Method and system for fluorescence detection based on time gating

Abstract

The present disclosure provides methods, devices, and systems for time gated based fluorescence detection. Time-based fluorescence analyses can be used in certain biochemical assays by measuring the flux of photons emitted by a fluorophore following a single excitation pulse.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of March 13, 2020, application number 202080036167.7, and invention name “Method and system for fluorescence detection based on time gating” (the application date of the corresponding PCT application is March 13, 2020, and the application number is PCT / US2020 / 022830).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 818,614, filed on March 14, 2019, which is incorporated herein by reference in its entirety. Background Art

[0004] Continuous wave (CW) fluorescence-based spectroscopy used in both heterogeneous and homogeneous biochemical assays can be used for life science research and in vitro diagnosis. For example, detection methods based on endpoint fluorescence can be widely used to detect and / or monitor the combination of capture probes and analytes in surface (solid phase) biochemical assays. Typically, the analyte may include a fluorophore construct that can emit light when excited by a light excitation source. Emission may occur at a wavelength longer than the excitation source. When the capture probe is attached to a specific and / or addressable coordinate on the surface, analyte capture may result in the generation of localized fluorescent signals; a phenomenon that can be detected by an optical detection device. Example optical detection devices may include a charge coupled device (CCD) or a complementary metal oxide semiconductor (CMOS) camera.

[0005] Time-gated fluorescence (TGF) analysis is a variant of fluorescence spectroscopy that can be used in certain biochemical assays. Unlike CW fluorescence methods, in TGF the excitation light may not be continuous and may be applied only for a short period of time, i.e. it may be time-gated. Summary of the invention

[0006] In TGF, the response of the analyte to a series of finite-time light excitation pulses is analyzed after each excitation pulse is turned off. In conventional TGF, the photon flux emitted from the fluorophore can be measured after each individual excitation pulse. One way to measure the photon flux is to quantify the photoinduced charge ( Figure 1 ). When combined with a fluorophore label of the capture probe and / or analyte, such a measured signal can in turn be used to assess the presence, abundance, and occasionally characteristics of the target analyte.

[0007] In certain applications, TGF may offer advantages over CW fluorescence. For example, when the fluorophore copy number is relatively low, TGF may provide a higher signal-to-background ratio. With a sufficiently fast photoexcitation switching source, there may be little background from the excitation signal during detection. In addition, if a long-lived fluorophore (e.g., lanthanide chelates) is used, short-lived autofluorescence background emission from surrounding materials and / or biomolecular structures may also be eliminated. Examples of sources of autofluorescence may include plastics, organic polymers, or intercellular debris.

[0008] While TGF may have advantages, its practical implementation may be very challenging. The first set of challenges may be related to the speed at which pulsed excitation and detection can occur. In a traditional TGF configuration, optical and electronic systems with pulse frequencies >100MHz may be required. The second set of challenges may stem from the inherently low number of photons emitted after each excitation pulse, with the total count being less than or equal to the number of fluorophores. Finally, TGF may offer limited "multicolor" capabilities compared to CW fluorescence. In TGF, distinguishing fluorophores based on their lifetimes may require higher speed and lower noise performance of the optical and electronic equipment.

[0009] In the present disclosure, apparatus and methods are provided to create high-performance, highly integrated and cost-effective TGF systems using semiconductor biochip devices and technologies. The methods and apparatus of the present disclosure can be used in life sciences and molecular diagnostics in genomics and proteomics, particularly massively parallel DNA and protein analysis and DNA sequencing.

[0010] One aspect of the present disclosure provides a device for detecting the presence or absence of an analyte in a solution, comprising: a chip including a sensor, the sensor including an electronic shutter, wherein the sensor is configured to (i) collect a signal generated from the solution when the solution is exposed to an excitation pulse within a first time period, (ii) remove, with the aid of the electronic shutter, photoinduced charges generated in the sensor by the excitation pulse within a second time period, wherein the second time period is different from the first time period, and (iii) generate an output signal at least partially derived from the signal after removing the photoinduced charges, wherein the output signal indicates the presence or absence of the analyte.

[0011] In some embodiments, the second time period is before the first time period. In some embodiments, the second time period is greater than the duration of the excitation pulse. In some embodiments, the chip includes a plurality of individually addressable locations, wherein the sensor including the electronic shutter is disposed at a first location of the plurality of individually addressable locations; and wherein additional sensors including additional electronic shutters are disposed at additional locations of the plurality of individually addressable locations.

[0012] In some embodiments, the signal comprises an electrical signal, and wherein the sensor further comprises at least one transducer configured to convert the optical signal from the solution into the electrical signal. In some embodiments, the electronic shutter comprises an electronic shutter switch operably coupled to the at least one transducer, the electronic shutter switch being configured to facilitate the removal of the light-induced charge from the at least one transducer when a voltage is applied to the electronic shutter switch. In some embodiments, the sensor further comprises at least one integrator configured to integrate the electrical signal. In some embodiments, the sensor further comprises at least one integrating switch disposed between the at least one transducer and the at least one integrator and operably coupled to the at least one transducer and the at least one integrator, wherein the at least one integrating switch is configured to transmit the electrical signal from the at least one transducer to the at least one integrator. In some embodiments, the sensor further comprises at least one additional transducer operably coupled to the at least one integrator, the at least one additional transducer being configured to convert the electrical signal integrated by the at least one integrator into the output signal. In some embodiments, the signal comprises the light-induced charge, and wherein the output signal comprises a voltage. In some embodiments, the chip is included in a complementary metal oxide semiconductor (CMOS) integrated circuit (IC).

[0013] In some embodiments, the chip further comprises a biosensing layer adjacent to the sensor, and wherein the biosensing layer comprises at least one probe specifically bound to the analyte. In some embodiments, the signal is at least partially derived from an optical signal generated by a label associated with the analyte when the analyte binds to the at least one probe. In some embodiments, the label is a fluorophore. In some embodiments, the signal is at least partially derived from an optical signal or a change thereof from the at least one probe or the analyte when the analyte binds to the at least one probe. In some embodiments, the at least one probe comprises an energy donor and the analyte comprises an energy acceptor. In some embodiments, the energy donor is a fluorophore, and wherein the energy acceptor is an additional fluorophore or a quencher. In some embodiments, the biosensing layer comprises at least one control probe, and wherein the sensor is configured to collect a control signal from the at least one control probe and to standardize the collected signal using the control signal. In some embodiments, the at least one control probe does not bind or interact with the analyte. In some embodiments, the device further comprises a reaction chamber, a controllable fluid unit, a temperature control unit, and a digital unit. In some embodiments, the reaction chamber is configured to dock the solution with the chip, and wherein the docking includes an interaction between the analyte and the biosensing layer of the chip. In some embodiments, the controllable fluid unit is configured to transfer at least a portion of the solution into or out of the reaction chamber. In some embodiments, the digital unit is configured to receive or store the output signal from the chip. In some embodiments, the chip is configured to repeat (i)-(ii) multiple times before (iii). In some embodiments, the output signal is a single output.

[0014] One aspect of the present disclosure provides a method for detecting the presence or absence of an analyte in a solution, comprising:

[0015] (a) activating a chip including a sensor, the sensor including an electronic shutter, wherein the sensor is configured to (i) collect a signal generated when the solution is exposed to an excitation pulse during a first time period, (ii) remove, by means of the electronic shutter, photoinduced charges generated in the sensor by the excitation pulse during a second time period, wherein the second time period is different from the first time period, and (iii) generate an output signal derived at least in part from the signal after removing the photoinduced charges, wherein the output signal indicates the presence or absence of the analyte;

[0016] (b) removing the photoinduced charges generated in the sensor during the second time period by the excitation pulse with the aid of the electronic shutter;

[0017] (c) collecting said signal generated when said solution is exposed to said excitation pulse during said first period of time; and

[0018] (d) after removing the photoinduced charge, generating the output signal derived at least in part from the signal, the output signal being indicative of the presence or absence of the analyte.

[0019] In some embodiments, the sensor is a time-gated fluorescence (TGF) light sensor. In some embodiments, the method further comprises integrating the signal collected in (c) using the sensor. In some embodiments, the method further comprises repeating (b)-(c) one or more times. In some embodiments, the one or more times comprises greater than or equal to about 100 times.

[0020] Another aspect of the present disclosure provides a device for detecting a signal, comprising: a chip including a sensor and an electronic shutter, wherein the sensor is configured to (i) detect the signal within a given time period, and (ii) generate data indicating a charge generated by the signal, and wherein the electronic shutter is configured to remove photoinduced charges, the photoinduced charges including charges generated by an excitation pulse within a time period before the given time period; and a readout circuit operably coupled to the sensor, wherein the readout circuit is configured to transfer the data from the sensor to a memory.

[0021] In some embodiments, the readout circuit is part of the chip. In some embodiments, the memory is external to the readout circuit. In some embodiments, the signal is a fluorescent signal. In some embodiments, the chip includes a sensor array, the sensor array includes a plurality of individually addressable positions; the sensor and the electronic shutter are arranged at a first position in the plurality of individually addressable positions; the second sensor and the second electronic shutter are arranged at a second position in the plurality of individually addressable positions. In some embodiments, the sensor is also configured to integrate the charge generated by the signal. In some embodiments, the sensor includes an integrating switch. In some embodiments, the sensor includes at least one photon charge transducer and at least one charge integrator, and the at least one integrating switch is located between the at least one photon charge transducer and the at least one charge integrator. In some embodiments, the chip is included in a complementary metal oxide semiconductor (CMOS) integrated circuit (IC). In some embodiments, the CMOS IC also includes a heater and a temperature control system. In some embodiments, the heater and the temperature control system control the temperature at the plurality of individually addressable positions.

