is not streptavidin protein (for example, it may be an avidin protein or an azide portion).

[0141] After removing the first aliquot from the reaction chamber of the integrated device, step 320 can proceed to operation 326. In operation 326, the surface of the reaction chamber is recoated by loading additional coating molecules (e.g., biotin, streptavidin) into the reaction chamber in preparation for loading a second aliquot.

[0142] In operation 328, a second aliquot of the sample may be reloaded into the reaction chamber of the integrated device. After operation 328, the analyte of the second aliquot may be sampled. The above steps of loading and unloading the integrated device via a technique for stripping the reaction chamber coating may be repeated as needed.

[0143] Enzymatic digestion of analytes A third exemplary technique for removing a portion of a sample from the reaction chamber of an integrated device involves enzymatic digestion of the analyte (e.g., a peptide) present in the reaction chamber after the sampling step. Enzymatic digestion of the analyte after the sampling step makes it possible to remove the previously sampled analyte from the reaction chamber without requiring the destruction of coating molecules and coupling sites, or recoating the surface of the reaction chamber of the integrated device.

[0144] For example, Figure 3C shows an example method for regenerating the chamber surface of the integrated device of Figure 1A, which, based on some embodiments, involves enzymatic digestion of previously sampled analytes. Step 330 begins in operation 332, in which a first aliquot of analyte is sampled while it is present in the reaction chamber of the integrated device. For example, excitation light emitted from at least one light source may be delivered to the reaction chamber, and a signal emitted from the reaction chamber (e.g., from the first aliquot of analyte and / or a fluorescent label linked thereto) may be detected in the photodetector region of the integrated device.

[0145] After the sampling process, in operation 334, the first aliquot may be removed from the reaction chamber. For example, the first aliquot may be removed from the integration device, allowing the loading of a second aliquot of the sample without signal interference from the first aliquot. Operation 334 provides a second example of unloading the first aliquot from the integration device, which involves completely enzymatically digesting the previously sampled analyte present in the reaction chamber of the integration device.

[0146] In particular, in operation 335A, the first aliquot analyte can be enzymatically digested. For example, a cutting enzyme, also known as a cutter and commonly used in protein and DNA / RNA sequencing, as shown in Figure 2A, can be used to cut the analyte from its fixed position on the surface of the reaction chamber. Generally, cutting enzymes are used to cut a single peptide from a peptide chain in order to perform sequencing. However, the inventors have recognized that by immersing the reference chamber in a solution containing a certain concentration of cutting enzyme for an extended period, an entire peptide chain can be cut from its fixed point on the surface of the reaction chamber. The enzymatically digested analyte can then be flushed and / or dissolved to remove the first aliquot analyte from the accumulation device.

[0147] In some embodiments, each reaction chamber may contain multiple coating molecules that coat the surface of the reaction chamber. Therefore, even if the previously bound coating molecules are occupied by the previously bound analyte (which is enzymatically digested in operation 335A of step 330), additional coating molecules can remain in the reaction chamber and bind to the coupling portion of the subsequent aliquot of the sample. In some embodiments, the additional coating molecules may be loaded into a tip after operation 335 to recoat the surface of the reaction chamber.

[0148] After removing the first aliquot from the reaction chamber of the integration device, step 330 can proceed to operation 336. In operation 336, the reaction chamber of the integration device may be recoated with a second aliquot of the sample. The analyte of the second aliquot may bind to any remaining coating molecules present in the reaction chamber via a coupling portion. After operation 336, the analyte of the second aliquot may be sampled. The above steps of loading and unloading the integration device via techniques for enzymatic digestion of the previously sampled analytes may be repeated as needed.

[0149] Melting of analyte-coupling subunit bonds A fourth exemplary technique for unloading a portion of a sample from the reaction chamber of an integrated device involves melting the bond between the sample analyte and a coupling portion attached to it while the sample analyte is present in the reaction chamber. For example, Figure 3D shows an exemplary method for regenerating the chamber surface of the integrated device shown in Figure 1A, which, according to some embodiments, involves melting the bond formed between the sample analyte and the coupling portion. Step 340 begins in operation 342, and a sample is taken while a first aliquot of analyte is present in the reaction chamber of the integrated device. For example, excitation light emitted from at least one light source may be delivered to the reaction chamber, and a signal emitted from the reaction chamber (e.g., from the first aliquot of analyte and / or a fluorescent label attached to it) may be detected in the photodetector region of the integrated device. In some embodiments, the bond formed between the sample analyte and the coupling portion includes a double-stranded nucleic acid molecule (e.g., a DNA molecular linker in protein sequencing applications), and the melting of the bond includes a step of denaturing the double-stranded nucleic acid molecule to a single strand to release the sample analyte.

[0150] After the sampling step, in operation 344, the first aliquot may be removed from the reaction chamber. For example, the first aliquot may be removed from the integration device to allow loading of a second aliquot of the sample without signal interference from the first aliquot. Operation 344 provides a second example of the process of unloading the first aliquot from the integration device, which involves melting the bond formed between the sample analyte and the coupling portion configured to bond to the coating molecules coating the surface of the reaction chamber.

[0151] In particular, in operation 335A, the bond between the sample analyte and the coupling moiety may be melted. In some embodiments, operation 335A includes denaturing the DNA strand linking the sample analyte to the coupling moiety from double-stranded DNA to single-stranded DNA, thereby releasing the analyte. The step of denaturing the DNA molecular linker formed between the sample analyte and the coupling moiety may include injecting energy, such as heat, into the system to melt the DNA strand. In some embodiments, this step includes adding any regeneration solution as disclosed above, and / or attaching any other chemical solution.

[0152] As described herein, the sample analyte is configured to be fixed to the surface of the reaction chamber via a bond formed between a coating molecule coating the surface of the reaction chamber and a coupling portion attached to the sample analyte. Step 340 aims to break the bond between the coupling portion and the sample analyte, thereby preventing the analyte from being fixed to the surface of the reaction chamber. Thus, after melting the covalent bond between the coupling portion and the sample analyte, the analyte of the first aliquot may be released from the surface of the reaction chamber in operation 345B. The released component may be flushed out of the reaction chamber in operation 345C. In some embodiments, the released component may be additionally and / or alternatively dissolved.

[0153] After removing the first aliquot from the reaction chamber of the integrated device, step 340 can proceed to operation 346. In operation 346, the reaction chamber of the integrated device may be reloaded with a second aliquot of the sample. The analyte of the second aliquot may be bonded to the remaining coating molecules present in the reaction chamber via a coupling portion. After operation 346, the analyte of the second aliquot may be sampled. The above process of loading and unloading the integrated device via a technique for melting the coupling portion-analyte bond can be repeated as required.

[0154] In some embodiments, the step of melting the bond between the sample analyte and the coupling portion includes the step of melting the bond between the analyte and a plurality of types of coupling portions, for example, first and second coupling portions. In some embodiments, the first and second coupling portions are different. In some embodiments, the first coupling portion is a biotin portion, and the second coupling portion is not a biotin portion (for example, a SNAP tag or an azide portion). [Examples]

[0155] The effectiveness of techniques for removing portions of a sample from integrated devices is further demonstrated herein, for example, as shown in Figures 4A–4J. In particular, Figures 4A–4J show data accumulated using a chip regeneration technique to break the bond between the coating molecule and the coupling moiety. The regeneration solution used in the experiments that produced the following data contained ammonium acetate, water, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Specifically, the regeneration solution contained 9 mL of HFIP, 0.5 mL of 5 M ammonium acetate, 1 mL of water, and biotin-PEG3 at a concentration between 100 μM and 200 μM.

[0156] Figures 4A–D show exemplary graphs illustrating the presence of analytes obtained during chip loading and regeneration processes according to some embodiments. For example, Figure 4A shows three consecutive graphs 411–413. Graph 411 shows signal acquisition when the integrated device reaction chamber is loaded with 93.6% analytes. Graph 412 shows signal acquisition obtained 15 minutes after the analytes were removed from the reaction chamber according to the destruction technique described herein (at 22.5% load). Graph 413 shows signal acquisition after reloading the integrated device reaction chamber with an additional sample at 68% load.

[0157] Figures 4B–4D show further exemplary graphs illustrating the presence of analytes obtained during the loading and unloading processes of the integrated device. As shown in Figure 4B, graph 421 shows signal acquisition after a first load at 58.7%; graph 422 shows signal acquisition after subsequent unloading (at 8.5% load); graph 423 shows signal acquisition after a second load at 27.7% load; graph 424 shows signal acquisition after subsequent unloading (at 3.8% load); and graph 425 shows signal acquisition after a third reload at 23.4% load. As shown in Figure 4C, Graph 431 shows the signal acquisition after the first load was applied at 30.4%, Graph 432 shows the signal acquisition after subsequent unloading (at 13.8%), and Graph 433 shows the signal acquisition after the second load was applied at 57.7%. As shown in Figure 4D, Graph 441 shows the signal acquisition after the first load was applied at 27.9%, Graph 442 shows the signal acquisition after subsequent unloading (at 7.2%), and Graph 443 shows the signal acquisition after the second load was applied at 64.9%.

[0158] Figure 4E shows spectra of samples immersed in regeneration solution under different conditions, obtained using high-performance liquid chromatography according to some embodiments. In particular, curve 451 shows signal acquisition from a sample first diluted with water and then mixed with detachment buffer; curve 452 shows signal acquisition from a sample incubated with detachment buffer for 10 minutes and then diluted with water; and curve 453 shows signal acquisition from a sample incubated with detachment buffer for 50 minutes and then diluted with water.

[0159] Figure 4F shows exemplary graphs illustrating signals acquired from the reaction chamber of the integrated device between the regeneration and loading processes in some embodiments. In particular, the first graph shows signal acquisition in response to recognition pulses delivered to the integrated device during unloading. The second graph shows signal acquisition after the first regeneration and loading processes. The third graph shows signal acquisition after additional loading of the integrated device. The fourth graph shows signal acquisition after unloading and regeneration of the integrated device.

[0160] Figures 4G to 4I show additional exemplary graphs illustrating signals acquired from the integrated device reaction chamber between the regeneration and loading steps in some embodiments. As shown in Figure 4G, graphs 461A and 461B show signal acquisition from the integrated device reaction chamber loaded with 28% sample. Graphs 462A and 462B show signal acquisition from the integrated device reaction chamber loaded with 7% sample. Graphs 463A and 463B show signal acquisition from the integrated device reaction chamber loaded with 65% sample.

[0161] Figure 4J shows additional exemplary graphs illustrating signals acquired from the reaction chamber of the integrated device between the regeneration and loading processes in some embodiments. Specifically, the first row of the graph shows signal acquisition after the first sample loading of the integrated device, the second row shows signal acquisition after the first unloading of the integrated device, and the third row shows signal acquisition after reloading the integrated device.

[0162] IV. Appearances relating to chip reuse technology The inventors have further developed additional techniques that can be used in combination with the chip recycling techniques described herein. For example, Figure 5A shows an exemplary process, according to some embodiments, for determining whether a sample is present in the chamber of the integrated device of Figure 1. The inventors have recognized that it is advantageous to determine whether the unloading techniques described herein have been successfully performed before loading additional samples into the integrated device.

[0163] Step 500, shown in Figure 5A, begins with operation 502, in which a first portion of the sample is removed from the reaction chamber of the integrated device. For example, operation 502 may be carried out according to any of the techniques for removing a sample from the reaction chamber of an integrated device described herein. The first portion of the sample may include a first aliquot of a sample divided into multiple aliquots.

[0164] After removing the first portion of the sample from the integrated device, a recognition process can be performed so that in operation 502, a recognition pulse of excitation light is used to determine whether any sample remains in the reaction chamber of the integrated device or whether the sample has been sufficiently removed. Thus, in operation 504, excitation light from at least one light source is delivered to the reaction chamber of the integrated device.

[0165] If any analyte from the first portion of the sample remains in the reaction chamber after operation 502, the remaining analyte and / or any fluorescent labels attached thereto may be excited in operation 504 in response to the delivery of excitation light from at least one light source. The excited analyte and / or any fluorescent labels attached thereto emit photons in response to the excitation, which can be collected by the photodetector region of the integrated device in operation 504. Furthermore, the coating molecules, coupling moieties, and / or fluorescent labels attached thereto may emit signals in response to the excitation light. Therefore, in some embodiments, any signal received from the reaction chamber by the photodetector region of the integrated device can be used to determine whether any of the coating molecules and / or coupling moieties are present in the reaction chamber of the integrated device.

[0166] In operation 508, it is determined whether any of the first parts of the sample are present in the reaction chamber of the integration device based on the signal collected in operation 506. For example, the emitted signal may include information about the analyte, coating molecules, coupling moieties and / or fluorescent labels attached to the object from which the signal was emitted (e.g., emission intensity, emission lifetime, wavelength, pulse duration, pulse interval duration). The integration device may be configured to use the emitted information to determine what is present in the reaction chamber, if any.

[0167] If, in operation 508, it is determined that at least a portion of the first part of the sample is still present in the reaction chamber, step 500 may proceed through this branch to operation 502, and the removal process to remove the first part of the sample from the reaction chamber is repeated. In some embodiments, the removal process performed when returning to operation 502 through this branch may differ from the initial removal process performed at the beginning of step 500.

[0168] In operation 508, if it is determined, based on the signal collected in operation 506, that the first portion of the sample is no longer present in the reaction chamber, step 500 may proceed through this branch to operation 510, in which a second portion of the sample is loaded into the reaction chamber of the integrated device. The second portion of the sample may include a second aliquot of a plurality of aliquots from which the sample has been divided.

[0169] Therefore, step 500 may be used to determine when reloading of the integration device with an additional sample should be performed. As described herein, the inventors have recognized that previously sampled analytes present in the reaction chamber when the newly loaded analyte is sampled may interfere with signal collection from the newly loaded analyte. Therefore, step 500 may be used to ensure that previously sampled analytes have been sufficiently removed before proceeding with reloading of the integration device.

[0170] In some embodiments, the step in operation 508 to determine whether a first portion of the sample is present in the reaction chamber includes the step of determining the amount of the first sample remaining in the reaction chamber. In such embodiments, the determined amount can be compared to a threshold. If the determined amount is below the threshold, step 500 can proceed to operation 510, in which additional sample is loaded into the reaction chamber of the integrated device. Alternatively, if the determined amount is not below the threshold in operation 508, step 500 can refrain from loading additional sample into the reaction chamber.

[0171] As described herein, the signal collected in operation 506 may be used to determine the presence of coating molecules and / or coupling portions in the reaction chamber. In such embodiments, the collected signal may be used, for example, to determine whether to load additional coating molecules and / or coupling portions into the reaction chamber of the integrated device before loading a second portion of the sample into the reaction chamber of the integrated device.

[0172] The inventors have further developed a technique that can be used in conjunction with the chip reuse technique described herein for determining whether to continue sampling of subsequent aliquots of a sample divided into multiple aliquots. For example, the inventors have recognized that some samples may contain the information of interest, while others do not need to be sampled at all. Therefore, information about the sample can be collected from the first aliquot of the multiple aliquots of the sample and used to determine whether to continue processing the sample by loading the second aliquot of the sample onto an integration device.