[0022] In some embodiments, the chip further comprises a biosensing layer adjacent to the sensor, and the biosensing layer comprises a surface comprising a plurality of probes. In some embodiments, the probes in the plurality of probes are identical. In some embodiments, the sensor receives fluorescence from a fluorescence source associated with the biosensing layer. In some embodiments, the fluorescence source is a fluorophore. In some embodiments, the fluorophore is attached to at least one of the plurality of probes. In some embodiments, the plurality of probes comprises at least one control probe. In some embodiments, the at least one control probe does not bind to or interact with a target molecule. In some embodiments, each of the plurality of probes specifically binds to or interacts with a target molecule. In some embodiments, the target molecule comprises a target molecule marker. In some embodiments, the target molecule marker comprises a target fluorophore. In some embodiments, each of the plurality of probes further comprises a molecular marker. In some embodiments, the molecular marker comprises a fluorophore. In some embodiments, the specific binding or interaction between the probe and the target molecule changes the fluorescence emitted from the fluorophore. In some embodiments, the device further comprises a reaction chamber, a controllable fluid system, a temperature control system, and a digital system. In some embodiments, the reaction chamber docks the sample with the biochip, and the docking includes interaction between the sample and the biosensing layer of the chip. In some embodiments, the controllable fluid system transfers at least one reagent into and / or out of the reaction chamber. In some embodiments, the at least one reagent includes the sample. In some embodiments, the temperature control system sets a first temperature of the reaction chamber at a first time point. In some embodiments, the digital system sends instructions to the chip and the temperature control system. In some embodiments, the digital system further stores data from the chip. In some embodiments, the digital system also receives data from the chip.

[0023] Another aspect of the present disclosure provides a method for detecting a signal, comprising activating a chip including a sensor and an electronic shutter, wherein the sensor is configured to (i) detect the signal within a given time period, and (ii) generate data indicating a charge generated by the signal, and wherein the electronic shutter is configured to remove photoinduced charges, the photoinduced charges including charges generated by an excitation pulse within a time period before the given time period; (b) using the electronic shutter to remove the photoinduced charges within the time period before the given time period; (c) using the sensor to detect the signal within the given time period and generate the data indicating the charge generated by the signal; and (d) transferring the data to a memory.

[0024] In some embodiments, the sensor is a time-gated fluorescence (TGF) light sensor. In some embodiments, (c) further comprises integrating the charge generated by the signal using the sensor. In some embodiments, the method further comprises repeating (a)-(c) one or more times. In some embodiments, one or more times comprises greater than or equal to about 10 times. In some embodiments, one or more times comprises greater than or equal to about 50 times. In some embodiments, one or more times comprises greater than or equal to about 100 times. In some embodiments, the method further comprises generating an output signal using the chip. In some embodiments, the output signal is a single output signal. In some embodiments, the chip comprises a plurality of individually addressable positions. In some embodiments, the chip further comprises an additional sensor and an additional electronic shutter, wherein the sensor and the electronic shutter are disposed at a first position in the plurality of individually addressable positions, and wherein the additional sensor and the additional electronic shutter are disposed at a second position in the individually addressable positions. In some embodiments, the first position is different from the second position. In some embodiments, the method further comprises removing additional light-induced charge using the additional electronic shutter in the time period prior to the given time period. In some embodiments, the method further comprises using the additional sensor to detect additional charge generated by the additional signal within the given time period and generating additional data indicating the additional charge generated by the additional signal. In some embodiments, the method further comprises integrating the additional charge using the sensor. In some embodiments, the plurality of independently addressable locations comprises greater than or equal to about 100 locations. In some embodiments, the plurality of independently addressable locations comprises greater than or equal to about 1,000 locations. In some embodiments, the plurality of independently addressable locations comprises greater than or equal to about 100,000 locations. In some embodiments, the plurality of independently addressable locations comprises greater than or equal to about 100 pixels.

[0025] Another aspect of the present disclosure provides a method for operating time-gated fluorescence (TGF) detection, comprising (a) activating a chip including a surface and an integrated circuit (IC) including at least one photosensor, wherein the IC includes an electronic shutter; (b) directing an excitation light pulse from an excitation light source to the surface; (c) during a first time period, removing a first photoinduced charge from the photosensor using the electronic shutter, wherein the first photoinduced charge includes a charge generated by the excitation light pulse during the first time period; (d) during a second time period after the first time period, measuring a second photoinduced charge generated in the photosensor, wherein the surface is not exposed to the excitation pulse during the second time period; and (e) integrating the second photoinduced charge measured in (d) during the second time period.

[0026] In some embodiments, the excitation pulse is generated by a laser. In some embodiments, the integration is performed by using a subcircuit included in the chip. In some embodiments, the method further comprises repeating (a)-(e) one or more times. In some embodiments, the one or more times include greater than or equal to about 10 times. In some embodiments, the one or more times include greater than or equal to about 50 times. In some embodiments, the one or more times include greater than or equal to about 100 times. In some embodiments, the method further comprises generating an output signal. In some embodiments, the output signal is a single output. In some embodiments, the method further comprises resetting the subcircuit once. In some embodiments, the subcircuit is not reset during the repetition. In some embodiments, the subcircuit is not reset between each repetition. In some embodiments, the method further comprises resetting the subcircuit before (b). In some embodiments, there is a gap between the first time period and the second time period. In some embodiments, the surface comprises a biosensing layer comprising at least one probe. In some embodiments, the at least one probe comprises a fluorophore. In some embodiments, when excited by the excitation light, the fluorophore emits a fluorescent signal. In some embodiments, the surface comprises a target molecule. In some embodiments, at least one target molecule includes a fluorophore. In some embodiments, the fluorophore emits a fluorescent signal when excited by excitation light. In some embodiments, the at least one probe specifically binds or interacts with the target molecule, thereby regulating the fluorescent signal emitted from the fluorophore contained in the at least one probe. In some embodiments, the integration includes an integrated photocurrent. In some embodiments, the method further includes converting the integrated photocurrent from an analog format to a digital format.

[0027] Another aspect of the present disclosure provides a device comprising: a chip operably coupled to a light source, the chip comprising a sensor, the sensor being configured to: (a) periodically detect one or more signals from an analyte associated with a surface of the chip, wherein the one or more signals are generated during or after subjecting the analyte to the light source; (b) integrate at least a subset of the one or more signals detected in (a) to generate an integrated signal; and (c) generate an output signal based on the integrated signal.

[0028] In some embodiments, the chip comprises an integrated complementary metal oxide semiconductor (CMOS) chip. In some embodiments, the output signal is a single output signal. In some embodiments, the sensor is a time-gated fluorescence (TGF) sensor. In some embodiments, the device does not include a filter disposed adjacent to the chip. In some embodiments, the output signal indicates a characteristic of the analyte. In some embodiments, the chip comprises a sensor array comprising a plurality of sensors. In some embodiments, each of the plurality of sensors is disposed at a position of the sensor array that can be individually addressed. In some embodiments, the analyte comprises a fluorophore. In some embodiments, the output signal is used to measure the lifetime of the fluorophore. In some embodiments, the analyte is fixed on the surface. In some embodiments, the analyte is part of a molecule fixed on the surface. In some embodiments, the analyte is fixed on the surface via a joint. In some embodiments, the one or more signals comprise fluorescent photons. In some embodiments, the sensor comprises a transducer configured to convert the fluorescent photons into electrical signals. In some embodiments, the sensor comprises a transducer configured to convert the fluorescent photons into electric charges. In some embodiments, the sensor further comprises an integrator configured to integrate the one or more signals. In some embodiments, the sensor comprises a switch operably coupled to the transducer and the integrator. In some embodiments, the switch transfers the charge from the transducer to the integrator. In some embodiments, the integrator is operably coupled to an additional transducer. In some embodiments, the additional transducer converts the charge into an electrical signal, thereby generating the output signal comprising the electrical signal. In some embodiments, the electrical signal comprises a voltage. In some embodiments, the light source is a pulsed light source. In some embodiments, the pulsed light source is a laser or a light emitting diode. In some embodiments, the pulsed light source is periodically modulated at a predetermined frequency.

[0029] Another aspect of the present disclosure provides a method comprising: (a) activating a chip comprising a sensor, wherein the sensor is configured to (i) periodically detect one or more signals from an analyte associated with a surface of the chip, wherein the one or more signals are generated during or after subjecting the analyte to the light source; (ii) integrate at least a subset of the one or more signals detected in (i) to produce an integrated signal; (iii) generate an output signal based on the integrated signal; (b) direct the light source to the chip to generate the one or more signals; (c) periodically detect the one or more signals from the analyte during or after subjecting the analyte to the light source; (d) integrate at least the subset of the one or more signals to generate the integrated signal; and (e) generate an output signal based on the integrated signal.

[0030] In some embodiments, the light source is a pulsed light source. In some embodiments, (c) is performed periodically at given intervals. In some embodiments, (c) occurs during or after each pulsed light source is turned off. In some embodiments, the output signal is a single output signal. In some embodiments, (d) is performed using an integrator. In some embodiments, (c) or (e) is performed using a transducer. In some embodiments, the output signal is an electrical signal. In some embodiments, the one or more signals are detected by the sensor without passing through an optical filter.

[0031] Other aspects and advantages of the present disclosure will become readily apparent to those skilled in the art through the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details are capable of modification in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are to be regarded as illustrative in nature, rather than restrictive.

[0032] Incorporation by reference

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the present invention are utilized, and the accompanying drawings (also referred to herein as "Figures"), in which:

[0035] Figure 1 An example timing diagram and waveform of time-gated fluorescence detection are illustrated.

[0036] Figure 2 The architecture and functional blocks of an example time-gated fluorescence (TGF) system are illustrated.

[0037] Figure 3 The architecture and functional blocks of an example time-gated fluorescence (TGF) complementary metal oxide semiconductor (CMOS) biochip are illustrated.

[0038] Figure 4 The architecture of an example CMOS integrated circuit (IC) for a TGF CMOS biochip system is illustrated.

[0039] Figure 5 The topology of an example TGF pixel with electronic shutter and periodic charge integration (PCI) capabilities is shown.

[0040] Figure 6 A timing diagram of a conventional TGF system is shown.

[0041] Figure 7 A timing diagram of an example periodic charge integration (PCI) TGF pixel is shown.

[0042] Fig. 8A FIG. 4 shows a planar configuration of a biosensing layer structure for a TGF CMOS biochip according to one embodiment of the present disclosure. Figure 8B The planar configuration of the biosensing layer structure of the TGF CMOS biochip with a thin film barrier according to one of the embodiments of the present disclosure is shown. Figure 8C A configuration of a 3D and permeable matrix coated on the surface of a biosensing layer for a TGF CMOS biochip according to one of the embodiments of the present disclosure is shown. Fig.8D A configuration of a 3D and permeable matrix coated on the surface of a biosensing layer with a thin film barrier for a TGF CMOS biochip according to one of the embodiments of the present disclosure is shown. Fig. 8E The microwell configuration of the biosensing layer used in the TGF CMOS biochip according to one embodiment of the present disclosure is shown. Fig.8F A microwell configuration for a biosensing layer having a 3D and permeable matrix coating the microwells of a TGF CMOS biochip according to one of the embodiments of the present disclosure is shown. Figure 8G A configuration of a combination of microwells with immobilized beads and microbeads for a TGF CMOS biochip according to one embodiment of the present disclosure is shown.

[0043] Fig. 9An example TGF CMOS biochip module and its TGF pixel structure according to one of the embodiments of the present disclosure are shown.

[0044] Fig. 10A The architecture of an example biochip according to one of the embodiments of the present disclosure is shown. Fig. 10B An example TGF pixel and its dedicated decimation cell according to one of the embodiments of the present disclosure are shown.

[0045] Fig.11 An exemplary TGF mode photosensor pixel schematic and timing diagram according to one of the embodiments of the present disclosure is shown.

[0046] Fig.12 Measured main electrical and optical properties of an example TGF pixel according to one of the embodiments of the present disclosure are shown.

[0047] Fig.13 Signal-to-background ratio and kinetic measurements of CWF and TGF modes according to one of the embodiments of the present disclosure are shown.

[0048] Fig.14 A die micrograph of an example TGF CMOS biochip according to one of the embodiments of the present disclosure is shown.

[0049] Fig.15A A circuit schematic of an example six-transistor (6T) PCI-TGF pixel according to one embodiment of the present disclosure is illustrated. Fig. 15B shows the submicron size Fig.15A The layout of pixels.

[0050] Fig.16 FIG. 1 shows a timing diagram of an example PCI-TGF pixel according to one embodiment of the present disclosure, wherein V is read during a reset cycle and after N PCI cycles. S to implement correlated double sampling (CDS). DETAILED DESCRIPTION

[0051] Although various embodiments of the present invention have been shown and described herein, it is readily understood by those skilled in the art that such embodiments are provided only as examples. Without departing from the present invention, those skilled in the art may conceive of various variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be adopted.

[0052] As used herein, the term "analyte" or "target" generally refers to the molecular species to be detected. Examples include small molecules, such as organic compounds, drugs, hormones, lipids, steroids, or metabolites; polynucleotides, such as deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, and peptide nucleic acids (PNA); polypeptides, such as proteins, peptides, antibodies, antigens, enzymes, and receptors; and tissue, organelle, and other receptor probes.

[0053] As used herein, the term "probe" or "capture probe" generally refers to a molecular species and / or other marker that can bind to a specific analyte or target. The probe can include a molecule and can be bound to a substrate, molecule or other solid surface directly or through a joint. Non-limiting examples of joints include amino acids, polypeptides, nucleotides, oligonucleotides and chemical joints. Multiple probes can be fixed on a substrate, molecule or other solid surface and can be referred to as a probe array. Multiple probes in a probe array can be arranged uniformly, for example as a spot arrangement, or unevenly.

[0054] As used herein, the terms "about" or "almost" generally mean within + / - 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a specified amount.

[0055] As used herein, the term "label" refers to a molecular structure that can be attached to a molecule (e.g., a target and / or a probe) to make the molecule detectable, distinguishable, and / or traceable by providing a characteristic that is not inherent to the target molecule. Examples of labels can include luminescent molecules (e.g., fluorophores), reduction-oxidation (redox) substances, or enzymes. In some cases, the label may include a fluorophore with a long lifetime, such as lanthanide chelates and transition metal chelates that emit or phosphoresce.

[0056] As used herein, the term "nucleotide" generally refers to a molecule that can serve as a monomer or subunit of a nucleic acid (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid RNA). Nucleotide can be a deoxynucleotide triphosphate (dNTP) or its analog, such as a molecule having multiple phosphates (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphates) in a phosphate chain. Nucleotide can generally include adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. Nucleotide can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits can be A, C, G, T or U, or any other subunit that is specific to one or more complementary A, C, G, T or U or complementary to purine (i.e., A or G, or variants thereof) or pyrimidine (i.e., C, T or U, or variants thereof). Subunits can enable single nucleic acid bases or base groups (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA or their uracil counterparts) to be resolved. Nucleotides can be labeled or unlabeled. Labeled nucleotides can produce detectable signals, such as optical, electrostatic or electrochemical signals.

[0057] As used herein, the terms "polynucleotide," "oligonucleotide," "nucleotide," "nucleic acid," and "nucleic acid molecule" generally refer to polymeric forms of nucleotides of various lengths (polynucleotides), either ribonucleotides (RNA) or deoxyribonucleotides (DNA). Examples of nucleotide sequences are sequences corresponding to natural or synthetic RNA or DNA, including genomic DNA and messenger RNA. The length of the sequence can be any length that can be amplified into a nucleic acid amplification product or amplicon, for example, up to about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000 or more 10,000 nucleotides in length, or at least about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1200, 1500, 2000, 5000, 10000 or 10000 nucleotides in length.

[0058] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and generally refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A polypeptide may include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. Examples of polypeptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptides and variants thereof, modified polypeptides, derivatives, analogs, fusion proteins, or combinations thereof. The polypeptide may be a natural peptide, a recombinant peptide, or a combination thereof.

[0059] As used herein, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

[0060] As used herein, the term "detector" generally refers to a device, typically including optical and / or electronic components, that can detect a signal.

[0061] Overview of the TGF system

[0062] The present disclosure provides methods, devices, reagents and systems based on time-gated fluorescence (TGF). The system may include a biochip based on TGF. The TGF biochip may be semiconductor integrated. In some cases, the semiconductor platform and manufacturing process for creating the system is a complementary metal oxide semiconductor (CMOS).

[0063] The methods and systems disclosed herein can be used to detect, analyze and / or quantify a variety of analytes present in aqueous samples by TGF transduction methods. The TGF CMOS biochip can be a monolithic integrated biosensor array with addressable locations. See, for example, U.S. Patent No. 9,708,647, U.S. Patent No. 9,499,861 and U.S. Patent No. 10,174,367, each of which is fully incorporated herein by reference. Each addressable location may include an independently operated TGF light sensor that detects TGF signals from its dedicated sensing area. Sensing / detection can be performed in real time in the presence of an aqueous sample, or when such a sample is washed away. The TGF light sensor can adopt a periodic charge integration (PCI) method, in which periodic signal accumulation is performed by applying multiple time-gated excitation pulses. The TGF CMOS biochip system can be physically docked with an aqueous sample and apply a physicochemical process to the sample, including, for example, applying a time-varying temperature distribution, biochemical reagents or pulsed excitation photon flux to the sample.

[0064] TGF CMOS biochip system, such as Figure 2 As shown, the components may include, but are not limited to, the following:

[0065] 1. TGF CMOS biochip, which can identify and detect analytes interfaced to its top surface through TGF transduction methods in a two-dimensional array format;

[0066] 2. A reaction chamber that can interface a sample fluid (e.g., a fluid aqueous sample containing an analyte) with the TGF CMOS biochip;

[0067] 3. An excitation source that can introduce a photon flux of a specific wavelength into the reaction chamber and / or the surface of the TGF CMOS biochip in a controlled manner and synchronized with the operation of the TGF CMOS biochip; 4. A controllable fluid system that is configured to move reagents and / or samples into the reaction chamber, and / or remove and / or hold reagents and / or samples from the reaction chamber, and remove and / or hold reagents and / or samples to the reaction chamber in a controlled manner and synchronized with the operation of the TGF CMOS biochip;

[0068] 5. A temperature controller that can set the temperature of the fluid within the reaction chamber in a controlled manner and synchronized with the TGF CMOS biochip operation; and

[0069] 6. TGF reagents and reporter constructs that can detect analytes and targets via the TGF CMOS biochip within the reaction chamber and according to a specific assay method.