[0173] For example, Figure 5B is an exemplary process for determining whether to continue processing a sample using the integration device of Figure 1A, according to some embodiments. Step 500 shown in Figure 5B begins with operation 522, in which a first portion of the sample is loaded into the reaction chamber of the integration device. For example, the first portion of the sample may include a first aliquot of the sample, and the sample is divided into multiple aliquots. The first portion of the sample may include multiple analytes to be sampled. The analytes can be bonded to the coupling portion described herein to fix the analytes to the surface of the reaction chamber via a bond between the coupling portion and a coating molecule that coats the surface of the reaction chamber. In some embodiments, the analytes may be labeled with a fluorescent dye.

[0174] In operations 524-526, a first portion of the sample loaded into the reaction chamber in operation 522 can be sampled. For example, in operation 524, excitation light from at least one light source can be delivered to the first portion of the sample while it is present in the reaction chamber of the integrated device. As a result, the analyte and / or fluorescent label attached to the first portion of the sample are excited and can emit photons collected by the photodetector region of the integrated device in operation 526.

[0175] In operation 528, based on the signal collected in operation 526, it is determined whether to load a second portion of the sample into the reaction chamber of the integration device. For example, as described herein, the emission signal may include information that can be extracted by the integration device, such as characteristic intensity, wavelength, emission lifetime, pulse duration, and inter-pulse duration. In some embodiments, the extracted information may be used to determine the identity of the analyte present in the first portion of the sample. Based on the identity of the analyte, it can be determined that the sample as a whole is of interest and that further processing of additional aliquots of the sample is desirable. In such cases, step 520 may proceed to operation 530 via this branch.

[0176] In other examples, based on the determined identity of the analyte, it can be determined that the sample as a whole is not of interest and that time and other resources do not need to be spent on further processing of the sample. Therefore, if it is determined in operation 528 that the second portion of the sample will not be loaded, step 520 can proceed to operation 532 via the no branch. In operation 532, the user may cancel loading the second portion of the sample into the reaction chamber of the integrated device. Instead, the first portion of the sample may be removed from the reaction chamber of the integrated device. In some embodiments, in operation 532, the first portion of a second additional sample may be loaded into the reaction chamber of the integrated device for sampling. In some embodiments, the sensor chip of the integrated device may be disposed of additionally or alternatively.

[0177] If it is determined in operation 528 that a second portion of the sample should be loaded, step 520 may proceed through this branch to operation 530, in which the first portion of the sample is removed and the second portion of the sample is loaded into the reaction chamber of the integration device, for example, according to any of the techniques described herein. Step 520 may be repeated as required by returning to operation 524, in which the second portion of the sample is processed, following operation 530.

[0178] Increased lifespan of V-chips As described herein, the inventors have recognized that when integrated devices are reused in multiple experiments, the components of the integrated device may degrade due to the long-term use of the sensor chip. Therefore, some aspects of the techniques described herein relate to techniques for increasing the service life of integrated devices. For example, the components of an integrated device may become susceptible to corrosion due to repeated immersion of the reaction chamber in the regeneration solution. Therefore, in some embodiments, the components of the integrated device may be configured to withstand such degradation (e.g., including corrosion-resistant coatings). In some embodiments, the components of the integrated device may be configured to withstand interference with luminescence signals generated by sequencing workflows that may result from the sequential regeneration of the chip and / or flow cell.

[0179] For example, in some embodiments, increasing the lifespan of an integrated device chip may be possible by maintaining the stability of the surface coating of the reaction chamber of the integrated device. An example of a suitable technique for maintaining the stability of the surface coating of the reaction chamber of an integrated device is described in U.S. Patent Application No. 17 / 067,184, filed on 9 October 2020 under Agent Reference Number R0708.70066US01, entitled “Surface Modification in the Vapor Phase,” which is incorporated herein by reference in its entirety. In some embodiments, the pH value of the regeneration solution described herein may be maintained at 7 to increase the stability of the reaction chamber surface coating.

[0180] VI. Additional Device Components In some embodiments, the integrated devices described herein may be configured to have one or more additional components. For example, Figure 6A is a block diagram of an analytical instrument including a compact mode-locked laser module according to one embodiment. As shown in Figure 6A, the portable advanced analytical instrument 6-100 may comprise one or more pulsed light sources 6-106 that are mounted as interchangeable modules within the instrument 6-100 or otherwise coupled to the instrument 6-100. The portable analytical instrument 6-100 may include an optical coupling system 6-115 and an analytical system 6-160. The optical coupling system 6-115 may include several combinations of optical components (e.g., any of the following components: lenses, mirrors, optical filters, attenuators, beam steering components, beam shaping components, etc., or may include one or more of these components) and may be configured to act on the output optical pulse 6-122 from the pulsed light source 6-106 and / or to couple the output optical pulse 6-122 from the pulsed light source 6-106 to the analytical instrument 6-160. The analysis system 6-160 may include a plurality of components configured to direct an optical pulse for sample analysis into at least one reaction chamber, receive one or more optical signals (e.g., fluorescence, backscatter) from at least one reaction chamber, and generate one or more electrical signals representing the received optical signals. In some embodiments, the analysis system 6-160 may include one or more photodetectors and may also include signal processing electronics (e.g., one or more microcontrollers, one or more field-programmable gate arrays, one or more microprocessors, one or more digital signal processors, logic gates, etc.) configured to process the electrical signals from the photodetectors. The analysis system 6-160 may also include data transmission hardware configured to transmit and receive data to and from external devices (e.g., one or more external devices on a network to which the instrument 6-100 can be connected via one or more data communication links).In some embodiments, the analysis system 6-160 may be configured to receive a bio-optoelectronic chip 6-140 that holds one or more samples to be analyzed.

[0181] Figure 6B shows a more detailed example of the portable analytical instrument 6-100, including a compact pulsed light source 6-108. In this example, the pulsed light source 6-108 comprises a compact passive mode-locked laser module 6-113. A passive mode-locked laser can autonomously generate optical pulses without the application of an external pulse signal. In some implementations, the module can be mounted on the instrument's chassis or frame 6-103 and can be located inside the instrument's outer casing. According to some embodiments, the pulsed light source 6-106 may include additional components that can be used to manipulate the light source and the output beam from the light source 6-106. The mode-locked laser 6-113 may include elements (e.g., saturable absorbers, acousto-optic modulators, Kerr lenses) in or coupled to the laser cavity that induce phase locking of the laser's longitudinal frequency modes. The laser cavity can be partially defined by cavity end mirrors 6-111, 6-119. This frequency mode locking results in pulsed operation of the laser (for example, intracavity pulses 6-120 travel back and forth between the cavity end mirrors), generating a stream of output light pulses 6-122 from one end mirror 6-111 which is partially transparent.

[0182] In some cases, the analytical instrument 6-100 is configured to accept a removable, packaged bio-optoelectronic or optoelectronic chip 6-140 (also known as a “disposable chip”). A disposable chip may include, for example, a bio-optoelectronic chip comprising multiple reaction chambers, integrated optics configured to deliver photoexcitation energy to the reaction chambers, and an integrated photodetector configured to detect fluorescence emission from the reaction chambers. In some implementations, the chip 6-140 may be disposable after a single use, while in other implementations, the chip 6-140 may be reused two or more times. Once the chip 6-140 is received by the instrument 6-100, it can communicate electrically and optically with the pulsed light source 6-106 and the devices within the analytical system 6-160. Electrical communication may be performed, for example, through electrical contacts on the chip package.

[0183] In some embodiments, referring to Figure 6B, the disposable chip 6-140 can be mounted (e.g., via socket connections) on an electronic circuit board 6-130, such as a printed circuit board (PCB), which may include additional electronic equipment. For example, PCB 6-130 may include circuitry configured to provide power, one or more clock signals, and control signals to the optoelectronic chip 6-140, as well as signal processing circuitry configured to receive signals representing fluorescence emission detected from the reaction chamber. The data returned from the optoelectronic chip may be processed partially or entirely by electronic equipment on device 6-100, although in some implementations, the data may be transmitted to one or more remote data processors via a network connection. PCB 6-130 may also include circuitry configured to receive feedback signals from the chip related to the optical coupling and power levels of the optical pulse 6-122 coupled to the waveguide of the optoelectronic chip 6-140. The feedback signals may be provided to one or both of the pulse light source 6-106 and the optical system 6-115 to control one or more parameters of the output beam of the optical pulse 6-122. In some cases, PCB 6-130 can supply or route power to the pulsed light source 6-106 in order to operate the light source and related circuits in the light source 6-106.

[0184] According to some embodiments, the pulsed light source 6-106 comprises a compact mode-locked laser module 6-113. The mode-locked laser may comprise a gain medium 6-105 (which may be a solid material in some embodiments), an output coupler 6-111, and a laser cavity end mirror 6-119. The optical cavity of the mode-locked laser can be coupled to the output coupler 6-111 and the end mirror 6-119. The optical axis 6-125 of the laser cavity may have one or more folds (bends) to lengthen the laser cavity and provide a desired pulse repetition rate. The pulse repetition rate is determined by the length of the laser cavity (e.g., the time it takes for an optical pulse to travel back and forth within the laser cavity).

[0185] In some embodiments, additional optical elements (not shown in Figure 6B) may be present within the laser cavity for beam shaping, wavelength selection, and / or pulse formation. In some cases, the end mirror 6-119 comprises a saturable absorbing mirror (SAM) that induces passive mode locking of longitudinal cavity modes, resulting in pulsed operation of the mode-locked laser. The mode-locked laser module 6-113 may further include a pump source (e.g., a laser diode, not shown in Figure 6B) for exciting the gain medium 6-105. Further details of the mode-locked laser module 6-113 can be found in U.S. Patent Application No. 15 / 844,469, “Compact Mode-Locked Laser Module,” filed on 15 December 2017 under Agent Reference Number R0708.70025US01, which is incorporated herein by reference.

[0186] When laser 6-113 is mode-locked, the cavity pulse 6-120 can circulate between the end mirror 6-119 and the output coupler 6-111, and a portion of the cavity pulse can be transmitted as output pulse 6-122 via the output coupler 6-111. Thus, the train of output pulse 6-122 can be detected at the output coupler as the cavity pulse 6-120 travels back and forth between the output coupler 6-111 and the end mirror 6-119 within the laser cavity.

[0187] In some implementations, the beam steering module 6-150 can receive output pulses from the pulsed light source 6-106 and is configured to adjust at least the position and incident angle of the optical pulses onto the optical coupler (e.g., a grating coupler) on the optoelectronic chip 6-140. In some cases, the output pulses 6-122 from the pulsed light source 6-106 can be manipulated by the beam steering module 6-150 to additionally or alternatively modify the beam shape and / or beam rotation at the optical coupler on the optoelectronic chip 6-140. In some implementations, the beam steering module 6-150 can further provide beam focusing and / or polarization adjustment of the output pulses onto the optical coupler. An example of a beam steering module is described in U.S. Patent Application No. 15 / 161,088, entitled “Pulsed Laser and Bioanalytic System,” filed on 20 May 2016 under Agent Reference Number R0708.70010US02, which is incorporated herein by reference. Another example of a beam steering module is described in a separate U.S. Patent Application No. 15 / 842,720, entitled “Compact Beam Shaping and Steering Assembly,” filed on 14 December 2017 under Agent Reference Number R0708.70024US01, which is incorporated herein by reference.

[0188] Figure 6C shows an example of a parallel reaction chamber that can be optically excited by a pulsed laser via one or more waveguides, according to some embodiments. Referring to Figure 6C, the output pulse 6-122 from the pulsed light source can be coupled to one or more optical waveguides 6-312 on, for example, a bio-optoelectronic chip 6-140. In some embodiments, the optical pulse can be coupled to one or more waveguides via a grating coupler 6-310, while in some embodiments, coupling to the ends of one or more optical waveguides on the optoelectronic chip can be used. According to some embodiments, a quad detector 6-320 can be placed on a semiconductor substrate 6-305 (e.g., a silicon substrate) to assist in the alignment of the beam of the optical pulse 6-122 to the grating coupler 6-310. One or more waveguides 6-312 and the reaction chamber or reaction chamber 6-330 can be integrated on the same semiconductor substrate with a dielectric layer (e.g., a silicon dioxide layer) interposed between the substrate, waveguide, reaction chamber, and photodetector 6-322.

[0189] Each waveguide 6-312 may include a tapered section 6-315 below the reaction chamber 6-330 to homogenize the optical power coupled to the reaction chamber along the waveguide. The reduction in taper forces more optical energy outward from the waveguide core, increasing coupling to the reaction chamber and compensating for optical losses along the waveguide, including losses of optical coupling to the reaction chamber. A second lattice coupler 6-317 may be placed at the end of each waveguide to guide the optical energy to an integrated photodiode 6-324. The integrated photodiode can detect the amount of power coupled below the waveguide and provide the detected signal to a feedback circuit that controls, for example, a beam steering module 6-150.

[0190] The reaction chamber 6-330 or reaction chamber 6-330 may be aligned with the tapered portion 6-315 of the waveguide and embedded in tab 6-340. A photodetector 6-322 may be placed on the semiconductor substrate 6-305 for each reaction chamber 6-330. In some embodiments, a semiconductor absorber (shown as an optical filter 6-530 in Figure 6-5) may be placed between the waveguide and the photodetector 6-322 at each pixel. A metal coating and / or multilayer coating 6-350 may be formed around the reaction chamber and on the waveguide to prevent photoexcitation of fluorophores that are not inside the reaction chamber (e.g., dispersed in a solution above the reaction chamber). The metal coating and / or multilayer coating 6-350 may extend beyond the ends of tab 6-340 to reduce the absorption loss of light energy within the waveguide 6-312 at the input and output ends of each waveguide.

[0191] Multiple rows of waveguides, reaction chambers, and time-binning photodetectors may be present on the optoelectronic chip 6-140. For example, one implementation may have 128 rows, each with 512 reaction chambers, for a total of 65,536 reaction chambers. Other implementations may include fewer or more reaction chambers and other layout configurations. Optical power from the pulsed light source 6-106 can be distributed to the multiple waveguides via one or more star couplers or multimode interference couplers located between the optical coupler 6-310 and the multiple waveguides 6-312 relative to the chip 6-140, or by any other means.

[0192] A non-limiting example of a biological reaction occurring within reaction chamber 6-330 is shown in Figure 6D. This example demonstrates the sequential incorporation of a nucleotide or nucleotide analog into an elongated chain complementary to a target nucleic acid. Sequential incorporation can occur within reaction chamber 6-330 and can be detected by advanced analytical instruments for DNA sequencing. The reaction chamber can have a depth between approximately 150 nm and 250 nm, and a diameter between approximately 80 nm and 160 nm. A metallization layer 6-540 (e.g., metallization for an electrical reference potential) can be patterned on top of the photodetector 6-322 to provide an aperture or diaphragm that blocks stray light from adjacent reaction chambers and other unwanted light sources. According to some embodiments, polymerase 6-520 can be located within reaction chamber 6-330 (e.g., mounted at the bottom of the chamber). Polymerase can take up target nucleic acids 6-510 (e.g., a portion of nucleic acid derived from DNA) and sequence the elongated strands of complementary nucleic acids to generate the elongated strands of DNA 6-512. Nucleotides or nucleotide analogs labeled with different fluorophores can be dispersed on top of the reaction chamber and in the solution within the reaction chamber.