[0070] 7. A digital system that can coordinate the operation of one or more components included in the system, collect data and / or transmit data to a processing and / or data analysis unit.

[0071] TGFCMOS biochip

[0072] like Figure 3 As shown, the TGF CMOS biochip may include components including but not limited to the following:

[0073] A. CMOS integrated circuit (IC), which may include the following functional blocks embedded in its monolithically integrated semiconductor substrate:

[0074] i. TGF photosensor arrays, which include multiple detectors in a two-dimensional array format. Individual detectors (e.g., "biosensing elements" or "pixels") can measure the photon flux emitted by fluorophores (Fe) at their addressable locations in parallel, simultaneously, and independently. The detectors can also employ the periodic charge integration (PCI) TGF method;

[0075] ii. Readout circuitry that can acquire data from individual TGF pixels and transfer them sequentially, in parallel, or in combination to an off-chip unit (external destination); and

[0076] iii. On-chip passive resistive heater and temperature sensor.

[0077] B. A biosensing layer, which can be located on the surface of a CMOS IC and can utilize a TGF approach to create an analyte-specific localized TGF signal coupled to a TGF pixel. The biosensing layer can include multiple probes at independently (and / or individually) addressable locations on a solid surface. Each pixel can include multiple identical or different probe molecules that can specifically bind or interact with a specific target / analyte or reagent in a reaction chamber;

[0078] CMOS Integrated Circuit (IC)

[0079] The architecture of the integrated CMOS IC for the TGF biochip is Figure 4 . The CMOS die includes a 2D light sensor array with a common readout circuit architecture similar to other biosensor arrays. See, for example, U.S. Pat. No. 9,708,647, U.S. Pat. No. 9,499,861, and U.S. Pat. No. 10,174,367, each of which is fully incorporated herein by reference. The light sensor array of the same CMOS embedded TGF pixels can be read sequentially (i.e., one pixel at a time) using row and column decoders. The chip output sent off-chip through the output buffer can be analog or digital.

[0080] The chip may also include a resistive heater and a temperature sensor to accommodate temperature control of the reaction chamber (e.g., Hassibi, A. et al., "A fully integrated CMOS fluorescence biochip for DNA and RNA testing," IEEE Journal of Solid-State Circuits, 52(11):2857-2870, 2017). In addition, the CMOS IC may also include a control block that can be programmed and accessed by the user off-chip to set the chip's functions and manage data acquisition.

[0081] The general topology of an example TGF pixel is shown in Figure 5 TGF receives F from addressable locations on its biosensing layer. e and F x, and photons can be converted into charge by using a photon charge transducer (PCT). Examples of PCTs in CMOS processes include lateral photodiodes (e.g., Cauwenberghs, G. et al., "Which photodiode to use: A comparison of CMOS-compatible structures," IEEE sensors journal, 9(7): 752-760, 2009), or pinned photodiode devices (e.g., Hondongwa, DB et al., "A review of the pinnedphotodiode for CCD and CMOS image sensors," IEEE J. Electron Devices Soc., 2(3): 33-43, 2014). The PCT device may include two switches connected to it. The first may be an electronic shutter switch (Ss), which completely removes the charge from the PCT by connecting the PCT to an electronic shutter voltage source (Vs). The second may be an integration switch (S1), which transfers the generated charge to a charge integrator element (CIE). The CIE device can be continuously connected to a charge voltage transducer (CVT) to produce a TGF pixel output. Additionally, the CIE may have a reset switch (S R ) can be used to remove the integrated charge at any time and essentially “reset” the CIE output value to V R .

[0082] The TGF pixels of the present disclosure may be different from conventional detectors for TGF or time-resolved fluorescence. One difference is that there is no S S and S I and the ability to selectively discard or integrate the charges generated by PCT. Figure 6 An example timing diagram of the operation of a conventional TGF system is shown in FIG. Figure 6 As shown, by quantizing the photoinduced charge during the integration time interval, at each N individual F x Measure F after the pulse e . Then the N outputs (X OUT [1] to X OUT [N]) average to estimate F e There are many challenges and non-ideal situations that may exist in this system. For example:

[0083] • N consecutive measurements (readings) may need to be made to estimate Fe or each pixel. Since Fe may be low, extensive averaging may be required, for example, values ​​of N>100 may be required in such TGF systems.

[0084] · Due to low signal levels (e.g., every Fe For example, if the gain of a CVT pulse is 10 total electrons per pulse), the CVT may require very high gain (e.g., >20 μV / e) and the analog quantization noise is equal to less than a few electrons per read.

[0085] When large biosensor arrays can achieve pixel counts of M>1000, the number of reads per frame becomes N×M, which quickly becomes overwhelming. For example, if the lifetime of the fluorophore used in TGF is τ L = 100ns, then you can create a period of 1ms = 10τ L F x Pulse sequence. If N = 100 and M = 1000, the readout speed will be 10 5 Readings / millisecond or 100 million samples / second. Given the noise requirements of the system, this may require very complex readout circuitry and require a lot of power. Therefore, one can consider reducing the pulse train frequency and radically slowing down the TGF measurement.

[0086] In this disclosure, by using Figure 4 With the topology shown, the above challenges can be solved. Figure 7 Depicted is a timing diagram of a TGF pixel of the present disclosure, which employs an intra-pixel periodic charge integration (PCI) scheme to improve the speed and performance of TGF measurements. Figure 7 As shown, by using S S and S I And applying an electronic shutter, the response of the N pulses of PCI can be integrated into the CIE which can produce a single output. This can enable each of the N pulses to be read out once, with an amplitude N times greater than that of conventional TGF. Other advantages of this approach may include but are not limited to: · One readout in PCI-TGF may be equal to N readouts in conventional TGF.

[0087] The accumulated charge and output amplitude signal of PCI-TGF can be N times larger than that of conventional TGF, and the readout speed can be N times slower. Therefore, PCI-TGF can use a more relaxed readout circuit with lower speed and higher signal chain quantization noise.

[0088] When using large biosensor arrays with M>1000 elements, the required readout and pixel scanning speed requirements may be N times less than traditional TGF. 6 Arrays of this number may become very feasible, and this number may be necessary for the massively parallel arrays used in life science research.

[0089] Challenges in PCI-TGF implementations may revolve around circuit and device implementation of the switches, and efficient methods of transferring charge within time intervals that are compatible with TGF and CIE.

[0090] Biosensing layer

[0091] The biosensing layer provided herein can include an organic layer that can be created on top of a CMOS IC and interfaced with a reaction chamber to: (a) form addressable locations for probes on top of pixels; and (b) achieve TGF transduction by first capturing the target and then creating a TGF signal as a function of the probe-target interaction and / or the structure of the captured target.

[0092] The biosensing layer can be created by various methods. For example, specific probe structures can be physically printed, fixed or spotted on the surface, or chemically synthesized. In some cases, the probes are first randomly distributed within the array 2D surface and then identified before detecting the target by alternative methods known in the art. In some cases, the surface of the IC (usually made of SiO2 or Si3N4) can be chemically modified with connectors and / or thin film structures to be compatible with probe accessories.

[0093] Figure 8 shows an example of a biosensing structure that is compatible with CMOS IC and TGF transduction methods (including PCI-TGF). Fig. 8A and Figure 8B In , a flat surface can be implemented to immobilize probes and addressable arrays can be created with and without a thin film barrier, respectively. Figure 8C and Fig.8D In , a 3D and permeable matrix can be coated on a surface to allow probes to be immobilized in close proximity to the surface. Fig. 8E and Fig.8F In the microwells, better separation of immobilized probes and separation of TGF pixels can be achieved. Figure 8G In , the combination of microwells and microbeads with immobilized beads can be used to create addressable arrays.

[0094] Reaction Chamber

[0095] A reaction chamber as provided herein can be a fluid chamber that interfaces with a CMOS TGF biochip and contains a fluid sample with analytes, targets, and other biochemical reagents required to perform a TGF assay.

[0096] The volume of the reaction chamber may be between about 0.1 μL and 10,000 μL, such as between about 1 μL and 100 μL.

[0097] The reaction chamber may include multiple inlets and outlets to accommodate interfacing with a controllable fluid system to insert or remove fluids.

[0098] To accommodate TGF, the fluid system can be pulsed F x Provides a transparent optical transmission path through the fluid and reaches the biosensing layer.x The transmittance over the wavelength range can be from 1% to 99.9%, but is typically from 5% to 80%.

[0099] The reaction chamber can be constructed using a variety of materials, for example, polymers, glasses, semiconductors, crystalline or ceramic materials, or combinations thereof.

[0100] Excitation source

[0101] The excitation sources provided herein may include a source capable of generating a wavelength-selective photon flux (F) with a controllable and time-varying amplitude. x ) The light source can illuminate the biosensing layer of the system and the coordinates where TGF transduction occurs.

[0102] The excitation source center wavelength may be anywhere between about 200 nm and 1500 nm, for example, between about 300 nm and 800 nm.

[0103] The excitation source spectral span (bandwidth) may be from about 1 nm to 500 nm, for example from about 10 nm to 100 nm.

[0104] The excitation source photon flux can be directional and can be optically collimated.

[0105] The excitation source peak output power may be from about 10 mW to 100 W, for example, from about 100 mW to 10W.

[0106] The excitation source power may be controllable and modulated with a bandwidth up to about 1 GHz, for example, up to about 1 MHz.

[0107] The excitation source may be turned off and on as fast as about 0.1 nanoseconds (ns), such as as fast as about 1 microsecond (μs).

[0108] Controllable fluid systems

[0109] The controllable fluid system introduces and / or removes and / or confines aqueous media, which may include samples and assay reagents and / or TGF transduction reagents, into the reaction chamber in a manner controlled by the user. The workflow and sequence of each fluid operation may be defined by the assay method and may be, for example, flow-through and one-way or closed tube.

[0110] Controllable fluid systems can use fluid components such as pumps, valves, and pipes to perform workflows.

[0111] Temperature Controller

[0112] The temperature controller system can establish a specific temperature for the fluid in the reaction chamber, and / or produce a temperature distribution that requires heating and / or cooling. The temperature controller can include a feedback control system that measures temperature using a temperature sensor within the CMOS biochip IC and / or a sensor device (e.g., a thermistor or thermocouple) coupled to the reaction chamber, and based on the measured temperature, adds or removes heat from the reaction chamber using a CMOS biochip IC heater and / or a thermal device (e.g., a Peltier device or a resistive heater). The temperature controller can include a heat sink for removing heat. The temperature controller can have components within the CMOS IC, including a resistive heater and / or a temperature sensor.

[0113] The temperature controller can change the temperature of the substrate, reaction chamber or array pixel. The temperature change rate can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ° C / minute. The temperature change rate can be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ° C / minute. The rate of temperature change can be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C / minute. The temperature controller can change the temperature at a linear rate (e.g., 5 °C / second). Alternatively, the temperature controller can change the temperature at a non-linear rate. The temperature controller can increase or decrease the temperature.

[0114] Digital System

[0115] Digital systems are essentially computing and controlling digital hardware, as opposed to embedded software, which controls and coordinates the functions of system components.

[0116] TGF Reagents and Reporter Constructs

[0117] To achieve TGF transduction, we can use molecular structures and constructs that exhibit fluorescent activity. Such molecular structures are sometimes called fluorophores (or fluorescent dyes, similar to chromophores), which are compounds that can re-emit light upon light excitation, with a lifetime of about 10ps to 10ms, such as about 1ns to 100ns.

[0118] As provided herein, the TGF system can employ various types of fluorophores. In some cases, fluorophores with longer lifetimes, such as greater than about 100 ns, can be used. In the case of fluorophores with longer lifetimes:

[0119] Longer-lived fluorophores may require slower PCI-TGF systems in CMOS biochip ICs;

[0120] The excitation source switching speed can be more controlled, and a more economical light source can be used; and

[0121] • If the lifetime is shorter than that of the employed TGF fluorophore, the negative impact of background autofluorescence from the biological sample and / or materials in the fluid chamber and / or biological layer can be mitigated.

[0122] The TGF system may not require an excitation and emission filter set or other wavelength filters to transmit the desired signal of the analyte and / or remove background fluorescence from the signal of the analyte. In some cases, the emission filter can filter out violet, blue, green, yellow, orange, and red light, or any combination thereof.

[0123] Various types of fluorophores may allow multicolor capabilities. In TGF, fluorophores can be distinguished by differences in fluorescence lifetimes after excitation. In some cases, these fluorophores can be reactive and / or conjugated dyes, nucleic acid dyes, fluorescent proteins, and cell function dyes. Once the emission light is pulsed in the direction of the substrate containing the fluorophore species, the shutter can separate the detection device from the emission light and the reflected emission light. The shutter can be removed to let the desired fluorescence in. If the shutter is opened shortly after the emission stops, the first fluorophore with a shorter lifetime can be detected in the detected signal. If the shutter is opened after waiting longer after the emission stops, a second fluorophore with a longer lifetime can be detected. In this case, the second fluorophore (longer lifetime) can be detected with little or no interference from the first fluorophore (shorter lifetime). In addition, the signal reading corresponding to the first fluorophore (shorter lifetime) in the presence of the second fluorophore (longer lifetime) can be estimated or calculated by calibrating the detected signal using information about the second fluorophore (longer lifetime) detected and / or determined later. Other experimental designs for multiplex detection of multiple fluorophores can be performed with or without emission and excitation filter sets. Thus, in the absence of excitation and emission filter sets, multiple fluorophores can be detected in a single experiment by opening and closing the shutter after emission. By varying the shutter delay profile and the time the shutter is open, individual species of fluorophores can be detected based on differences in their decay rates. Multicolor capability may be limited by the overlap between the shutter speed and the fluorescence decay rates of the fluorophores to be detected.

[0124] For example, in some examples, metal chelates, such as lanthanide chelates, can be used as TGF fluorophores. In some cases, TGF fluorophores may be used primarily as molecular reporters in TGF assays, either as stand-alone reporters or as elements (donors or acceptors) in fluorescence energy transfer moieties. Examples include, but are not limited to Resonance energy transfer (FRET) technology. See Song, Y. et al., "Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions," Annals of biomedical engineering 39(4): 1224-1234, 2011. The effect of the TGF fluorophore can include promoting the generation of a specific TGF signal, which can be associated with the presence or absence of a molecular reaction or the presence or absence of a specific target molecule.

[0125] TGF fluorophores can be used as labels for specific target analytes, in which case the target can be chemically modified to incorporate the TGF fluorophore. Examples include, but are not limited to, Northern blot hybridization, Southern blot hybridization, DNA microarrays, quantitative polymerase chain reaction (PCR), digital PCR, and diagnostic assays.

[0126] • In microarray and Northern blot hybridization, mRNA target analytes can be converted to fluorophore-labeled complementary DNA (cDNA), for example, by reverse transcription.

[0127] In Southern blot hybridization, fluorophore-labeled cDNA can be used to identify target sequences.

[0128] In quantitative polymerase chain reaction (PCR) and digital PCR (dPCR), fluorophores can be incorporated into amplified nucleic acid sequences or primer sequences to demonstrate accumulation of target sequences (see, e.g., Y. Wong et al., “Applications of digital PCR in precision medicine,” Expert Review of Precision Medicine and Drug Development 2(3):177-186, 2017).

[0129] • In diagnostic assays, the device can be used to isolate target nucleic acids and fluorophore-labeled cDNA can be used for direct detection.

[0130] TGF fluorophores can also be used as labels for probe detection in sandwich assays. Examples include, but are not limited to, protein immunoblot hybridization, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELISPOT) including FluoroSpot (see, e.g., G. Kesa et al., "Comparison of ELISpot and FluoroSpot in the Analysis of SwineFlu-Specific IgG and IgA Secretion by in Vivo Activated Human B Cells," Cells 1(2):27-34, 2012) and protein arrays.

[0131] • In these methods, TGF fluorophores can be used as direct detection methods, where the fluorophore is coupled to the primary detection antibody.

[0132] • In these methods, TGF fluorophores can also be used as an indirect detection method, where the fluorophore is conjugated to a secondary antibody.

[0133] ELISPOT is an assay that quantitatively measures the frequency of cytokine secretion by individual cells. ELISPOT assay is also a form of immunostaining that uses antibodies to detect analytes, including but not limited to any biological or chemical being identified or measured, such as protein analytes.

[0134] FluoroSpot assay is a variation of ELISpot assay. FluoroSpot assay uses fluorescence to analyze multiple analytes. It can detect the secretion of more than one type of protein or other analyte.

[0135] TGF fluorophores can be used as labels in cell sorting, counting and detection methods. An example could be flow cytometry, where cells are labeled with fluorophores.

[0136] In this method, cells can be classified and counted by their fluorescence distribution. In this method, specific cell characteristics and / or functions can be identified by their fluorescence distribution.

[0137] TGF fluorophore can be used to label solid phase and immobilize probe applications. An example is a reverse fluorophore assay (e.g., A. Hassibi et al., "Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip," Nature biotechnology, 36(8): 738-745, 2018)

[0138] The TGF fluorophore can be used to monitor an assay for a chemical reaction while introducing a target molecule into a reagent. The target molecule and / or the reagent can include a TGF fluorophore. Examples are Sanger sequencing, next generation sequencing (NGS) assays such as sequencing by synthesis (SBS), (see Ansorge; Metzker; and Pareek et al., "Sequencing technologies and genome sequencing," J. Appl. Genet., 52(4): 413-435, 2011), sequencing by hybridization (SBH) (see, Qin, Schneider and Brenner, "Sequencing by Hybridization of Long Targets," PLoS One., 7(5): e35819, 2012) and pyrophosphate sequencing.

[0139] In this method, single-molecule real-time (SMRT) sequencing and Illumina sequencing can use nucleotides labeled with TGF fluorophores to determine the sequence of nucleic acids

[0140] Sequence information of nucleic acids can be used to improve people's lives. (See, e.g., Ansorge, W., "Next-generation DNA sequencing techniques," New Biotech. 25(4): 195-203, 2009). Several DNA sequencing platforms are already commercially available. The availability of parallel NGS technology enables comprehensive analysis of biological targets, including but not limited to genomes, transcriptomes, and interactomes. (See, e.g., Shendure, J. and Ji, H., "Next-generation DNA sequencing," Nature Biotech. 26: 1135-45, 2008). However, although NGS technologies may produce comprehensive results, their turnaround time may be too slow to cope with the rapid progression of infectious processes in critically ill patients. In addition, although it is possible to multiplex a large number of target amplification reactions (e.g., multiplex PCR), it is not simple to detect multiple amplicons simultaneously.

[0141] Commercially available NGS sequencing platforms may include the Illumina Genome Analyzer, the Roche (454) Genome Sequencer, the Life Technologies SOLiD platform, and real-time sequencers such as the sequencer from Pacific Biosciences. These platforms may require the construction of a set of DNA fragments from a biological sample. In most cases, the DNA fragments are flanked by platform-specific adapters.