[0193] VII. Applications of Chip Recycling Technology Having described several techniques for reusing sensor chips to increase the number of samples processed by a single device, we now describe an exemplary application of chip reuse techniques. For example, we have recognized that the identification of one or more molecules in a sample under analysis can be carried out in combination with the techniques described herein. In particular, measurements of one or more properties of emitted light can be obtained by a device such as an integrated device described herein, and the collected measurements can be compared with known values ​​of the measured properties of fluorescent markers to determine which fluorescent marker is most likely to be the emission source. Then, by identifying the fluorescent markers, the identity of the molecules to which the fluorescent markers are attached can be determined based on specific types of molecules to which fluorescent markers are known to adhere.

[0194] The technologies described herein include, for example, U.S. Patent Application No. 16 / 686,028, titled “Methods and Compositions for Protein Sequencing,” filed on November 15, 2019, under Agent Reference Number R0708.70042US02; PCT Application No. PCT / US19 / 61831, also titled “Methods and Compositions for Protein Sequencing,” filed on November 15, 2019, under Agent Reference Number R0708.70042WO00; and “Integrated Sensor for Multi-Dimensional Signal Analysis,” filed on March 2, 2021, under Agent Reference Number R0708.70090US01. This technology can be used in combination with techniques for detecting and / or identifying molecules in a sample, including the technique described in U.S. Patent Application No. 17 / 190,331, entitled “ANALYSIS,” each of which is incorporated herein by reference. Such techniques may be used, for example, in applications of protein sequencing and / or nucleic acid (e.g., DNA and / or RNA) sequencing.

[0195] Nucleic acid sequencing Some aspects of this disclosure are useful for sequencing biological polymers such as nucleic acids and proteins. In some embodiments, the methods, compositions, and devices described herein may be used to identify a series of nucleotides or amino acid monomers incorporated into nucleic acids or proteins (for example, by detecting the time course of incorporation of a series of labeled nucleotides or amino acid monomers). In some embodiments, the methods and devices described herein may be used to identify a series of nucleotides incorporated into a template-dependent nucleic acid sequencing reaction product synthesized by a polymerase enzyme.

[0196] In some embodiments, nucleic acid sequencing involves providing the nucleic acid of interest to be coated on the surface of a solid support (e.g., attached to the bottom surface of a sample well) via coupling moieties and / or coating molecules. In some embodiments, the coupling moiety-coating molecule conjugate is formed by a linkage between a biotin coupling moiety and a streptavidin coupling moiety coated on the surface of the well.

[0197] In some embodiments, a method for sequencing a target nucleic acid is provided. In some embodiments, the method for sequencing a target nucleic acid is a step of providing a mixture comprising (a) the target nucleic acid, (b) a primer complementary to the target nucleic acid, (c) a nucleic acid polymerase, and (d) nucleotides for incorporation into an elongated nucleic acid chain complementary to the target nucleic acid, wherein the nucleotides comprise different types of luminescently labeled nucleotides, the luminescently labeled nucleotides generate detectable signals during sequential incorporation into the elongated nucleic acid chain, and the detectable signals of the different types of luminescently labeled nucleotides are distinguishable from each other in the time domain (e.g., timing of the detectable signals and / or The method includes the steps of: (ii) subjecting the mixture of (i) to a polymerization reaction under conditions sufficient to generate the extended nucleic acid chain by the extension of the primers; (iii) measuring the detectable signal from the luminescently labeled nucleotides after sequential incorporation into the extended nucleic acid chain; and (iv) determining the timing and / or frequency of the measured detectable signal from the luminescently labeled nucleotides during sequential incorporation into the extended nucleic acid chain to identify a time series of incorporation of the luminescently labeled nucleotides into the extended nucleic acid chain, thereby determining the sequence of the target nucleic acid.

[0198] In some embodiments, a sequencing method is provided, comprising the steps of: (i) exposing a complex in a sample well of a target volume to one or more labeled nucleotides, the complex comprising a target nucleic acid or a plurality of nucleic acids present in the sample, at least one primer complementary to the target nucleic acid, and DNA polymerase; (ii) subjecting the complex to a polymerization reaction under conditions sufficient to generate an extended nucleic acid chain by extension of the primers; (iii) directing a series of pulses of excitation light to the target volume; (iv) detecting a plurality of photons emitted from one or more labeled nucleotides after the sequential incorporation of nucleotides into the extended nucleic acid containing the primers; and (v) identifying the sequence of the incorporated nucleotides by determining one or more properties of the emitted photons. These properties may be selected from luminescence lifetime, retention time, luminescence intensity, emission wavelength, pulse duration, and / or inter-pulse duration. In some embodiments, the property is luminescence lifetime.

[0199] In some embodiments, free nucleotides are conjugated to the same type of luminescent label such that the detectable signals for the luminescently labeled nucleotides are distinguishable from each other in the time domain (e.g., by determining the timing, intensity, and / or frequency of the detectable signals). In exemplary embodiments, free nucleotides are conjugated to the same type of label, and the difference in the intensity of the detectable signals is measured within the time domain. Nucleotides incorporated into an elongated nucleic acid chain complementary to the target nucleic acid by polymerase remain in the detection region for a longer duration than free nucleotides, and therefore their intensity is greater and / or longer than that of unincorporated nucleotides.

[0200] In some embodiments, free nucleotides (e.g., four types of nucleotides) are each conjugated to different types of luminescent labels such that the detectable signals for the luminescently labeled nucleotides are distinguishable from each other in the time domain (e.g., by determining the timing, intensity, and / or frequency of the detectable signals). Nucleotides incorporated into an elongated nucleic acid chain complementary to the target nucleic acid by polymerase remain in the detection region for a longer duration than free nucleotides, and therefore their intensity is greater and / or longer than that of unincorporated nucleotides.

[0201] Embodiments of chip regeneration methods provided herein are highly accurate and long read lengths, for example, with accuracy of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and / or about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 40 A single nucleic acid molecule can be sequenced with read lengths of 0 bp, 500 bp, 1000 bp, 2,500 bp, 5,000 bp, 6,000 bp, 7,000 bp, 7,500 bp, 7,750 bp, 8,000 bp, 8,500 bp, 9,000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or 100,000 bp or more. In exemplary embodiments, the disclosed method can sequence a single nucleic acid molecule with an accuracy of about 70%, about 72.5%, about 74%, about 75%, about 80%, about 85%, about 90%, or about 92%. In some embodiments, the disclosed method can sequence a single nucleic acid molecule with an accuracy of about 94%. In exemplary embodiments, the disclosed method can sequence a single nucleic acid molecule with a read length of approximately 8500 base pairs.

[0202] In some embodiments, the target nucleic acid or nucleic acid polymerase is attached to a support. In some embodiments, the incorporation time series is determined after subjecting the mixture of (i) to a polymerization reaction. In some embodiments, aspects of the present application may be used to assay a biological sample, for example, to determine the sequence of one or more nucleic acids or polypeptides in the sample, and / or to determine the presence or absence of variants of one or more nucleic acids or polypeptides (e.g., one or more mutations in a gene of interest) in the sample. In some embodiments, a patient sample (e.g., a human patient sample) may be tested to provide nucleic acid sequence information or to determine the presence or absence of one or more nucleic acids of interest for diagnostic, prognostic, and / or therapeutic purposes. In some examples, a diagnostic test may include sequencing nucleic acid molecules in a biological sample of interest, for example, by sequencing cell-free deoxyribonucleic acid (DNA) molecules and / or expression products (e.g., ribonucleic acid (RNA)) in the biological sample of interest.

[0203] In some embodiments, one or more analytes analyzed (e.g., investigated and / or identified) using luminescence lifetime and / or intensity may be labeled molecules (e.g., analytes labeled with one or more luminescent markers). In some embodiments, analytes containing one or more biomolecules may be identified using markers. In some examples, luminescent markers are used to identify individual subunits of biomolecules. Some embodiments use luminescent markers (also referred to herein as “markers”) which may be exogenous or endogenous markers. Exogenous markers may be external luminescent markers used as reporters and / or tags for luminescence labeling. Examples of exogenous markers include, but are not limited to, fluorescent molecules, fluorophores, fluorescent dyes, fluorescent stains, organic dyes, fluorescent proteins, species involved in fluorescence resonance energy transfer (FRET), enzymes, and / or quantum dots. Other exogenous markers are also known in the art. Such exogenous markers may be conjugated to probes or functional groups (e.g., molecules, ions, and / or ligands) that specifically bind to a particular target or component. By linking an exogenous tag or reporter to a probe, target identification becomes possible by detecting the presence of the exogenous tag or reporter. Examples of probes include protein, nucleic acid (e.g., DNA, RNA) molecules, lipids, and antibody probes. By combining exogenous markers with functional groups, any suitable probe, tag, and / or label used for detection can be formed, including molecular probes, labeled probes, hybridization probes, antibody probes, protein probes (e.g., biotin-conjugated probes), enzyme labels, fluorescent probes, fluorescent tags, and / or enzyme reporters.

[0204] In some embodiments, the luminescence label in a set of four nucleotides can be selected from dyes containing aromatic or heteroaromatic compounds, and may be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, phenanthidine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranylate, coumarin, fluorescein, rhodamine, or other similar compounds. Exemplary dyes include xanthene dyes, such as fluorescein, or rhodamine dyes, naphthalene dyes, coumarin dyes, acridine dyes, cyanine dyes, benzoxazole dyes, stilbene dyes, pyrene dyes, phthalocyanine dyes, phycobilin protein dyes, squaraine dyes, BODIPY dyes, etc.

[0205] In some embodiments, the luminescent label in a set of four nucleotides includes Alexa Fluor® 546, Cy® 3B, Alexa Fluor® 555, and Alexa Fluor® 555, as well as the FRET pair Alexa Fluor® 555 and Cy® 3.5. In some embodiments, the luminescent label in a set of four nucleotides includes Alexa Fluor® 555, Cy® 3.5, Alexa Fluor® 546, and DyLight® 554-R1. In some embodiments, the luminescent label in a set of four nucleotides includes Alexa Fluor® 555, Cy® 3.5, ATTO Rho6G, and DyLight® 554-R1. In some embodiments, the luminescent label in a set of four nucleotides includes Alexa Fluor® 555, Cy® 3B, ATTO Rho6G, and DyLight® 554-R1. In some embodiments, the luminescent label in a set of four nucleotides includes Alexa Fluor® 555, Cy® 3B, ATTO 542, and DyLight® 554-R1. In some embodiments, the luminescent label in a set of four nucleotides includes Alexa Fluor® 555, Cy® 3B, ATTO 542, and Alexa Fluor® 546. In some embodiments, the luminescent label in a set of four nucleotides includes Cy® 3.5, Cy® 3B, ATTO Rho6G, and DyLight® 554-R1.

[0206] In certain embodiments, at least one, at least two, at least three, or at least four types of luminescently labeled nucleotides are 6-TAMRA, 5 / 6-carboxyrhodamine 6G, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 610, Alexa Fluor® 647, Aberrior Star 635, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO Rho 6G, ATTO 542, ATTO 647N, ATTO Rho14, Chromis630, Chromis654A, Chromeo(trademark)642, CF(trademark)514, CF(trademark)532, CF(trademark)543, CF(trademark)546, CF(trademark)546, CF(trademark)555, CF(trademark)568, CF(trademark)633, CF(trademark)640R, CF(trademark)660C, CF(trademark)660R, CF(trademark)680R, Cy(registered trademark)3, Cy(registered trademark)3B, Cy(registered trademark)3.5, Cy(registered trademark)5, Cy(registered trademark)5.5, Dyomics-530, Dyomics-547P1, Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555, Dyomics-556, Dyomics-560, Dyomics-650, Dyomics-680, DyLight(registered trademark) 554-R1, DyLight(registered trademark) 530-R2, DyLight(registered trademark) 594, DyLight(registered trademark) 635-B2, DyLight(registered trademark) 650, DyLight(registered trademark) The luminescent dyes include those selected from the group consisting of (Registered Trademark) 655-B4, DyLight® 675-B2, DyLight® 675-B4, DyLight® 680, HiLyte® Fluor532, HiLyte® Fluor555, HiLyte® Fluor594, LightCycler® 640R, Seta® 555, Seta® 670, Seta® 700, Seta® u647, and Seta® u665, or luminescent dyes of formula (dye 101), (dye 102), (dye 103), (dye 104), (dye 105), or (dye 106) as described herein.

[0207] In some embodiments, at least one, at least two, at least three, or at least four types of luminescently labeled nucleotides each include a luminescent dye selected from the group consisting of Alexa Fluor® 546, Alexa Fluor® 555, Cy® 3B, Cy® 3.5, DyLight® 554-R1, Alexa Fluor® 546, Atto Rho6G, ATTO425, ATTO465, ATTO488, ATTO495, ATTO514, ATTO520, ATTO Rho6G, and ATTO542.

[0208] In some embodiments, the first type of luminescently labeled nucleotide comprises Alexa Fluor® 546, the second type of luminescently labeled nucleotide comprises Cy® 3B, the third type of luminescently labeled nucleotide comprises two Alexa Fluor® 555s, and the fourth type of luminescently labeled nucleotide comprises Alexa Fluor® 555 and Cy® 3.5.

[0209] In some embodiments, at least one, at least two, at least three, or at least four types of luminescently labeled nucleotides include a luminescent dye selected from the group consisting of Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 594, Alexa Fluor® 610, CF® 532, CF® 543, CF® 555, CF® 594, Cy® 3, DyLight® 530-R2, DyLight® 554-R1, DyLight® 590-R2, DyLight® 594, DyLight® 610-B1, or a luminescent dye of formula (dye 101), (dye 102), (dye 103), (dye 104), (dye 105), or (dye 106).

[0210] In some embodiments, the first and second types of luminescently labeled nucleotides include luminescent dyes selected from the group consisting of Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, CF® 532, CF® 543, CF® 555, Cy® 3, DyLight® 530-R2, and DyLight® 554-R1, while the third and fourth types of luminescently labeled nucleotides include luminescent dyes selected from the group consisting of Alexa Fluor® 594, Alexa Fluor® 610, CF® 594, DyLight® 590-R2, DyLight® 594, and DyLight® 610-B1, or luminescent dyes of formula (dye 101), (dye 102), (dye 103), (dye 104), (dye 105), or (dye 106).

[0211] In certain embodiments, at least one, at least two, at least three, or at least four of the luminescently labeled nucleotide molecules are TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomer Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, SuperfolderGFP, monomer Azami Green Green), TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomer Kusabira-Orange, mKOK, mKO2, mOrange, mOrange2, mRaspberry, mCherry mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red It contains a luminescent protein selected from the group consisting of Red), LSS-mKate (mKate)1, LSS-mKate (mKate)2, mBeRFP, PA-GFP, PAmCherry (PAmCherry)1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0212] Although this disclosure refers to luminescent markers, other types of markers may also be used with the devices, systems, and methods presented herein. Such markers may be mass tags, electrostatic tags, electrochemical labels, or any combination thereof.

[0213] Exogenous markers may be added to the sample, but endogenous markers may already be part of the sample. Endogenous markers may include any existing luminescent markers that can emit light or "autofluoresce" in the presence of excitation energy. The autofluorescence of endogenous fluorophores may provide label-free, non-invasive labeling that does not require the introduction of exogenous fluorophores. Examples of such endogenous fluorophores include, but are not limited to, hemoglobin, oxyhemoglobin, lipids, collagen and elastin crosslinks, reduced nicotinamide adenine dinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin, keratin, and / or porphyrins.

[0214] This disclosure presents a technique for detecting a single molecule using a set of luminescent tags (e.g., luminescent markers, luminescent labels) for labeling different molecules. Such a single molecule may be a nucleotide or amino acid with a tag. The tag may be detected when it is released from the single molecule while bound to it, or when it is bound to the single molecule and then released. In some cases, the tags are luminescent tags. Each luminescent tag in a selected set associates with each molecule. For example, a set of four tags may be used to "label" nucleic acid bases present in DNA—each tag in the set associates with a different nucleic acid base, for example, the first tag associates with adenine (A), the second tag associates with cytosine (C), the third tag associates with guanine (G), and the fourth tag associates with thymine (T). In addition, each luminescent tag in a set of tags has different properties that can be used to distinguish the first tag in the set from the other tags in the set. Thus, each tag can be uniquely identified using one or more of these distinguishing features. For example, and not limited to, tag features that could be used to distinguish one tag from another may include the emission energy and / or wavelength of light emitted by the tag in response to excitation energy, the wavelength of excitation light absorbed by a particular tag and which puts the tag into an excited state, and / or the emission lifetime of the tag.

[0215] In certain embodiments, the template-dependent nucleic acid sequencing product is carried out by a naturally occurring nucleic acid polymerase. In some embodiments, the polymerase is a mutant or modified variant of a naturally occurring polymerase. In some embodiments, the template-dependent nucleic acid sequencing product comprises one or more nucleotide segments complementary to the template nucleic acid chain. In one aspect, the disclosure provides a method for sequencing a template (or target) nucleic acid chain by sequencing the complementary nucleic acid chain.

[0216] As used herein, the term "polymerase" generally refers to any enzyme (or polymerase) capable of catalyzing polymerization reactions. Examples of polymerases include, but are not limited to, nucleic acid polymerases, transcriptases, or ligases. A polymerase may be a polymerase.

[0217] Embodiments relating to the elongation of single-molecule nucleic acids (e.g., for nucleic acid sequencing) may use any polymerase capable of synthesizing a nucleic acid complementary to the target nucleic acid molecule. In some embodiments, the polymerase may be DNA polymerase, RNA polymerase, reverse transcriptase, and / or one or more mutants or variants thereof.

[0218] Examples of polymerases include DNA polymerase, RNA polymerase, heat-stable polymerase, wild-type polymerase, modified polymerase, Escherichia coli (E. coli) DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase, φ29 (psi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase, and Platinum polymerase. Examples include, but are not limited to, Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragments, polymerases having 3'→5' exonuclease activity, and their variants, modified products, and derivatives. In some embodiments, the polymerase is a single-subunit polymerase. Non-limiting examples of DNA polymerases and their properties are described in detail, among other things, in *DNA Replication 2nd edition*, Kornberg and Baker, WH. Freeman, New York, NY (1991).

[0219] When a base pair is formed between the nucleic acid base of the target nucleic acid and a complementary dNTP, polymerase incorporates the dNTP into the newly synthesized nucleic acid chain by forming a phosphodiester bond between the 3' hydroxyl terminus of the newly synthesized chain and the alpha-phosphate of the dNTP. In examples where the luminescent tag conjugated to the dNTP is a fluorophore, its presence is signaled by excitation, and pulses of emission are detected during or after the integration process. In the case of a detection label conjugated to the terminal (gamma) phosphate of the dNTP, when the dNTP is incorporated into the newly synthesized chain, beta and gamma phosphates are released, and the detection label, which diffuses freely in the sample well, causes a decrease in the luminescence detected from the fluorophore.

[0220] In some embodiments, the polymerase is a highly processable polymerase. However, in some embodiments, the polymerase is a low processable polymerase. Processability of a polymerase generally refers to the polymerase's ability to sequentially incorporate dNTPs into a nucleic acid template without releasing the nucleic acid template. In some embodiments, the polymerase is a polymerase with low 5'→3' exonuclease activity and / or 3'→5' exonuclease (activity). In some embodiments, the polymerase is modified (e.g., by amino acid substitution) to have lower 5'→3' exonuclease activity and / or 3'→5' activity compared to the corresponding wild-type polymerase. Further non-limiting examples of DNA polymerases include 9°Nm(trademark) DNA polymerase (New England Biolabs) and the P680G mutant of Klenow exopolymerase (Tuske et al. (2000) JBC 275(31):23759-23768). In some embodiments, lower processability of the polymerase improves accuracy for sequencing templates containing one or more stretches of nucleotide repeats (e.g., two or more consecutive bases of the same type). In some embodiments, the polymerase has a higher affinity for labeled nucleotides than for unlabeled nucleic acids.

[0221] In other embodiments, the disclosure provides a method for sequencing a target nucleic acid by sequencing multiple nucleic acid fragments when the target nucleic acid comprises fragments. In certain embodiments, the method includes the step of combining multiple fragment sequences to provide the sequence or subsequence of the parent target nucleic acid. In some embodiments, the combining step is performed by computer hardware and software. The methods described herein may enable sequencing of a set of relevant target nucleic acids, such as an entire chromosome or genome.

[0222] During sequencing, polymerases may couple (e.g., ligate) to a priming site on the target nucleic acid molecule. The priming site may be a primer complementary to a portion of the target nucleic acid molecule. Alternatively, the priming site may be a gap or nick provided within a double-stranded segment of the target nucleic acid molecule. The gap or nick may be 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 nucleotides in length. The nick may provide a cleavage site within one strand of the double-stranded sequence (providing a priming site for polymerases, such as strand-displacement polymerase enzymes).

[0223] In some cases, sequencing primers can be annealed to target nucleic acid molecules, which may or may not be immobilized on a solid support. The solid support may, for example, have sample wells (e.g., nano-apertures, reaction chambers) on a microchip (or chip) used in nucleic acid sequencing. In some embodiments, sequencing primers can be immobilized on a solid support, but hybridization of the target nucleic acid molecule also immobilizes the target nucleic acid molecule on the solid support. In some embodiments, polymerase is immobilized on a solid support, and the soluble primer and target nucleic acid come into contact with the polymerase. However, in some embodiments, a complex containing polymerase, target nucleic acid, and primer is formed in solution, and the complex is immobilized on a solid support (e.g., via immobilization of polymerase, primer, and / or target nucleic acid).

[0224] Under appropriate conditions, a polymerase enzyme in contact with an annealed primer / target nucleic acid can add or incorporate one or more nucleotides into the primer, and several nucleotides can be added to the primer in a template-dependent manner, in the 5'→3' direction. Such incorporation of nucleotides into the primer (e.g., via the action of polymerase) can be commonly referred to as a primer extension reaction. Each nucleotide is detectable and identifiable during the nucleic acid extension reaction (e.g., based on its luminescence lifetime and / or other characteristics) and can associate with a detectable tag used to determine the sequence of each nucleotide incorporated into the extended primer, and thus the newly synthesized nucleic acid molecule. The sequence of the target nucleic acid molecule can also be determined via sequence complementarity of the newly synthesized nucleic acid molecule. In some cases, the annealing of the sequencing primer to the target nucleic acid molecule, and the incorporation of nucleotides into the sequencing primer, can occur under similar reaction conditions (e.g., the same or similar reaction temperatures) or different reaction conditions (e.g., different reaction temperatures).

[0225] Accordingly, in some embodiments, the Disclosure provides methods and compositions for determining nucleotide sequence information from nucleic acid biomolecules (e.g., for sequencing polynucleotides). In some embodiments, nucleotide sequence information can be determined for a single nucleic acid biomolecule. The sequencing methods of the Disclosure may include “synthetic sequencing” assay methods. The sequencing methods of the Disclosure may include time-course (or time-domain) measurement assay methods. In some embodiments, synthetic sequencing methods may include the steps of presenting a population of target nucleic acid molecules (e.g., copies of the target nucleic acid) and / or amplifying the target nucleic acid to realize the population of the target nucleic acid. However, in some embodiments, synthetic sequencing is used to determine the sequence of a single molecule in each reaction being evaluated (and nucleic acid amplification is not required in the preparation of the target template for sequencing). In some embodiments, according to some aspects of the Application, multiple single-molecule sequencing reactions are carried out in parallel (e.g., on a single chip). For example, in some embodiments, multiple single-molecule sequencing reactions are carried out in separate reaction chambers (e.g., nanoapertures, sample wells) fabricated on a single chip.

[0226] In some embodiments, the disclosed nucleic acid sequencing method is performed in sample wells fabricated on complementary metal oxide semiconductor (CMOS) chips. Each well may be aligned with a CMOS photodiode. In some embodiments, the disclosed nucleic acid sequencing method is performed on a CMOS chip in conjunction with a benchtop integrated device. In some embodiments, the sequencing method is performed on a CMOS chip in conjunction with a single-channel or multi-channel photodetector synthesis sequencing integrated device (see Figure 2A).

[0227] In some embodiments, the nucleic acid of the luminescently labeled nucleotide further comprises a third oligonucleotide chain that is annealed to at least one of the first and second oligonucleotide chains. In some embodiments, the nucleic acid further comprises a fourth oligonucleotide chain that is annealed to at least one of the first, second, and third oligonucleotide chains. In some embodiments, the oligonucleotide chains form a Holliday junction.

[0228] In some embodiments, the Disclosure provides a method for determining the sequence of a template nucleic acid. In some embodiments, the method includes a step of exposing a complex (the complex comprising a template nucleic acid, a primer, and a polymerase) in a target volume to several types of luminescently labeled nucleotides. In some embodiments, each type of luminescently labeled nucleotide contains one or more luminescent labels (e.g., containing the same luminescent label or different luminescent labels) linked to one or more nucleoside polyphosphates via the nucleic acid. In some embodiments, the nucleic acid includes a protective molecule. Thus, in some embodiments, the Disclosure provides a nucleic acid sequencing method utilizing any of the luminescently labeled nucleoside polyphosphate compositions described herein.

[0229] In some embodiments, the method further includes the step of directing a series of pulses of one or more excitation energies toward the vicinity of a target volume. In some embodiments, the method further includes the step of detecting a plurality of emitted photons from the luminescently labeled nucleotide during a period of sequential integration into the nucleic acid, including the primers. In some embodiments, the method further includes the step of identifying the sequence of the integrated nucleotides by determining the timing and, optionally, the frequency of the emitted photons.

[0230] In some embodiments, four different nucleotides in the reaction mixture (e.g., adenine, guanine, cytosine, and thymine / uracil) can each be labeled with one or more luminescent molecules (e.g., one or more luminescent labels). In some embodiments, each type of nucleotide can be bound to two or more identical luminescent molecules (e.g., two or more identical fluorescent dyes bound to the nucleotide). In some embodiments, each luminescent molecule can be bound to two or more nucleotides (e.g., two or more identical nucleotides). In some embodiments, two or more nucleotides can be bound to two or more luminescent molecules (e.g., via a nucleic acid linker containing one or more protective molecules). In some embodiments, all four nucleotides are labeled with luminescent molecules that absorb and emit within the same spectral range (e.g., 520–570 nm).

[0231] Some embodiments have the ability to sequence a single nucleic acid molecule with high accuracy and long read lengths, for example, accuracy of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and / or read lengths of about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or 100,000 bp or more. In some embodiments, the target nucleic acid molecule used in single-molecule sequencing is a single-stranded target nucleic acid (e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid (RNA), RNA derivatives) template that is added to or immobilized in a sample well (e.g., a nanoaperture) containing at least one additional component of the sequencing reaction (e.g., a polymerase such as DNA polymerase, sequencing primers) and immobilized or linked to a solid support, e.g., the bottom or sidewall of the sample well. The target nucleic acid molecule or polymerase can be linked directly or via a linker to the sample wall, e.g., the bottom or sidewall of the sample well. The sample well (e.g., nano-aperture) may contain any other reagents required for nucleic acid synthesis via primer extension reactions, such as suitable buffers, cofactors, enzymes (e.g., polymerase), and deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphodes (including deoxyadenosine triphodes (dATP), deoxycytidine triphodes (dCTP), deoxyguanosine triphodes (dGTP), deoxyuridine triphodes (dUTP), and deoxythymidine triphodes (dTTP)), including dNTPs, which may contain luminescent tags, such as fluorophores.In some embodiments, each class of dNTPs (e.g., adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs (e.g., dUTP), and thymine-containing dNTPs (e.g., dTTP)) is conjugated to different luminescent tags such that detection of light emitted from the tag indicates the identity of the dNTP incorporated into a newly synthesized nucleic acid. The light emitted from the fluorescent tag is detectable via any suitable device and / or method, including, for example, detection devices and methods as otherwise described herein, and is attributable to the corresponding fluorescent tag (and therefore the associated dNTP). The fluorescent tag may be conjugated to the dNTP at any position such that the presence of the fluorescent tag does not inhibit the incorporation of the dNTP into a newly synthesized nucleic acid chain or inhibit polymerase activity. In some embodiments, the fluorescent tag is conjugated to the terminal phosphate (e.g., gamma phosphate) of the dNTP.

[0232] In some embodiments, the single-stranded target nucleic acid template may come into contact with sequencing primers, dNTPs, polymerase, and other reagents necessary for nucleic acid synthesis. In some embodiments, all applicable dNTPs may come into contact with the single-stranded target nucleic acid template simultaneously (e.g., all dNTPs are present simultaneously) so that dNTP incorporation can occur sequentially. In other embodiments, the dNTPs may come into contact with the single-stranded target nucleic acid template sequentially, in which case the single-stranded target nucleic acid template comes into contact with each applicable dNTP individually, with washing steps provided between contacts of the single-stranded target nucleic acid template with different dNTPs. Such a cycle of contacting the single-stranded target nucleic acid template individually with each dNTP and then washing may be repeated for each consecutive base position of the single-stranded target nucleic acid template to be identified.

[0233] In some embodiments, a sequencing primer anneals to a single-stranded target nucleic acid template, and a polymerase sequentially incorporates dNTPs (or other deoxyribonucleoside polyphosphates) into the primer based on the single-stranded target nucleic acid template. A unique luminescent tag associated with each incorporated dNTP may be excited using suitable excitation light during or after the dNTPs are incorporated into the primer, and its emission may subsequently be detected using any suitable device(s) and / or method(s), including detection devices and methods described separately herein. By detecting specific emission of light (e.g., having a specific radiative (luminescence) lifetime, intensity, spectrum, and / or combination thereof), the detection can be attributed to the specific incorporated dNTP. Sequences obtained from the collection of detected luminescent tags may then be used to determine the sequence of the single-stranded target nucleic acid template via sequence complementarity.