[0142] Example 1

[0143] In this example, a fully integrated TGF CMOS biochip is demonstrated, which is a DNA and protein addressable array specifically designed for biotechnology. Fig. 9 As shown, the CMOS IC is assembled on a printed circuit board (PCB) substrate and then integrated with a fluidic module to create a biochip consumable. The biochip IC includes an array of 1024 biosensor pixels, each with an integrated emission filter with an optical density (OD) of ~5.8 and an addressable (unique) fixed probe (DNA). The pixel-level light sensor with a Nwell-Psub photodiode (acting as a PCT element) is designed to limit shot noise and provide a detection dynamic range (DDR) of >130dB. A temperature control and circulation system is also integrated in the biochip to accommodate thermal control. Therefore, the integration of the bandgap temperature sensor and the resistive heater can achieve a heating / cooling rate of + / -10℃ / s in the range of 25℃ to 100℃, with an overall accuracy of ±0.25℃.

[0144] The chip and the architecture of the 120μm-pitch biosensor pixel and extraction unit are shown in Figure 2. Fig. 10A and Fig. 10B The TGF pixels in the 32×32 array include a ΔΣ current detector that converts the photocurrent I ph as its input and produces a 1-bit digital output stream which is fed into the on-chip decimation array. Fig. 10B ) includes a current integrator (acting as CIE+CVT), a clocked comparator (ADC) and a programmable current source (DAC).

[0145] In CWF mode (i.e., without a pulsed excitation source or electronic shutter), the ΔΣ current detector is operated at f c The decimation unit operates continuously at the frequency of f by performing two stages of 32-bit accumulation and then s The frequency is downsampled and read out to realize sinc 2 In TGF mode, similar operation is performed, but periodic activation is performed to convert the I ph The chip then accumulates and measures fluorescence emission at preprogrammed time intervals.

[0146] In this chip, the TGF pixel, decimation array, bandgap temperature sensor, and reference voltage DAC are all operated and read by a single digital core module running at 50MHz and accessible through a serial peripheral interface (SPI) port ( Fig. 10A A single resistive heater can deliver up to 20W of power using an external power supply, has a serpentine structure, and is evenly distributed in the top metal layer. The chip can be fully operational using 14 pins (and bond wires) clustered on one side of the chip to facilitate efficient fluidic assembly and consumable manufacturing ( Fig. 9 ).

[0147] exist Fig.11 In Figure 1, a schematic diagram and timing diagram of a light sensing pixel in TGF mode are depicted. A capacitive transimpedance amplifier (CTIA) is used as a CIE+CVT, and a clocked comparator generates the pixel output D out The DAC is implemented using a current source that can be used to apply current pulses into the CTIA input with two adjustable durations (Φ1 and Φ2). The electronic shutter uses SH1, SH2, and SH3 to temporarily shut off the CTIA’s feedback capacitor, C f out of the circuit and use an op amp to short I ph To V d Due to transistor mismatch, each shutter operation injects a small amount of charge into C f, which is represented by the pixel-related electronic shutter offset current I s This current is related to the dark current I dc When added, the random offset current I of the pixel is formed o =I s +I dc , in CWF and TGF modes, this current is measured and extracted to estimate I ph This is done using the CDS method, where one frame is taken with excitation light and another without excitation light, and the measurement is then subtracted from the other.

[0148] The decimation array has a dedicated bit cell for each pixel. The bit cell consists of a 32-bit incrementer and a 32-bit adder, forming a two-stage accumulation unit ( Fig. 10B ). With T = 1 / f s The output of the adder is loaded into a 32-bit shift register for the interval. The data from the shift register is then passed to the digital unit in a serial scan chain.

[0149] The electrical and optical measurements of the biochip were Fig.12 The measured pixel signal-to-noise ratio (SNR) shows that the added sensor noise is ~30% of the shot noise when the quantization noise is not limited to the 100fA to 1nA input current region. s =1.667 Hz, and the total double depletion region (DDR) is 137 dB (1.33 fA–10 nA). The photodiode external quantum efficiency (QE) with and without an integrated emission filter is shown to be 0.4 and 3.69 × 10 -7 (OD ~ 5.8) passband and stopband QE. I measured at 100pA maximum amplitude (<1% of full scale) dc and I o The distribution verifies the expected randomness. Fig.12 The output of the temperature sensor as a function of temperature is also reported in , showing that a 2-point calibration accuracy of ±0.25°C can be achieved over the temperature range of 25°C-100°C.

[0150] exist Fig.13In, the results of two (2) biosensing experiments are reported and compared to demonstrate different modes of operation. In all experiments, the same surface functionalization and array-based DNA hybridization or ligand-receptor binding were performed. However, different molecular labels were attached to the targets of CWF and TGF, respectively. In CWF, the signal-to-background ratio (S / B) showed the lowest value using the R-phycoerythrin fluorophore. This is probably due to the non-ideal blocking of the excitation light. The S / B increased significantly when using TGF and DTBTA-Eu3+, which is a long-lived fluorophore based on europium (lanthanide) chelate. It is obvious that the background photon emission from the pulsed light emitting diode (LED) excitation source decays significantly within 100μs and the background becomes much smaller compared to the CWF mode.

[0151] exist Fig.14 , a micrograph of the implemented TGF biochip is shown.

[0152] Example 2

[0153] This example shows how to design PCI-TGF pixels in applications that require high-density biosensor pixel arrays, such as DNA SBS and DNA SBH systems. The example also shows how miniaturized PCI-TGF pixels can be incorporated into standard high-density image sensor arrays. As shown in the example, PCI can be added to the circuitry of multi-million-pixel CMOS image sensors that can have sub-micron pixel sizes.

[0154] Fig.15A Depicted is a circuit diagram example of a six-transistor (6T) pixel topology including a pinned photodiode (PPD) as a PCT and two (2) charge transfer gates; one charge transfer gate transfers charge to a sense node (TX) that acts as an integrating switch, and the other acts as an electronic shutter (SH). Charge is integrated on a floating diffusion (acting as CIE+CVT), generating a voltage V s Use source follower gate readout. In this description, we assume that the pixel is located at (i,j) coordinates within the photosensor array and that V s It can be accessed by the column signal (COL[j]) by activating the row select signal (SEL[i]). The charge is a floating diffusion and can be reset using RST[i].

[0155] exist Fig. 15B , the layout of this pixel is shown, which can be scaled down to sub-micron dimensions similar to equivalent CMOS image sensor pixels.

[0156] exist Fig.16 , a schematic diagram of a PCI-TGF pixel is shown. Fig.16As shown, correlated double sampling (CDS) can be achieved by reading V after a reset cycle and N PCI cycles. s As shown in the reset cycle, the output of the pixel is V DD -ΔV n -V th , where ΔV n and V th are the offset and threshold voltages of the source follower transistor, respectively. Now, at the end of the Nth integration period, V s =V DD -ΔV n -V th -NΔQ / Cend where ΔQ is the charge collected by the emission of a single excitation pulse and C is the floating diffusion effective capacitance. Therefore, by subtracting these two values ​​(i.e. CDS) we can obtain a value that follows the PCI scheme and is independent of the offset of the source follower which may vary from pixel to pixel within the array.

[0157] The present invention provides embodiments including but not limited to the following:

[0158] 1. A device for detecting the presence or absence of an analyte in a solution, comprising:

[0159] A chip comprising a sensor, the sensor comprising an electronic shutter, wherein the sensor is configured to (i) collect a signal generated from the solution when the solution is exposed to an excitation pulse during a first time period, (ii) remove, with the aid of the electronic shutter, photoinduced charges generated in the sensor by the excitation pulse during a second time period, wherein the second time period is different from the first time period, and (iii) generate an output signal at least partially derived from the signal after removing the photoinduced charges, wherein the output signal indicates the presence or absence of the analyte.

[0160] 2. The apparatus of embodiment 1, wherein the second time period precedes the first time period.

[0161] 3. The apparatus of embodiment 1, wherein the second time period is greater than a duration of the excitation pulse.

[0162] 4. The device of embodiment 1, wherein the chip comprises a plurality of individually addressable locations,

[0163] wherein said sensor including said electronic shutter is disposed at a first position of said plurality of individually addressable positions; and

[0164] Additional sensors including additional electronic shutters are disposed at additional locations in the plurality of individually addressable locations.

[0165] 5. The device of embodiment 1, wherein the signal comprises an electrical signal, and wherein the sensor further comprises at least one transducer configured to convert an optical signal from the solution into the electrical signal.

[0166] 6. A device according to embodiment 5, wherein the electronic shutter includes an electronic shutter switch operably coupled to the at least one transducer, and the electronic shutter switch is configured to facilitate the removal of the light-induced charge from the at least one transducer when a voltage is applied to the electronic shutter switch.

[0167] 7. The device according to embodiment 5, wherein the sensor further comprises at least one integrator configured to integrate the electrical signal.

[0168] 8. An apparatus according to embodiment 7, wherein the sensor further comprises at least one integrating switch disposed between and operably coupled to the at least one transducer and the at least one integrator, wherein the at least one integrating switch is configured to transmit the electrical signal from the at least one transducer to the at least one integrator.

[0169] 9. The device of embodiment 7, wherein the sensor further comprises at least one additional transducer operably coupled to the at least one integrator, the at least one additional transducer being configured to convert the electrical signal integrated by the at least one integrator into the output signal.

[0170] 10. The apparatus of embodiment 1, wherein the signal comprises photoinduced charge, and wherein the output signal comprises a voltage.