[0234] While this disclosure refers to dNTPs, the devices, systems, and methods presented herein may be used with various types of nucleotides, such as ribonucleotides and deoxyribonucleotides (e.g., deoxyribonucleoside polyphosphates having at least 4, 5, 6, 7, 8, 9, or 10 phosphate groups). Such ribonucleotides and deoxyribonucleotides may include various types of tags (or markers) and linkers. In some embodiments, this disclosure provides methods and compositions that can be advantageously utilized in the techniques described in U.S. Patent Applications No. 14 / 543,865, 14 / 543,867, 14 / 543,888, 14 / 821,656, 14 / 821,686, 14 / 821,688, 15 / 161,067, 15 / 161,088, 15 / 161,125, 15 / 255,245, 15 / 255,303, 15 / 255,624, 15 / 261,697, and 15 / 261,724, each of which is incorporated herein by reference.

[0235] The signal emitted when a nucleotide is incorporated can be stored in memory and later processed to determine the sequence of the target nucleic acid template. This may involve comparing the signal to a reference signal to determine the identity of the incorporated nucleotide as a function of time. Alternatively or additionally, the signal emitted when a nucleotide is incorporated can be collected and processed in real time to determine the sequence of the target nucleic acid template in real time (e.g., at the time of nucleotide incorporation).

[0236] As used herein, the term “nucleic acid” generally refers to a molecule containing one or more nucleic acid subunits. A nucleic acid may contain one or more subunits selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or their variants. In some cases, a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a derivative thereof. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may be cyclic.

[0237] As used herein, the term “nucleotide” generally refers to a nucleic acid subunit (which may include A, C, G, T, or U, or their variants or analogs). A nucleotide may include any subunit that can be incorporated into a growing nucleic acid chain. Such a subunit may be A, C, G, T, or U, or specific to one or more complementary A, C, G, T, or U, or any other subunit complementary to a purine (e.g., A or G, or its variant or analog) or a pyrimidine (e.g., C, T, or U, or its variant or analog). A subunit can enable the solubility of individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or their uracil counterparts).

[0238] Nucleotides generally consist of a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide may contain a nucleic acid base, a pentose sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose. Nucleotides can be nucleoside monophosphates or nucleoside polyphosphates. The nucleotides may be deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphophosphates, which can be selected from deoxyadenosine triphophosphate (dATP), deoxycytidine triphophosphate (dCTP), deoxyguanosine triphophosphate (dGTP), deoxyuridine triphophosphate (dUTP), and deoxythymidine triphophosphate (dTTP) dNTPs, which include detectable tags, such as luminescent tags or markers (e.g., fluorophores).

[0239] A nucleoside polyphosphate may have "n" phosphate groups, where "n" is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. Examples of nucleoside polyphosphates include nucleoside diphosphates and nucleoside triphosphates. A nucleotide may be a nucleoside with a terminally labeled phosphate group, such as a terminally labeled nucleoside polyphosphate. Such labels may be luminescent (e.g., fluorescent or chemiluminescent), fluorescence-induced, colored, chromogenic, mass tag, electrostatic, or electrochemical. The label (or marker) can be linked to the terminal phosphate group via a linker. The linker may comprise, for example, at least one hydroxyl group, sulfhydryl group, amino group, or haloalkyl group, which are considered suitable for forming phosphate esters, thioesters, phosphoramides, or alkylphosphonate bonds at the terminal phosphate of natural or modified nucleotides. The linker may be cleavable, for example, with the assistance of a polymerase, to separate the label from the terminal phosphate. Examples of nucleotides and linkers are presented in U.S. Patent No. 7,041,812, which is incorporated herein by reference.

[0240] Nucleotides (e.g., nucleotide polyphosphates) may contain methylated nucleic acid bases. For example, a methylated nucleotide may be a nucleotide containing one or more methyl groups linked to a nucleic acid base (e.g., directly linked to the ring of the nucleic acid base, or linked to a substituent on the ring of the nucleic acid base). Exemplary methylated nucleic acid bases include 1-methylthymine, 1-methyluracil, 3-methyluracil, 3-methylcytosine, 5-methylcytosine, 1-methyladenine, 2-methyladenine, 7-methyladenine, N6-methyladenine, N6, N6-dimethyladenine, 1-methylguanine, 7-methylguanine, N2-methylguanine, and N2, N2-dimethylguanine.

[0241] The term "primer," as used herein, generally refers to a nucleic acid molecule (e.g., an oligonucleotide) that may contain a sequence comprising A, C, G, T, and / or U, or variants or analogs thereof. A primer may be DNA, RNA, PNA, or a synthetic oligonucleotide containing variants or analogs thereof. A primer may be designed so that its nucleotide sequence is complementary to the target strand, or it may contain a random nucleotide sequence. In some embodiments, a primer may include a tail (e.g., a poly-A tail, an index adapter, a molecular barcode, etc.). In some embodiments, the primers may include 5-15 bases, 10-20 bases, 15-25 bases, 20-30 bases, 25-35 bases, 30-40 bases, 35-45 bases, 40-50 bases, 45-55 bases, 50-60 bases, 55-65 bases, 60-70 bases, 65-75 bases, 70-80 bases, 75-85 bases, 80-90 bases, 85-95 bases, 90-100 bases, 95-105 bases, 100-150 bases, 125-175 bases, 150-200 bases, or more than 200 bases.

[0242] Peptide sequencing In some embodiments, aspects of the present disclosure can be used to assay biological samples, for example, to determine the identity or sequence of one or more polypeptides in a sample and / or to determine the presence or absence of one or more variants (e.g., one or more amino acid substitutions in the polypeptide of interest) in a sample. In some embodiments, the compounds described herein can be subjected to peptide sequencing (also called "polypeptide sequencing") by detecting single-molecule bond interactions during the peptide degradation process. In some embodiments, the peptide of interest is covalently or non-covalently linked to a surface.

[0243] As used herein, polypeptide sequencing refers to determining sequence information for a polypeptide. In some embodiments, this may involve determining the identity of each consecutive amino acid for some (or all) of the polypeptide. However, in some embodiments, this may involve evaluating the identity of a subset of amino acids within a polypeptide (and, for example, determining the relative position of one or more types of amino acids without determining the identity of each amino acid within the polypeptide). In some embodiments, amino acid content information can be obtained from the polypeptide without directly determining the relative positions of different types of amino acids within the polypeptide. Amino acid content alone may be used to infer the identity of the polypeptides present (for example, by comparing the amino acid content with a polypeptide information database and determining which polypeptides have the same amino acid content).

[0244] In some embodiments, the methods for sequencing peptides presented herein may be carried out by identifying one or more amino acids of a polypeptide. In some embodiments, one or more amino acids of a polypeptide (e.g., terminal and / or internal amino acids) are labeled (e.g., directly or indirectly, using a binder, e.g., an amino acid recognition molecule, etc.), and the relative positions of the labeled amino acids within the polypeptide are determined. In some embodiments, the relative positions of amino acids within the polypeptide are determined using a series of amino acid labeling steps. In certain embodiments, the relative positions of amino acids within the polypeptide are determined using a series of labeling and cleavage steps. However, in some embodiments, the relative positions of labeled amino acids within a polypeptide can be determined not by removing amino acids from the polypeptide, but rather by translocating the labeled polypeptide through a pore (e.g., a protein channel), and detecting a signal (e.g., a FRET signal) from the labeled amino acid(s) during the translocating period to determine the relative positions of the labeled amino acids within the polypeptide molecule.

[0245] In some embodiments, the identity of terminal amino acids (e.g., N-terminal or C-terminal amino acids) is evaluated, then the terminal amino acids are removed, and the identity of the next terminal amino acid is evaluated, and this process is repeated until multiple consecutive amino acids in the polypeptide are evaluated. In some embodiments, the step of evaluating amino acid identity includes determining the types of amino acids present. In some embodiments, the determination of amino acid types includes determining the actual amino acid identity by determining, for example, which of the 20 naturally occurring amino acids are the terminal amino acids (e.g., using a binder that is specific to each terminal amino acid). In some embodiments, the types of amino acids are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.

[0246] However, in some embodiments, the step of evaluating the identity of terminal amino acid types may include the step of determining a subset of potential amino acids that may be present at the end of the polypeptide. In some embodiments, this can be achieved by determining that the amino acid is not one or more specific amino acids (and therefore could be any of the other amino acids). In some embodiments, this can be achieved by determining which of a particular subset of amino acids (e.g., based on size, charge, hydrophobicity, post-translational modification, binding properties) can be located at the end of the polypeptide (e.g., using a binder that binds to a particular subset of two or more terminal amino acids). In some embodiments, the step of evaluating the identity of terminal amino acid types may include the step of determining that the amino acid has post-translational modifications.

[0247] In some embodiments, the methods presented herein include contacting a polypeptide with a labeled amino acid recognition molecule that selectively binds to one type of terminal amino acid. In some embodiments, the labeled recognition molecule selectively binds to one type of terminal amino acid over other types of terminal amino acids. In some embodiments, the labeled recognition molecule selectively binds to one type of terminal amino acid over the same type of internal amino acids. In still other embodiments, the labeled recognition molecule selectively binds to one type of amino acid, such as an amino acid of the same type as terminal and internal amino acids, at any position of the polypeptide.

[0248] In some embodiments, polypeptide sequencing can proceed by contacting a polypeptide that associates with one or more terminal amino acids with one or more amino acid recognition molecules. In some embodiments, the labeled amino acid recognition molecule interacts with the polypeptide by associating with the terminal amino acid.

[0249] In some embodiments, one or more types of amino acids are identified by detecting one or more electrical features of a labeled recognition molecule. In some embodiments, the labeled recognition molecule includes a recognition molecule that selectively binds to one type of amino acid, and a conductivity label that associates with the recognition molecule. Thus, one or more electrical features (e.g., charge, current oscillation color, and other electrical features) may be associated with the selective binding of the recognition molecule to identify amino acids in a polypeptide. In some embodiments, multiple types of labeled recognition molecules may be used in the method according to this application, in which case each type includes a conductivity label that produces a change in electrical signal (e.g., a change in conductivity, e.g., a change in conductivity intensity or a characteristic pattern of conductivity transitions) that can be uniquely identified from among multiple types. In some embodiments, each of the multiple types of labeled recognition molecules includes a conductivity label having a different number of charged groups (e.g., a different number of load groups and / or positive charge groups). Thus, in some embodiments, the conductivity label is a charge label. Examples of charge labels include dendrimers, nanoparticles, nucleic acids, and other polymers having multiple charged groups. In some embodiments, the conductivity label can be uniquely identified by its net charge (e.g., net positive charge or net negative charge), its charge density, and / or the number of its charge groups.

[0250] Thus, in some embodiments, one or more types of amino acids are identified by detecting the luminescence lifetime or luminescence intensity of one or more labeled amino acid recognition molecules that selectively bind to one or more types of amino acids. In some embodiments, the methods presented herein include contacting a polypeptide with one or more labeled amino acid recognition molecules (or recognition molecules) that selectively bind to one or more types of terminal amino acids. As an illustrative and non-limiting example, if four labeled recognition molecules are used in the methods of the present application, any one reagent selectively binds to one type of terminal amino acid (different from another type of amino acid to which any of the other three reagents selectively binds) (e.g., the first reagent binds to the first type of terminal amino acid, the second reagent binds to the second type of terminal amino acid, the third reagent binds to the third type of terminal amino acid, and the fourth reagent binds to the fourth type of terminal amino acid).

[0251] In some embodiments, the recognition molecules can be engineered by one of ordinary skill in the art while using known techniques. In some embodiments, desirable properties can include the ability to have high affinity and selectively bind thereto only when one type of amino acid is located at the end (e.g., N-terminus or C-terminus) of the polypeptide. In still other embodiments, desirable properties can include the ability to have high affinity and selectively bind thereto when one type of amino acid is located at the end (e.g., N-terminus or C-terminus) of the polypeptide as well as when it is located at an internal position of the polypeptide. In some embodiments, desirable properties include the ability to selectively bind to two or more types of amino acids with low affinity (e.g., having a K D of, for example, greater than or equal to about 50 nM, for example, between about 50 nM and about 50 μM, between about 100 nM and about 10 μM, between about 500 nM and about 50 μM). For example, in some aspects, the present disclosure presents methods of sequencing by detecting reversible binding interactions. Advantageously, such methods can be carried out using recognition molecules that reversibly bind to two or more types of amino acids (e.g., a subset of amino acid types) with low affinity.

[0252] In some embodiments, polypeptide sequencing involves providing the polypeptide of interest coated on a surface body of a solid support (e.g., linked to the bottom surface of a sample well) via coupling moieties and / or coating molecules. In some embodiments, the coupling moiety-coating molecule conjugate is formed by a linkage between a biotin coupling moiety and a streptavidin coupling moiety coated on the surface of the well.

[0253] In some embodiments, the target polypeptide is immobilized on a surface at one end via a coupling moiety, while the other end is free to allow for detection and cleavage of the terminal amino acid during the sequencing reaction. Therefore, in some embodiments, the reagent used in a particular polypeptide sequencing reaction preferentially interacts with the terminal amino acid at the unimmobilized (e.g., free) end of the polypeptide. Thus, the polypeptide remains immobilized throughout repeated detection and cleavage cycles.

[0254] In some embodiments, polypeptide sequencing can be carried out by contacting the polypeptide with one or more amino acid recognition molecules that associate with one or more terminal amino acids. In some embodiments, the labeled amino acid recognition molecules interact with the polypeptide by associating with the terminal amino acids.

[0255] In some embodiments, the method further includes the step of identifying amino acids (terminal or internal amino acids) of a polypeptide by detecting a labeled amino acid recognition molecule. In some embodiments, the detection step includes the step of detecting luminescence emitted from the labeled amino acid recognition molecule. In some embodiments, the luminescence is uniquely associated with the labeled amino acid recognition molecule, thereby the luminescence is associated with the type of amino acid to which the labeled amino acid recognition molecule selectively binds. Thus, in some embodiments, the type of amino acid is identified by determining one or more luminescence properties of the labeled amino acid recognition molecule.

[0256] In other embodiments, polypeptide sequencing proceeds by removing terminal amino acids by contacting the polypeptide with an exopeptidase (which binds to and cleaves the terminal amino acids of the polypeptide). Once the terminal amino acids are removed by the exopeptidase, polypeptide sequencing proceeds by subjecting the polypeptide (having n-1 amino acids) to an additional cycle of terminal amino acid recognition and cleavage. In some embodiments, these steps may occur in the same reaction mixture as in the case of a dynamic peptide sequencing reaction, for example. In some embodiments, these steps may be carried out using other methods known in the art, such as peptide sequencing by Edman degradation.

[0257] In some embodiments, dynamic polypeptide sequencing is performed in real time by evaluating the binding interactions between terminal amino acids, labeled amino acid recognition molecules, and optionally cleavage reagents (e.g., exopeptidases). The labeled amino acid recognition molecules may associate (e.g., bind) with and dissociate from terminal amino acids, thereby eliciting a series of pulses in a signal output that can be used to identify the terminal amino acid. In some embodiments, the series of pulses provides a pulsing pattern (e.g., a characteristic pattern) that can be useful in diagnosing the identity of the corresponding terminal amino acid.

[0258] Edman degradation involves a repetitive cycle of modifying and cleaving the terminal amino acids of a polypeptide, with each sequentially cleaved amino acid being identified and the amino acid sequence of the polypeptide determined. Conventional Edman degradation peptide sequencing can be carried out by (1) contacting the target polypeptide with one or more amino acid recognition molecules that selectively bind to one or more terminal amino acids. In some embodiments, step (1) further includes removing one or more labeled amino acid recognition molecules that do not selectively bind to the polypeptide.

[0259] In some embodiments, dynamic peptide sequencing is performed by observing different association events, such as association events between an amino acid recognition molecule and an amino acid located at the terminal end of a peptide, in which case each association event generates a signal, such as a change in the intensity of a luminescence signal (which persists for a certain period of time).

[0260] In some embodiments, observing different association events, such as association events between an amino acid recognition molecule and an amino acid located at the terminal end of a peptide, is possible during the peptide degradation process. In some embodiments, a transition from one characteristic signal pattern to another indicates amino acid cleavage (e.g., amino acid cleavage resulting from peptide degradation). In some embodiments, amino acid cleavage refers to the removal of at least one amino acid from the terminal end of a polypeptide (e.g., removal of at least one terminal amino acid from a polypeptide). In some embodiments, amino acid cleavage is determined by estimation based on the duration between characteristic signal patterns. In some embodiments, amino acid cleavage is determined by detecting a change in signal generated by the association of a labeled cleavage reagent with an amino acid located at the terminal end of a polypeptide. In some embodiments, when a cleavage reagent (exopeptidase) is used, amino acids are sequentially cleaved from the terminal end of the polypeptide during the degradation process, so a series of intensity changes or a series of signal pulses are detected.

[0261] In some embodiments, signal pulse information may be used to identify amino acids based on characteristic patterns within a series of signal pulses. In some embodiments, the characteristic pattern comprises multiple signal pulses, each containing a pulse duration. In some embodiments, the multiple signal pulses may be characterized by summary statistics (e.g., mean, median, time decay constant) relating to the distribution of pulse durations in the characteristic pattern. In some embodiments, the mean pulse duration of the characteristic pattern is between approximately 1 millisecond and approximately 10 seconds (e.g., between approximately 1 ms and 1 second, between approximately 1 ms and 100 ms, between approximately 1 ms and 10 ms, between approximately 10 ms and 10 seconds, between approximately 100 ms and 10 seconds, between approximately 1 second and 10 seconds, between approximately 10 ms and 100 ms, or between approximately 100 ms and 500 ms). In some embodiments, different characteristic patterns corresponding to different types of amino acids within a single polypeptide may be distinguished from each other based on statistical significance differences in the summary statistics. For example, in some embodiments, one characteristic pattern may be distinguishable from another characteristic pattern based on a difference of at least 10 milliseconds in average pulse duration (e.g., between approximately 10 ms and 10 seconds, between approximately 10 ms and 1 second, between approximately 10 ms and 100 ms, between approximately 100 ms and 10 seconds, between approximately 1 second and 10 seconds, or between approximately 100 ms and 1 second). In some embodiments, to distinguish one pattern from another with statistical confidence, it should be recognized that the smaller the difference in average pulse duration between different characteristic patterns, the longer the pulse duration may be required for each characteristic pattern.

[0262] In some embodiments, the disclosure presents a method for immobilizing a peptide or amino acid recognition molecule on a surface by linking any one of the compounds described herein to the surface of a sample well. In some embodiments, the surface is functionalized using a coupling portion configured to link (e.g., covalently) to the functionalized end of the peptide. In some embodiments, the method includes the step of immobilizing a single peptide on the surface of each of a plurality of sample wells. In some embodiments, encapsulating a single peptide in each sample well is advantageous for single-molecule detection methods, such as single-molecule peptide sequencing.

[0263] In some embodiments, the peptide containing the functionalized terminus is in contact with a complementary coupling moiety. In some embodiments, the functionalized terminus and coupling moiety include a non-covalent binding partner (e.g., forming a non-covalent linkage between the peptide and the sample well surface). Examples of non-covalent binding partners include protein-protein binding partners (e.g., barnase and barstar) and protein-ligand binding partners (e.g., biotin and streptavidin).

[0264] Sequencing using luminescence lifetime measurement Individual pixels on an integrated photodetector can enable emission lifetime measurements used to identify fluorophores and / or reporters that label one or more targets, such as molecules or specific locations on molecules. While the following description discusses sample identification based on lifetime measurements, it should be understood that one or more additional or alternative characteristics of the signal emitted from the reaction chamber in response to excitation light may be used to identify the sample. For example, any of the techniques described in U.S. Patent Application No. 17 / 190,331, “Integrated Sensor for Multi-Dimensional Signal Analysis,” filed on 2 March 2021 under Agent Reference Number R0708.70090US01, which is incorporated in its entirety in this specification, can be used.

[0265] Any one or more molecules of interest, including proteins, amino acids, enzymes, lipids, nucleotides, DNA, and RNA, can be labeled with fluorophores. When combined with the detection of the emitted light spectrum or other labeling techniques, luminescence lifetime can increase the total number of fluorophores and / or reporters that can be used. Lifetime-based identification can be used in single-molecule analysis methods to provide information about the characteristics of molecular interactions in complex mixtures, which may include protein-protein interactions, enzyme activity, molecular dynamics, and / or diffusion on membranes. Furthermore, fluorophores with different luminescence lifetimes can be used to tag target components in various assay methods based on the presence of labeled components. In some embodiments, components can be separated, for example, by using a microfluidic system, based on the detection of a specific lifetime of a fluorophore.

[0266] Emission lifetime measurement can be used in combination with other analytical methods. For example, luminescence lifetime can be used in combination with fluorescence resonance energy transfer (FRET) techniques to identify the state and / or environment of donor and acceptor fluorophores located on one or more molecules. Such measurements can be used to determine the distance between donors and acceptors. In some cases, energy transfer from donor to acceptor may reduce the donor lifetime. In another example, luminescence lifetime measurement can be used in DNA sequencing applications, such as when four fluorophores with different lifetimes can be used to label four different nucleotides (A, T, G, C) in a DNA molecule with an unknown nucleotide sequence. Emission lifetime can be used instead of the emission spectrum of the fluorophores to identify the nucleotide sequence. By using luminescence lifetime instead of emission spectrum for certain techniques, artifacts from absolute intensity measurements are reduced, thus improving accuracy and measurement resolution. Furthermore, lifetime measurement can reduce system complexity and / or cost because it requires lower excitation energy wavelengths and / or detection of lower emission energy wavelengths.

[0267] In some embodiments, the integrated photodetectors described herein can measure or identify luminescence lifetime. Measuring luminescence lifetime is based on exciting one or more fluorescent molecules and measuring the time evolution of the emitted light. The probability that a fluorescent molecule will emit a photon after it has reached an excited state decreases exponentially over time. The rate at which this probability decreases may be characteristic of the fluorescent molecule and may differ between different fluorescent molecules. By detecting the time characteristics of the light emitted by a fluorescent molecule, it may be possible to identify the fluorescent molecule and / or distinguish fluorescent molecules from one another. The luminescent molecule is also referred to herein as a luminescent marker (or simply “marker”), label, and fluorophore.

[0268] After reaching the excited state, the marker can emit photons with a certain probability at a given time. The probability of photon emission from an excited marker may decrease over time after the marker is excited. The decrease in the probability of photon emission over time can be represented by an exponential decay function p(t)=e -t / τ where p(t) is the probability of photon emission at time t and τ is the time parameter of the marker. The time parameter τ indicates the time after excitation at which the probability that the marker emits photons becomes a constant value. The time parameter τ is a characteristic of the marker that may be different from its absorption and emission spectral characteristics. Such a time parameter τ is called the luminescence lifetime of the marker, or simply the "lifetime". In some embodiments, the marker may have a luminescence lifetime in the range of 0.1 to 20 ns. However, the techniques described herein are not limited with respect to the lifetime of the marker used.

[0269] The lifetime of the marker may be used to distinguish more than one marker and / or to identify the marker. In some embodiments, measurements of the luminescence lifetimes of multiple markers having different lifetimes excited by an excitation source may be performed. As an example, four markers having lifetimes of 0.5, 1, 2, and 3 nanoseconds, respectively, may be excited by a light source that emits light having a selected wavelength (e.g., 635 nm as an example). The markers may be identified or distinguished from each other based on measurements of the lifetimes of the light emitted by the markers.

[0270] Measurements of the luminescence lifetime can use relative intensity measurements by comparing how the intensity changes over time, in contrast to absolute intensity values. As a result, measurements of the luminescence lifetime can avoid some of the difficulties of absolute intensity measurements. Absolute intensity measurements may depend on the concentration of the fluorophore present and may require a calibration step for various fluorophore concentrations. In contrast, measurements of the luminescence lifetime may be insensitive to the concentration of the fluorophore.

[0271] When distinguishing markers by lifetime measurement, it may be possible to use fewer wavelengths of excitation light than when distinguishing markers by measuring the emission spectrum. In some embodiments, when using excitation light and / or luminescent light of fewer wavelengths, the number of sensors, filters, and / or diffractive optical elements can be reduced or eliminated. In some embodiments, labeling can be performed using markers having different lifetimes, and the markers can be excited by light having the same excitation wavelength or spectrum. In some embodiments, an excitation light source that emits light of a single wavelength or spectrum may be used, thereby reducing costs. However, since any number of excitation light wavelengths or spectra can be used, the techniques described herein are not limited in this regard. In some embodiments, an integrated photodetector can be used to determine both spectral and temporal information about the received light. In some embodiments, quantitative analysis of the types of molecules present can be performed by determining the temporal parameters, spectral parameters, or a combination of temporal and spectral parameters of the light emitted from the markers.

[0272] An integrated device having an integrated photodetector according to an aspect of the present application can be designed with functions suitable for various detection and imaging applications. Such an integrated photodetector can have the ability to detect light within one or more time intervals, or "time bins". To collect information about the arrival time of light, charge carriers are generated in response to incident photons and can be separated into respective time bins based on their arrival times.

[0273] Integrated photodetectors that detect the arrival time of incident photons can reduce the requirement for additional optical filtering (e.g., optical spectral filtering). As described below, the integrated photodetector according to this application may include a drain for removing photogenerating carriers at a specific time point. By removing the photogenerating carriers in this manner, unwanted charge carriers generated in response to the excitation light pulse can be discarded without requiring optical filtering to prevent reception of light from the excitation pulse. Such photodetectors can reduce the complexity of the overall design integration, optical and / or filtering components, and / or cost.

[0274] In some embodiments, the emission lifetime may be determined by measuring the time profile of the emitted emission by assembling charge carriers collected in one or more time bins of an integrated photodetector to detect emission intensity values ​​as a function of time. In some embodiments, the lifetime of a marker may be determined by performing a series of measurements in which the time it takes for the marker to be excited and then for a photon to be emitted is measured. For each measurement, the excitation source may generate a pulse of excitation light directed to the marker, and the time between the excitation pulse and the subsequent photon event from the marker can be determined. Additionally or alternatively, if the excitation pulses occur repeatedly and periodically, the time between the occurrence of a photon emission event and a subsequent excitation pulse can be measured, and the time of the photon absorption event can be determined by subtracting the measured time from the time interval between excitation pulses (i.e., the period of the excitation pulse waveform).

[0275] By repeating such experiments with multiple excitation pulses, the number of instances in which photons are emitted from a marker within a specific time interval after excitation can be determined, which represents the probability of photon emission within such a time interval after excitation. The number of collected photon emission events can be obtained based on the number of excitation pulses emitted to the marker. The number of photon emission events over the measurement period may range from 50 to 10,000,000 or more in some embodiments, but the techniques described herein are not limited to this. The number of instances in which photons are emitted from a marker within a specific time interval after excitation can be reflected in a histogram representing the number of photon emission events occurring within a series of discrete time intervals or time bins. The number of time bins and / or the time interval of each bin can be set and / or adjusted to identify a specific lifetime and / or a specific marker. The number of time bins and / or the time interval of each bin may depend on the sensor used to detect the emitted photons. The number of time bins may be 1, 2, 3, 4, 5, 6, 7, 8, or more, for example, 16, 32, 64, or more. Using a curve fitting algorithm, the curve can be fitted to the recorded histogram, resulting in a function that represents the probability of a photon being emitted after the excitation of a marker at a given time. The above decay function p(t)=e -t / τ Using this method, histogram data can be approximately fitted. From such curve fitting, time parameters or lifetimes can be determined. By comparing the determined lifetimes with the known lifetimes of the markers, the types of markers present can be identified.

[0276] The lifetime can be calculated from intensity values ​​acquired at two time intervals. In some embodiments, the photodetector measures intensity over at least two time bins. Photons emitting luminescence energy between times t1 and t2 are measured by the photodetector as intensity I1, and luminescence energy emitted between times t3 and t4 is measured as I2. Any suitable number of intensity values ​​can be acquired. Such intensity measurements can then be used to calculate the lifetime. If only one fluorophore is present at a time, the time-binned emission signal can be fitted to a single exponential decay. In some embodiments, only two time bins may be required to accurately identify the lifetime for a fluorophore. If more than one fluorophore is present, the individual lifetimes can be identified from the combined emission signal by fitting the emission signal to multiple exponential decays, such as a double or triple exponential decay. In some embodiments, more than two time bins may be required to accurately identify more than one emission lifetime from such a signal. However, in some cases using multiple fluorophores, the average emission lifetime can be determined by fitting a single exponential decay to the emission signal.

[0277] In some cases, the probability of a photon emission event, and therefore the lifetime of a marker, can vary depending on the surrounding environment and / or the state of the marker. For example, the lifetime of a marker confined within a volume having a diameter smaller than the wavelength of the excitation light may be shorter than that of a marker not confined within that volume. Lifetime measurements can be performed using known markers under similar conditions to those when the marker is used for labeling. The lifetime determined from such measurements using known markers can be used when identifying the marker. [Examples]

[0278] An on-chip evaluation of sequencing accuracy was performed after implementing an exemplary method of chip regeneration for DNA sequencing applications as described in this disclosure. Aliquots of a sample containing a known DNA molecule of interest were loaded onto an array of sample wells on a CMOS chip. This evaluation involved three sequential runs of terminal DNA sequencing performance on the sample aliquots: the first run was performed on a new, clean chip; the second run was performed after applying the exemplary regeneration solution of this disclosure; and the third run was performed after a second application of the regeneration solution to the chip. Therefore, the results of the third run represented an evaluation of the second regeneration step. The regeneration solution used in this experiment contained 9 mL of HFIP, 0.5 mL of 5 M ammonium acetate, 1 mL of water, and biotin-PEG3 (bis-biotin-PEG4) at a concentration between 100 μM and 200 μM.

[0279] The workup for the regeneration process in this experiment is as follows: After the first sequencing run, the residue was removed from the chip, and then the nucleoside digestion mix (NEB#M0649) was applied to the chip. The chip was then incubated at 37°C for 40 minutes. Subsequently, the regeneration solution was applied to the chip, and the chip was incubated at room temperature for a further 30 minutes. Finally, the residue was removed from the chip again.