[0171] 11. The device of embodiment 1, wherein the chip is included in a complementary metal oxide semiconductor (CMOS) integrated circuit (IC).

[0172] 12. The device according to embodiment 1, wherein the chip further comprises a biosensing layer adjacent to the sensor, and wherein the biosensing layer comprises at least one probe that specifically binds to the analyte.

[0173] 13. The apparatus of embodiment 12, wherein the signal originates at least in part from an optical signal generated by a label associated with the analyte when the analyte binds to the at least one probe.

[0174] 14. The device of embodiment 13, wherein the label is a fluorophore.

[0175] 15. The apparatus of embodiment 12, wherein the signal is derived at least in part from an optical signal or a change thereof from the at least one probe or the analyte when the analyte binds to the at least one probe.

[0176] 16. The device of embodiment 15, wherein the at least one probe comprises an energy donor and the analyte comprises an energy acceptor.

[0177] 17. The device of embodiment 16, wherein the energy donor is a fluorophore, and wherein the energy acceptor is an additional fluorophore or a quencher.

[0178] 18. The device of embodiment 12, wherein the biosensing layer comprises at least one control probe, and wherein the sensor is configured to collect a control signal from the at least one control probe and to normalize the collected signal using the control signal.

[0179] 19. The device of embodiment 18, wherein the at least one control probe does not bind to or interact with the analyte.

[0180] 20. The apparatus according to embodiment 12 further comprises a reaction chamber, a controllable fluid unit, a temperature control unit and a digital unit.

[0181] 21. The device of embodiment 20, wherein the reaction chamber is configured to dock the solution with the chip, and wherein the docking comprises an interaction between the analyte and the biosensing layer of the chip.

[0182] 22. The apparatus of embodiment 20, wherein the controllable fluid unit is configured to transfer at least a portion of the solution into or out of the reaction chamber.

[0183] 23. The device according to embodiment 21, wherein the digital unit is configured to receive or store the output signal from the chip.

[0184] 24. The device according to embodiment 1, wherein the chip is configured to repeat (i)-(ii) multiple times before (iii).

[0185] 25. An apparatus according to embodiment 24, wherein the output signal is a single output.

[0186] 26. A method for detecting the presence or absence of an analyte in a solution, comprising:

[0187] (a) activating a chip including a sensor, the sensor including an electronic shutter, wherein the sensor is configured to (i) collect a signal generated when the solution is exposed to an excitation pulse during a first time period, (ii) remove, by means of the electronic shutter, photoinduced charges generated in the sensor by the excitation pulse during a second time period, wherein the second time period is different from the first time period, and (iii) generate an output signal derived at least in part from the signal after removing the photoinduced charges, wherein the output signal indicates the presence or absence of the analyte;

[0188] (b) removing the photoinduced charges generated in the sensor during the second time period by the excitation pulse with the aid of the electronic shutter;

[0189] (c) collecting the signal generated when the solution is exposed to the excitation pulse during the first time period; and

[0190] (d) after removing the photoinduced charge, generating the output signal derived at least in part from the signal, the output signal being indicative of the presence or absence of the analyte.

[0191] 27. The method of embodiment 26, wherein the sensor is a time-gated fluorescence (TGF) light sensor.

[0192] 28. The method of embodiment 26 further comprises integrating the signal collected in (c) using the sensor.

[0193] 29. The method according to embodiment 26 also includes repeating (b)-(c) one or more times.

[0194] 30. The method of embodiment 29, wherein the one or more times comprises greater than or equal to about 100 times.

[0195] 31. A device for detecting a signal, comprising:

[0196] a chip including a sensor and an electronic shutter, wherein the sensor is configured to (i) detect the signal within a given time period, and (ii) generate data indicating a charge generated by the signal, and wherein the electronic shutter is configured to remove light-induced charge, the light-induced charge including charge generated by an excitation pulse within a time period prior to the given time period; and

[0197] A readout circuit is operably coupled to the sensor, wherein the readout circuit is configured to transfer the data from the sensor to a memory.

[0198] 32. An apparatus according to embodiment 31, wherein the readout circuit is part of the chip.

[0199] 33. An apparatus according to embodiment 31 or 32, wherein the memory is external to the readout circuit.

[0200] 34. The device of any one of embodiments 31-33, wherein the chip comprises a sensor array comprising a plurality of individually addressable locations;

[0201] wherein said sensor and said electronic shutter are disposed at a first position of said plurality of individually addressable positions; and

[0202] The second sensor and the second electronic shutter are arranged at a second position among the plurality of individually addressable positions.

[0203] 35. The device of any one of embodiments 31-34, wherein the sensor is further configured to integrate the charge generated by the signal.

[0204] 36. The apparatus of any one of embodiments 31-35, wherein the sensor comprises an integral switch.

[0205] 37. An apparatus according to embodiment 36, wherein the sensor includes at least one photon charge transducer and at least one charge integrator, and wherein the at least one integrating switch is located between the at least one photon charge transducer and the at least one charge integrator.

[0206] 38. The apparatus of any one of embodiments 31-37, wherein the chip is included in a complementary metal oxide semiconductor (CMOS) integrated circuit (IC).

[0207] 39. The device of any one of embodiments 31-38, wherein the chip further comprises a biosensing layer adjacent to the sensor, and wherein the biosensing layer comprises a surface comprising a plurality of probes.

[0208] 40. The device of embodiment 39, wherein a fluorophore is attached to at least one of the plurality of probes.

[0209] 41. The apparatus of any one of embodiments 39-40, wherein the plurality of probes comprises at least one control probe.

[0210] 42. The apparatus of any one of embodiments 39-41, further comprising a reaction chamber, a controllable fluid system, a temperature control system, and a digital system.

[0211] 43. The apparatus of embodiment 42, wherein the reaction chamber docks the sample with the biochip, and wherein the docking comprises an interaction between the sample and the biosensing layer of the chip.

[0212] 44. An apparatus according to embodiment 42 or 43, wherein the controllable fluid system transports at least one reagent into and / or out of the reaction chamber.

[0213] 45. A method for detecting a signal, comprising:

[0214] (a) activating a chip including a sensor and an electronic shutter, wherein the sensor is configured to (i) detect the signal within a given time period, and (ii) generate data indicating charges generated by the signal, and wherein the electronic shutter is configured to remove light-induced charges including charges generated by an excitation pulse within a time period prior to the given time period;

[0215] (b) removing the photoinduced charges using the electronic shutter during the time period before the given time period;

[0216] (c) detecting the signal within the given time period using the sensor and generating the data indicative of the charge generated by the signal; and

[0217] (d) transferring the data to a memory.

[0218] 46. ​​A method according to embodiment 45, wherein the sensor is a time-gated fluorescence (TGF) light sensor.

[0219] 47. A method according to embodiment 45 or 46, wherein (c) also includes integrating the charge generated by the signal using the sensor.

[0220] 48. The method of any one of embodiments 45-47, further comprising repeating (a)-(c) one or more times.

[0221] 49. A method according to embodiment 48, wherein the one or more times includes greater than or equal to about 10 times, 50 times or 100 times.

[0222] 50. The method according to any one of embodiments 48-49 also includes using the chip to generate an output signal.

[0223] 51. A method according to embodiment 50, wherein the output signal is a single output signal.

[0224] 52. A method according to any one of embodiments 45-51, wherein the chip comprises a plurality of individually addressable locations.

[0225] 53. A method according to embodiment 52, wherein the chip further comprises an additional sensor and an additional electronic shutter, wherein the sensor and the electronic shutter are arranged at a first position among the plurality of individually addressable positions, and wherein the additional sensor and the additional electronic shutter are arranged at a second position among the individually addressable positions.

[0226] 54. The method according to embodiment 53 further includes using the additional electronic shutter to remove additional photoinduced charge during the time period before the given time period.

[0227] 55. The method of embodiment 54 further comprises using the additional sensor to detect additional charge generated by the additional signal within the given time period and to generate additional data indicative of the additional charge generated by the additional signal.

[0228] 56. The method of embodiment 55 further comprises integrating the additional charge using the sensor.

[0229] 57. A method according to any one of embodiments 52-56, wherein the plurality of independently addressable locations includes greater than or equal to about 100 locations, 1,000 locations, or 100,000 locations.

[0230] 58. A method according to any one of embodiments 52-57, wherein the plurality of independently addressable locations includes locations greater than or equal to about 100 pixels.

[0231] 59. A method for operating a time-gated fluorescence (TGF) assay, comprising:

[0232] (a) activating a chip comprising a surface and an integrated circuit (IC) comprising at least one light sensor, wherein the IC comprises an electronic shutter;

[0233] (b) directing an excitation light pulse from an excitation light source toward the surface;

[0234] (c) removing first light-induced charge from the photosensor using the electronic shutter during a first time period, wherein the first light-induced charge includes charge generated by the excitation light pulse during the first time period;

[0235] (d) measuring a second light-induced charge generated in the photosensor during a second time period after the first time period, wherein the surface is not exposed to the excitation pulse during the second time period; and

[0236] (e) integrating the second photoinduced charge measured in (d) during the second time period.

[0237] 60. A method according to embodiment 59, wherein the integration is performed by using a subcircuit contained in the chip.

[0238] 61. The method of any one of embodiments 59-60, further comprising repeating (a)-(e) one or more times.

[0239] 62. A method according to embodiment 61, wherein the one or more times includes greater than or equal to about 10 times, 50 times or 100 times.

[0240] 63. The method according to any one of embodiments 61-62 also includes generating an output signal.