[0280] Applying the regeneration solution before the second and third runs appeared to successfully dissociate the streptavidin-biotin interaction on the surface of the sample wells. The read length and accuracy of the nucleotide sequencing for each of the three runs were measured and plotted in the graphs shown in Figures 7A and 7B. As shown in Figure 7B, an average accuracy of approximately 74% and a maximum (or "best") accuracy of over 94% were observed in the second and third runs. Surprisingly, the second and third runs showed comparable sequencing performance to the first run. Therefore, in this experiment, chip regeneration and reuse may have improved readouts. Figure 7A shows the average read length between signals obtained from the three runs, which includes a read length of approximately 8500 bp during the third run. Figure 7B also shows the "alignment score" for each sequence readout against the known sequence of the molecule.

[0281] Next, an on-chip evaluation of sequencing accuracy was performed after implementing an exemplary method of chip regeneration in peptide sequencing applications according to this disclosure. Aliquots of a sample containing the known peptide molecule of interest were loaded onto an array of sample wells on a CMOS chip. This evaluation involved eight sequential runs of the sample aliquots. The first run was performed on a new, clean chip. Each subsequent run was performed after applying the regeneration solution as described above. In this experiment, the presence of the peptide of interest was detected using a labeled amino acid recognition molecule bound to terminal amino acids. The luminescence of the labeled recognition molecule generates a series of pulses in the signal output. The series of pulses provides a characteristic pulse pattern that diagnoses the identity of the corresponding terminal amino acids. This experiment was repeated using two different chips, S42906-16-14 and S42906-16-19.

[0282] The workup for the regeneration process in this experiment is as follows: After the first sequencing run, the residue is removed from the chip, then the regeneration solution is applied to the chip, and the chip is incubated at room temperature for 30 minutes. Finally, the residue is removed from the chip again.

[0283] As demonstrated in the signal output results shown in Figures 8A to 8H, the accuracy and read length results for peptide molecules for runs after the first run exceeded expectations. The percentage of loaded wells for each run is plotted in Figure 8A. The loading percentage for runs 5 to 8 exceeded 25%. The percentage of actively loaded and pulsed sample wells (activity %) out of the total number of wells on the chip is plotted in Figure 8B. This figure shows that between approximately 2.4% and 17.5% of the sample showed active pulse between runs 2 and 8. The frequency of activity % between runs 5 and 8 was approximately 15%.

[0284] The accuracy of the sequencing results for read lengths 1, 2, and 3 (R1, R2, and R3) is plotted in Figures 8C–8E for each of the eight runs. The y-axis reflects the count of sample wells (apertures) providing sequencing readouts for the plotted read lengths. These counts decrease as the read length progresses from 1 to 3. Figure 8E shows the median signal-to-noise ratio (SNR) across each of the eight runs. Figure 8G shows the average cutting depth across all read lengths. Figure 8H shows the percentage of active wells in the chip for each run with read lengths greater than 3.

[0285] The results of both experiments show that the regeneration process did not produce significant interference resulting from signals emitted from previously loaded aliquots when sampling later loaded aliquots, thus presenting a viable solution for removing the sample from the tip.

[0286] VIII. Calibration of integrated devices using photobleaching information Embodiments of the techniques described herein further provide techniques for calibrating an integrated device and / or one or more components interacting with an integrated device using photobleaching information obtained from a reference dye. For example, the integrated device may be part of a system further comprising at least one excitation source. The calibration techniques described herein may be used to calibrate a system comprising an integrated device. Figure 9 shows an exemplary process 900 for calibrating a system comprising an integrated device using photobleaching information, according to some embodiments.

[0287] In operation 902, the sample molecule may be labeled with a reference dye. For example, the reference dye may include a fluorescent molecule that emits light in response to excitation by light from at least one light source. The reference dye molecule may be of a type that binds to a specific sample molecule such that the properties of the light emitted by the reference dye molecule can be used to identify the sample molecule to which the reference dye molecule is bound.

[0288] In operation 904, a dye-labeled sample is loaded into one or more reaction chambers. For example, in some embodiments, multiple reference dyes of the same type are loaded into multiple reaction chambers to evaluate the difference in signals received from multiple reaction chambers. In some embodiments, the aspects of the technique described herein are not limited thereto, and the sample molecules may be labeled with the reference dye after the sample molecules have been loaded into multiple reaction chambers.

[0289] Figure 10-1 shows a sample molecule 1009 labeled with a reference dye molecule 1010 attached to a surface 1006, according to one embodiment. Figure 10-2 illustrates a dye-labeled sample attached inside a reaction chamber, according to one embodiment. In particular, in Figure 10-2, a sample molecule 1009 consisting of an amino acid chain (phenylalanine (F), tryptophan (W), tyrosine (Y), leucine (L), and serine (S)) is loaded into the reaction chamber 1008. The sample molecule 1009 is labeled with a reference dye molecule 1010. A CMOS chip 1014 may be provided, including a photonic component 1012 for collecting the signal emitted by the reference dye molecule 1010, as described herein with reference to the overview of the integrated device.

[0290] Operation 904 can be performed while one or more reaction chambers are not irradiated with excitation light. It should be understood that in some embodiments, sample molecules labeled with multiple dyes may be loaded into multiple reaction chambers during operation 904.

[0291] In some embodiments, the sample molecule having a reference dye may be loaded into the reaction chamber alone, and the molecule to be sequenced may be loaded into the reaction chamber afterward. In some embodiments, the molecule having a reference dye may be loaded into the reaction chamber together with the molecule to be sequenced after a calibration process has been performed. In some embodiments, the reference dye may be bound to the molecule to be sequenced itself, or the reference dye may be cleaved before sequencing. In some embodiments, the reference dye may be a free-diffusing dye molecule that does not need to be attached or otherwise immobilized in the reaction chamber. In some embodiments, the reference dye may be loaded into the reaction chamber as part of the surface chemistry preparation of the chip. Therefore, embodiments of this technique are not limited to any particular configuration of the reference dye in the reaction chamber. According to some embodiments, operation 902 may include the techniques described in U.S. Patent Application No. 17 / 082,906, “METHODS OF PREPARING SAMPLES FOR MULTIPLEX POLYPEPTIDE SEQUENCING,” filed on 28 October 2019 (Agent Reference Number R0708.70077US01), which is incorporated by reference in its entirety.

[0292] In operation 906, one or more reaction chambers are irradiated with excitation light from a light source (e.g., a laser). In particular, in operation 906, one or more reaction chambers may be periodically irradiated with excitation light (e.g., by unblocking the light source, reconnecting the light source to the chip, and / or increasing the output supplied by the light source in a time fast relative to the characteristic bleaching time of the system). For example, Figure 11 shows an exemplary graph 1100 showing the light source excitation output over time according to some embodiments. In some embodiments, the excitation light may be irradiated onto the reaction chambers until the reference dye is bleached. In some embodiments, the irradiation of the reaction chambers with excitation light may be controlled to avoid bleaching of the reference dye molecule. A further description of photobleached reference dye molecules is provided herein.

[0293] In operation 908, a time trace is recorded showing the fading step for the reaction chamber containing one or more sample molecules irradiated in operation 906. For example, Figure 12-1 shows an exemplary graph 1200 showing the signal measured over time from a reference dye, according to one embodiment. As shown in Figure 12-1, the measured signal increases in intensity and then decreases after a certain period. The period between the increase and decrease in the intensity of the measured signal may correspond to the fading time of the reference dye molecule.

[0294] In operation 910, it is determined whether there are any additional excitation pathways to be excited. If it is determined in operation 910 that there are additional excitation pathways to be excited, process 900 returns to operation 906 via the "yes" branch and irradiates an additional reaction chamber via the additional excitation pathway. If it is determined in operation 910 that there are no additional excitation pathways, process 900 may proceed to operation 912 via the "no" branch.

[0295] In operation 912, reference metrics may be stored. For example, the reference metrics may include a time trace showing the fading steps acquired in operation 908. For example, Figure 12-2 shows exemplary histograms of measured fading time and dye intensity for the collection of a single molecule in a reaction chamber according to some embodiments. Graph 1902 shows a histogram of measured fading time for the collection of a single molecule reference dye loaded in the reaction chamber of the integrated device. As shown in Figure 12-2, the median fading time for the collection of a single molecule reference dye is 5.8 seconds. Graph 1904 shows a histogram of measured reference dye signal intensity for the collection of a single molecule reference dye loaded in the reaction chamber of the integrated device. In some embodiments, the fading time may be determined at least in part based on the measured signal intensity. For example, the measured signal intensity may show a peak. The period from when the peak intensity is reached until it decreases from the peak intensity may correspond to the fading time.

[0296] The reference metric may include information derived from information acquired in operation 908. In some embodiments, the reference metric may include a reference excitation intensity for molecules(s) in the reaction chamber(s)(s)(s), which can be determined based on the characteristic fade time. For example, the exponential distribution of fade time is given by exp(-t / τ), where τ is the characteristic fade time. For single-molecule collection, the median fade time divided by ln(2) provides an estimate of the characteristic fade time τ. Faster characteristic fade times may result from higher excitation intensities, and vice versa.

[0297] In operation 914, sequencing parameters may be adjusted based on reference metrics (e.g., characteristic fade time, excitation intensity). In some embodiments, the output of the laser irradiating the reaction chamber with excitation light may be adjusted. For example, in some embodiments, the laser output may be reduced to account for a relatively fast characteristic fade time. In some embodiments, the laser output may be reduced to ensure that the excitation intensity does not exceed a target value during subsequent sequencing applications. Thus, the read length may be improved for sequencing applications where photodamage could be a factor in the lifetime of the sequencing reaction.

[0298] In some embodiments, the configuration of the integrated device may be adjusted based on a reference metric. For example, the inventors have recognized that differences in the reaction chambers can cause the characteristic fading time of the reference dye to vary between different reaction chambers. Therefore, the configuration of the reaction chamber, photodetector, and / or light source that irradiates the pixels with excitation light may be adjusted based on a reference metric. For example, in the case of a reaction chamber with a relatively fast fading time, the intensity and / or duration of the excitation light irradiated onto the reaction chamber may be reduced.

[0299] In some embodiments, fading information can be used to identify the number of reference dye molecules in the reaction chamber. For example, in embodiments in which excitation light is irradiated onto one or more reference dye molecules until they photobleach, the fading step can be observed by observing the change in the intensity of the signal collected from the reaction chamber (e.g., emitted from one or more reference dye molecules) over time. Photobleaching of a single reference dye molecule may be represented by a single step in intensity (e.g., a characteristic decrease in the signal intensity at a given point in time). In some embodiments, the characteristic decrease in the signal intensity is substantially equal to the intensity of the signal emitted by the reference dye molecule before fading. Specifically, after fading, the reference dye molecule no longer emits a signal, resulting in a single-step decrease in intensity that is substantially equal to the intensity of the signal emitted by the reference dye molecule before fading.

[0300] If a single fading step is observed (for example, as represented by a change in signal intensity observed over time), it can be determined that a single reference dye molecule has faded, and that a single reference dye molecule is present in the reaction chamber from which the signal is collected. If two or more fading steps are observed, it can be determined that multiple reference dye molecules have been loaded into the reaction chamber. The number of reference dye molecules determined to be loaded into the reaction chamber can be considered a surrogate for determining the number of biomolecules of the sample present in the reaction chamber. In some embodiments, an indicator of the number of sample molecules in the reaction chamber may be used to include or exclude a particular reaction chamber from subsequent analysis. Further explanations for determining an indicator of quantitative loading using photofading are provided herein.

[0301] Following the execution of calibration process 900, the integration device may be used to collect and analyze signals from molecules placed in one or more reaction chambers. For example, Figure 13 shows a sequencing reaction in a reaction chamber that may be performed following calibration process 900 according to some embodiments. The sequencing reaction shown in Figure 6 may involve the attachment of reference dye molecules 1010A and 1010B to a sample 1009 in reaction chamber 1008. As described herein, the reference dye molecules may be of a specific type and may bind to a specific type of sample molecule. For example, reference dye molecule 1010A selectively binds only to a first type of amino acid (e.g., leucine, denoted as L), and reference dye molecule 1010B selectively binds to a second type of amino acid (e.g., phenylalanine, tyrosine, and tryptophan, denoted as F, Y, and W, respectively). Following sampling of the sample molecule 1009 (for example, by irradiating the reaction chamber 1008 with excitation light and collecting the signals emitted from the bound reference dye molecules 1010A and 1010B), the sample molecule and / or the bound reference dye molecules 1010A and 1010B may be cleaved using the cleavage molecule 316. The sample molecule may be identified based on the signals emitted by the reference molecules. The processes of binding, receiving the emitted signals, and cleavage may be repeated multiple times for a single sample molecule and / or reaction chamber. In some embodiments, the calibration techniques described herein may be used to calibrate a system including an integrated device configured for use in protein and / or DNA / RNA sequencing applications.

[0302] The calibration techniques described herein may be used in combination with any preferred sampling techniques that follow the calibration. For example, in some embodiments, the calibration techniques described herein may be used in combination with techniques for sample multiplexing, such as those described in U.S. Patent Application No. 17 / 5085,783, “SYSTEMS AND METHODS FOR SAMPLE PROCESS SCALING,” filed October 22, 2021 (Agent Reference Number R0708.70112US02), which is incorporated herein by reference in whole.

[0303] IX. Quantitative load Several embodiments relate to systems and methods for determining an index of quantitative loading in one or more reaction chambers of an integrated device. For example, the inventors have recognized that it is advantageous to determine an index of quantitative loading in one or more reaction chambers of an integrated device in real time (e.g., during the loading process). The index of quantitative loading may include an indication of how many biomolecules are present in the reaction chamber (e.g., whether the reaction chamber is empty, single-loaded, double-loaded, or multiple-loaded with three or more biomolecules). In some embodiments, the index of quantitative loading may include an indication of the percentage of reaction chambers that are single-loaded (or loaded with any other desired number).

[0304] Figure 14 shows an exemplary process 1400 for quantifying the load in one or more reaction chambers according to some embodiments. Process 1400 may be initiated by operation 1402 in which a sample is loaded into an integrated device. The sample may contain multiple biomolecules. The multiple biomolecules may be of any suitable type. For example, the multiple biomolecules may contain biomolecules that are desired to be identified through sequencing. In some embodiments, the multiple biomolecules include peptides. In some embodiments, the multiple biomolecules include nucleic acids (e.g., ribonucleic acid, deoxyribonucleic acid).

[0305] Multiple biomolecules may be labeled with fluorescently labeled molecules (also referred to herein as reference dye molecules). In some embodiments, multiple biomolecules may be labeled before loading the sample onto the integration device. In some embodiments, multiple biomolecules may not be labeled at loading, and fluorescently labeled molecules that are loaded onto the integration device before or after may be bound to the multiple biomolecules.

[0306] In some embodiments, the fluorescently labeled molecule may be directly attached to the biomolecule itself. The fluorescently labeled molecule may be covalently or non-covalently bonded to the biomolecule (e.g., via a linker or streptavidin). In some embodiments, the fluorescently labeled molecule may be loaded into the reaction chamber as part of the interfacial chemical preparation of the chip. In some embodiments, the fluorescently labeled molecule may be reversibly bound to the biomolecule, as further described herein. Thus, embodiments of the art are not limited to any particular configuration of the fluorescently labeled molecule in the reaction chamber. According to some embodiments, operation 1402 may include the technique described in U.S. Patent Application No. 17 / 082,906, “METHODS OF PREPARING SAMPLES FOR MULTIPLEX POLYPEPTIDE SEQUENCING,” filed October 28, 2020 (Agent Reference Number R01408.70077US01), which is incorporated by reference in its entirety.

[0307] In some embodiments, the fluorescently labeled molecule consists of an oligonucleotide. The oligonucleotide can be covalently bonded to a biomolecule. In some embodiments, the oligonucleotide is hybridized to a complementary oligonucleotide that is covalently bonded to the biomolecule.

[0308] Fluorescently labeled molecules can be separated from biomolecules by a minimum distance (e.g., greater than 1 nm, greater than 2 nm, greater than 5 nm, 5–10 nm, greater than 10 nm, 10–15 nm, greater than 15 nm, 15–90 nm, greater than 90 nm) (e.g., via one or more spacers). By separating the fluorescently labeled molecules from biomolecules by a minimum distance, damage to the biomolecules can be prevented during quantitative loading determination (e.g., during photobleaching).

[0309] In operation 1404, excitation light may be irradiated onto one or more reaction chambers of the integrated device. The excitation light may be generated by at least one excitation source (e.g., a laser) as described herein. The excitation light may be irradiated onto all or part of the reaction chambers of the integrated device.

[0310] The excitation light excites any fluorescently labeled molecule bound to a biomolecule in the reaction chamber, causing it to emit excitation light. In operation 1406, signals emitted from one or more reference dye molecules in response to the excitation light can be acquired from each reaction chamber.

[0311] For example, emitted light may be collected by one or more photodetector regions of the integrated device. In some embodiments, the integrated device may include one photodetector region for each reaction chamber. In some embodiments, multiple photodetector regions may be provided for a single reaction chamber. In some embodiments, multiple reaction chambers may correspond to a single photodetector region.

[0312] In operation 1408, the quantitative loading index may be determined based on the release signal obtained in operation 1406. As described herein, the quantitative loading index may include an indication of how many biomolecules are present in the reaction chamber (e.g., whether the reaction chamber is empty, single-loaded, double-loaded, or multiple-loaded with three or more biomolecules). In some embodiments, the quantitative loading index may include an indication of the percentage of reaction chambers that are single-loaded (or loaded with any other desired number).

[0313] In some embodiments, irradiating one or more reaction chambers of an integrated device with excitation light includes photobleaching one or more fluorescently labeled molecules, and the quantitative loading index is determined based on the number of photobleaching steps represented in the emission signal (e.g., the number of photobleaching steps that can be observed in the emission signal). For example, one or more reaction chambers may be irradiated with excitation light according to a step function (e.g., by unblocking the light source, reconnecting the light source to the chip, and / or increasing the output supplied by the light source in a time fast relative to the characteristic bleaching time of the system). The excitation light may be irradiated onto the reaction chamber until the reference dye is bleached. Figures 15-1, 15-2, and 15-3 show exemplary traces obtained from a chamber having a fluorescently labeled molecule that is photobleached in operation 1404. In other embodiments, the signal emitted by one or more reference dyes in the reaction chamber may be obtained without photobleaching.

[0314] Figure 15-1 shows an exemplary trace 1500A from a chip loading process representing a single loaded well, according to one embodiment. As shown in Figure 15-1, the signal intensity over a period of time is plotted. When excitation light is first irradiated onto the reaction chamber, the signal intensity received from the reaction chamber may rise to a peak. After irradiating the reaction chamber with excitation light (e.g., by turning on a light source such as a laser), a single step in intensity (e.g., a characteristic decrease in signal intensity at a certain point in time) is observed in Figure 15-1. In some embodiments, as shown in Figure 15-1, the characteristic decrease in signal intensity is substantially equal to the signal intensity emitted by the reference dye molecule before fading. After fading, the reference dye molecule no longer emits a signal, resulting in a single step decrease in intensity, as shown in Figure 15-1. Thus, the signal shown in Figure 15-1 indicates the presence of a single fluorescent label, and therefore a single biomolecule, in the reaction chamber.

[0315] Figure 15-2 shows an exemplary trace 1500B from a tip loading process representing a double-loaded well according to one embodiment. As shown in Figure 15-2, the signal includes two steps of signal intensity indicating the presence of two fluorescently labeled molecules, and therefore two biomolecules in the reaction chamber.

[0316] Figure 15-3 shows an exemplary trace of 1500C from a chip loading process representing a multiple-loaded well according to one embodiment. As shown in Figure 15-3, the signal includes three steps in signal intensity (three characteristic drops in the intensity of the received signal) indicating the presence of three fluorescently labeled molecules, and therefore three biomolecules in the reaction chamber.

[0317] In some cases, the photobleaching step may not be observed. Therefore, based on the absence of observed photobleaching, it may be determined that biomolecules are not present in the reaction chamber. In some embodiments, the signal can be obtained from the reaction chamber without photobleaching the fluorescently labeled molecules present in the reaction chamber. In such embodiments, the number of fluorescently labeled molecules, and therefore the number of biomolecules, present in the reaction chamber can be determined by the relative intensity of the signal obtained from the reaction chamber.

[0318] In some embodiments, the fluorescently labeled molecule can be reversibly bound to a biomolecule or a secondary biomolecule attached to a biomolecule. For example, the secondary biomolecule may include an N-terminal amino acid recognition agent (e.g., ClpS, UBR box, etc.). In some embodiments, the secondary biomolecule is an oligonucleotide. In some embodiments, the oligonucleotide can be reversibly hybridized to a biomolecule or an oligonucleotide attached to a biomolecule.

[0319] The on and off states of binding of a fluorescently labeled molecule to a biomolecule can be represented by the on and off pulsation of the fluorescently labeled molecule. In particular, when a fluorescently labeled molecule binds to a biomolecule and is excited by excitation light, the fluorescently labeled molecule emits light that can be collected by one or more photodetector regions. When not bound, the fluorescently labeled molecule cannot emit light. Fluorescently labeled molecules can exhibit periodic binding patterns to biomolecules. Therefore, the fluorescent label generates a periodic pulsation pattern that can be detected by collecting the light emitted from the fluorescent label when it binds to a biomolecule.

[0320] Figure 16 shows a periodic pulsation pattern of a fluorescent molecule reversibly bound to a biomolecule, according to some embodiments. Plots 1602 and 1604 show examples of pulses detected by one or more photodetector regions during a single pulse period T.

[0321] Plots 1602 and 1604 show examples of double-loaded wells having two biomolecules and individual fluorescent labels reversibly bound to them. Thus, two pulses are detected during pulse period T. In the illustrated embodiment of plot 1602, the two fluorescently labeled molecules bind to the individual biomolecules substantially simultaneously during period T. Thus, a single composite pulse with intensity 2I, consisting of individual pulses from each of the two fluorescently labeled molecules, is detected within period T. In the illustrated embodiment of plot 1604, the two fluorescently labeled molecules bind to the individual biomolecules at discrete time intervals during period T. Thus, two pulses with intensity I are detected within period T.

[0322] Although the illustrated embodiment shows an example in which two biomolecules are doubly loaded into the reaction chamber, the techniques described herein can similarly be used to detect the presence of a single biomolecule, the presence of more than two biomolecules, or the absence of any biomolecule in the reaction chamber by determining the intensity of the pulses emitted from the reaction chamber during the pulsation period T. The pulsation period and intensity of the pulses can be determined based on the characteristic pulse width and characteristic intensity of a fluorescently labeled molecule reversibly bound to the biomolecule.

[0323] Figure 16 shows a method for detecting and using the timing and intensity of pulses to determine the number of fluorescently labeled molecules in the reaction chamber bound to biomolecules during each pulse period T, and therefore the number of biomolecules currently present in the reaction chamber. Thus, operation 1408 of process 1400 can be carried out using reversibly bound molecules as described herein.

[0324] Returning to process 1400, process 1400 may proceed to operation 1410 after determining a quantitative load index based on the emitted signal. In operation 1410, a decision is made as to whether to continue the load. The decision to continue or terminate the load may be based on the quantitative load index determined in operation 1410.

[0325] For example, in some embodiments, the operation of the integrated device may be optimized based on a quantitative loading index. In some embodiments, the quantitative loading index may be used to adjust the method of loading the sample onto the integrated device. In some embodiments, the quantitative loading index can be used to determine whether the desired number of biomolecules have been loaded into the optimal number of reaction chambers. In some embodiments, the desired number of biomolecules is 1, and therefore, the quantitative loading index is used to determine whether the optimal number (e.g., maximum) of reaction chambers are single-loaded.

[0326] If the optimal number of reaction chambers being loaded has not been reached, as indicated by the quantitative loading indicator, additional loading may be performed. For example, process 1400 may be looped back to operation 1402, in which additional sample is loaded into the integration device.

[0327] In some embodiments, a quantitative loading index can be used to adjust the amount of additional sample loaded in subsequent loading steps. For example, the sample may contain biomolecules at a specific concentration. The quantitative loading index can be used to determine whether to increase or decrease the concentration of biomolecules. For example, if it is determined that the optimal number of single-load reaction chambers is not met, additional loading can be performed using an additional sample with a higher concentration of biomolecules than the initial sample.

[0328] In some embodiments, a quantitative loading index may be used to adjust how signals from reaction chambers are processed for subsequent analysis. For example, if a reaction chamber is determined to be empty, double-loaded, or multiple-loaded, it may be determined to ignore signals from these reaction chambers by either not performing sequencing on them or not processing the signals received from them.

[0329] In some embodiments, quantitative loading indicators may be used to inform future operation of the integrated device. For example, quantitative loading indicators may be used to adjust how the sample is loaded onto the integrated device in the future (e.g., the concentration of biomolecules present in the sample, the time to perform the loading, the rate at which the loading is performed, etc.).

[0330] If it is decided in operation 1410 not to continue loading, process 1400 proceeds to operation 1412, where loading is terminated. For example, in some embodiments, termination may be carried out by removing, washing, and / or replacing the solution containing unimmobilized (e.g., unbound) peptides. In some embodiments, termination may be carried out autonomously.

[0331] In some embodiments, terminating the loading may involve removing the fluorescently labeled molecule from the reaction chamber. In some embodiments, the fluorescently labeled molecule may be removed by a chemical cleavage process. In some embodiments, the fluorescently labeled molecule may be removed by an enzymatic cleavage process.

[0332] As described herein, process 1400 may be executed continuously. In some embodiments, process 1400 may be executed autonomously, for example, using software. This makes it possible to continuously monitor the number of labeled biomolecules in each reaction chamber. The software analysis process can monitor the load in real time. For example, the software analysis may detect signal pulse / photobleaching steps, as described herein.

[0333] In some embodiments, one or more different types of fluorescently labeled molecules are provided to the biomolecule. For example, a first type of fluorescently labeled molecule and a second type of fluorescently labeled molecule may be provided for binding to the biomolecule.

[0334] In some embodiments, a "heatmap" of the reaction chamber can be generated to visually show the percentage of the integrated device (e.g., the percentage of the reaction chamber) to which one or more biomolecules are loaded. Figure 17 shows an exemplary heatmap of a reaction chamber illustrating the loading percentage according to some embodiments.

[0335] In some embodiments, the quantitative load index may be obtained for only a portion of the integrated device (e.g., a portion of all reaction chambers of the integrated device). The quantitative load index may be extrapolated to the remaining reaction chambers of the integrated device to obtain an extrapolated quantitative load index for the entire integrated device (or, therefore, the other portion).

[0336] In some embodiments, sample sequencing may be performed after performing calibration and quantitative loading techniques described herein (for example, in operation 1414 of process 1400). For example, calibration and quantitative loading techniques may be used in combination with techniques for sample identification using machine learning, such as those described in U.S. Patent Application No. 16 / 900,582, “TECHNIQUES FOR PROTEIN IDENTIFICATION USING MACHINE LEARNING AND RELATED SYSTEMS AND METHODS,” filed June 12, 2020 (Agent Reference Number R0708.70063US01), which is incorporated by reference in its entirety.

[0337] The techniques described herein for obtaining real-time measurements related to an integrated device (e.g., related to the occupancy rate of one or more reaction chambers of the integrated device) can be implemented at any time while the integrated device is in use (e.g., during sample preparation, loading, sequencing, sample removal, etc.), and such techniques are not limited to use during loading of the integrated device.

[0338] X. Equivalents and range While some aspects and embodiments of the technology of this disclosure have been described herein, it should be understood that various modifications, changes, and improvements will be readily conceivable to those skilled in the art. Such modifications, changes, and improvements are intended to be within the spirit and scope of the technology described herein. Accordingly, it should be understood that the embodiments described above are presented only as examples, and that embodiments of the present invention may be practiced in a manner different from those described in detail, within the scope of the appended claims and their equivalents. In addition, any combination of two or more features, systems, articles, materials, kits, and / or methods described herein is included within the scope of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually contradictory.

[0339] Furthermore, as described, some embodiments may be embodied in one or more methods. The actions performed as part of the method can be ordered in any preferred manner. Accordingly, embodiments can be constructed in which the actions are performed in a different order than shown, including performing some actions simultaneously, even if they are shown as a series of actions in the exemplary embodiments.

[0340] All definitions defined and used herein should be understood to encompass both dictionary definitions, definitions in references cited by reference, and the common meanings of the terms defined, or either of these.

[0341] In this specification and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless otherwise clearly indicated.

[0342] When phrases such as "and / or" are used in this specification and in the claims of this application, they should be understood to mean "one or both" of the elements thus linked, that is, elements that exist conjunctively in some cases and disjunctively in other cases.

[0343] When used in this specification and in the claims, the phrase “at least one” in relation to an enumeration of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the enumeration of elements, but not necessarily including at least one of every element specifically enumerated in the enumeration of elements, nor excluding any combination of elements in the enumeration of elements. This definition also allows for the optional presence of elements other than those specifically identified in the enumeration of elements referred to by the phrase “at least one,” whether relating to or not relating to those specifically identified elements.

[0344] In the claims and the above specification, all transitional phrases such as “equipped with,” “include,” “carry,” “possess,” “contain,” “accompany,” “hold,” and “composed of” should be understood as open-ended, meaning they include but are not limiting. The transitional phrases “consist of” and “essentially consist of” should be considered closed or semi-closed transitional phrases, respectively.

Claims

[Claim 1] 1. A method for reusing an integrated device for processing a sample, the sample being divided into a plurality of aliquots, the sample comprising analytes, the analytes comprising biomolecules including one or more luminescently labeled molecules, the method comprising: loading a first aliquot of the plurality of aliquots into at least a portion of a plurality of chambers of the integrated device; sampling the first aliquot of analyte while the analyte is present in at least a portion of the plurality of chambers, the sampling step including determining a luminescence lifetime, luminescence intensity, wavelength, pulse duration, and / or inter-pulse duration of a signal emitted by the one or more luminescent labeled molecules; removing the first aliquot from at least a portion of the plurality of chambers of the integrated device; loading a second aliquot of the plurality of aliquots into at least a portion of the plurality of chambers of the integrated device.

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