[0241] 64. A method according to embodiment 63, wherein the output signal is a single output.

[0242] 65. The method according to any one of embodiments 60-64 also includes resetting the sub-circuit once.

[0243] 66. A method according to embodiment 65, wherein the subcircuit is not reset during the repetition.

[0244] 67. A method according to embodiment 65, wherein the subcircuit is not reset between each of the repetitions.

[0245] 68. The method of embodiment 65 further comprises resetting the subcircuit before (b).

[0246] 69. A method according to any one of embodiments 59-68, wherein there is a gap between the first time period and the second time period.

[0247] 70. A method according to any one of embodiments 59-69, wherein the integration includes integrating photocurrent.

[0248] 71. The method of embodiment 70 further comprises converting the integrated photocurrent from an analog format to a digital format.

[0249] 72. An apparatus comprising:

[0250] A chip operably coupled to a light source, the chip comprising a sensor configured to:

[0251] (a) periodically detecting one or more signals from an analyte associated with a surface of the chip, wherein the one or more signals are generated during or after subjecting the analyte to the light source;

[0252] (b) integrating at least a subset of the one or more signals detected in (a) to generate an integrated signal; and

[0253] (c) generating an output signal based on the integrated signal.

[0254] 73. The apparatus of embodiment 72, wherein the chip comprises an integrated complementary metal oxide semiconductor (CMOS) chip.

[0255] 74. An apparatus according to embodiment 72 or 73, wherein the output signal is a single output signal.

[0256] 75. The device of any one of embodiments 72-74, wherein the sensor is a time-gated fluorescence (TGF) sensor.

[0257] 76. The device of any one of embodiments 72-75, wherein the device does not include an optical filter disposed adjacent to the chip.

[0258] 77. The device of any one of embodiments 72-76, wherein the chip comprises a sensor array comprising a plurality of sensors.

[0259] 78. An apparatus according to embodiment 77, wherein each of the plurality of sensors is disposed at an individually addressable location of the sensor array.

[0260] 79. The device of any one of embodiments 72-77, wherein the analyte comprises a fluorophore.

[0261] 80. The apparatus of embodiment 79, wherein the output signal is used to measure the lifetime of the fluorophore.

[0262] 81. The device of any one of embodiments 72-80, wherein the analyte is immobilized on the surface.

[0263] 82. An apparatus according to any one of embodiments 72-81, wherein the one or more signals include fluorescent photons.

[0264] 83. An apparatus according to embodiment 82, wherein the sensor comprises a transducer configured to convert the fluorescent photons into electrical signals.

[0265] 84. The device of embodiment 82 or embodiment 83, wherein the sensor comprises a transducer configured to convert the fluorescent photons into electrical charge.

[0266] 85. The device of any one of embodiments 72-84, wherein the sensor further comprises an integrator configured to integrate the one or more signals.

[0267] 86. An apparatus according to embodiment 85, wherein the sensor includes a switch operably coupled to the transducer and the integrator.

[0268] 87. An apparatus according to embodiment 86, wherein the switch transfers the charge from the transducer to the integrator.

[0269] 88. The apparatus of any one of embodiments 84-87, wherein the integrator is operably coupled to an additional transducer.

[0270] 89. An apparatus according to embodiment 88, wherein the additional transducer converts the electric charge into an electrical signal, thereby generating the output signal comprising the electrical signal.

[0271] 90. The apparatus of embodiment 89, wherein the electrical signal comprises a voltage.

[0272] 91. An apparatus according to any one of embodiments 72-90, wherein the light source is a pulsed light source.

[0273] 92. A method comprising:

[0274] (a) activating a chip including a sensor configured to (i) periodically detect one or more signals from an analyte associated with a surface of the chip, wherein the one or more signals are generated during or after subjecting the analyte to the light source; (ii) integrate at least a subset of the one or more signals detected in (i) to generate an integrated signal; and (iii) generate an output signal based on the integrated signal;

[0275] (b) directing the light source to the chip to generate the one or more signals;

[0276] (c) periodically detecting the one or more signals from the analyte during or after subjecting the analyte to the light source;

[0277] (d) integrating the at least a subset of the one or more signals to generate the integrated signal; and

[0278] (e) generating an output signal based on the integrated signal.

[0279] 93. A method according to embodiment 92, wherein the light source is a pulsed light source.

[0280] 94. A method according to any one of embodiments 92-93, wherein (c) is performed periodically at given intervals.

[0281] 95. A method according to any one of embodiments 93-94, wherein (c) occurs during or after each time the pulsed light source is turned off.

[0282] 96. A method according to any one of embodiments 92-95, wherein (d) is performed using an integrator.

[0283] 97. The method of any one of embodiments 92-96, wherein (c) or (e) is performed using a transducer.

[0284] 98. A method according to any one of embodiments 92-97, wherein the output signal is an electrical signal.

[0285] 99. The method of any one of embodiments 92-98, wherein the one or more signals are detected by the sensor without passing through a filter.

[0286] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. The present invention is not intended to be limited to the specific embodiments provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not meant to be interpreted as restrictive. Without departing from the present invention, those skilled in the art will now think of many variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various substitutions to the embodiments of the present invention described herein can be adopted when practicing the present invention. It is therefore contemplated that the present invention should also encompass any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and are therefore intended to encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A device for detecting the presence or absence of an analyte in a solution, comprising: A chip comprising a sensor, the sensor comprising an electronic shutter, wherein the sensor is configured to (i) collect a signal generated from the solution when the solution is exposed to an excitation pulse during a first time period, (ii) remove, with the aid of the electronic shutter, photoinduced charges generated in the sensor by the excitation pulse during a second time period, wherein the second time period is different from the first time period, and (iii) generate an output signal at least partially derived from the signal after removing the photoinduced charges, wherein the output signal indicates the presence or absence of the analyte. 2 . The apparatus of claim 1 , wherein the second time period precedes the first time period.

3. The apparatus of claim 1, wherein the second time period is greater than a duration of the excitation pulse.

4. The device of claim 1, wherein the chip comprises a plurality of individually addressable locations, wherein said sensor including said electronic shutter is disposed at a first position of said plurality of individually addressable positions; and Additional sensors including additional electronic shutters are disposed at additional locations in the plurality of individually addressable locations.

5. A method for detecting the presence or absence of an analyte in a solution, comprising: (a) activating a chip including a sensor, the sensor including an electronic shutter, wherein the sensor is configured to (i) collect a signal generated when the solution is exposed to an excitation pulse during a first time period, (ii) remove, by means of the electronic shutter, photoinduced charges generated in the sensor by the excitation pulse during a second time period, wherein the second time period is different from the first time period, and (iii) generate an output signal derived at least in part from the signal after removing the photoinduced charges, wherein the output signal indicates the presence or absence of the analyte; (b) removing the photoinduced charges generated in the sensor during the second time period by the excitation pulse with the aid of the electronic shutter; (c) collecting the signal generated when the solution is exposed to the excitation pulse during the first time period; as well as (d) after removing the photoinduced charge, generating the output signal derived at least in part from the signal, the output signal being indicative of the presence or absence of the analyte.

6. A device for detecting a signal, comprising: a chip including a sensor and an electronic shutter, wherein the sensor is configured to (i) detect the signal within a given time period, and (ii) generate data indicating charge generated by the signal, and wherein the electronic shutter is configured to remove light-induced charge, the light-induced charge including charge generated by the excitation pulse within a time period prior to the given time period; and A readout circuit is operably coupled to the sensor, wherein the readout circuit is configured to transfer the data from the sensor to a memory.

7. A method for detecting a signal, comprising: (a) activating a chip including a sensor and an electronic shutter, wherein the sensor is configured to (i) detect the signal within a given time period, and (ii) generate data indicating charges generated by the signal, and wherein the electronic shutter is configured to remove light-induced charges including charges generated by an excitation pulse within a time period prior to the given time period; (b) removing the photoinduced charges using the electronic shutter during the time period before the given time period; (c) detecting the signal within the given time period using the sensor and generating the data indicative of the charge generated by the signal; and (d) transferring the data to a memory.

8. A method for operating a time-gated fluorescence (TGF) assay, comprising: (a) activating a chip comprising a surface and an integrated circuit (IC) comprising at least one light sensor, wherein the IC comprises an electronic shutter; (b) directing an excitation light pulse from an excitation light source toward the surface; (c) removing first light-induced charge from the photosensor using the electronic shutter during a first time period, wherein the first light-induced charge includes charge generated by the excitation light pulse during the first time period; (d) measuring a second light-induced charge generated in the photosensor during a second time period after the first time period, wherein the surface is not exposed to the excitation pulse during the second time period; as well as (e) integrating the second photoinduced charge measured in (d) during the second time period.

9. A device comprising: A chip operably coupled to a light source, the chip comprising a sensor configured to: (a) periodically detecting one or more signals from an analyte associated with a surface of the chip, wherein the one or more signals are generated during or after subjecting the analyte to the light source; (b) integrating at least a subset of the one or more signals detected in (a) to generate an integrated signal; as well as (c) generating an output signal based on the integrated signal.

10. A method comprising: (a) activating a chip including a sensor configured to (i) periodically detect one or more signals from an analyte associated with a surface of the chip, wherein the one or more signals are generated during or after subjecting the analyte to the light source; (ii) integrating at least a subset of the one or more signals detected in (i) to produce an integrated signal; (iii) generating an output signal based on the integrated signal; (b) directing the light source to the chip to generate the one or more signals; (c) periodically detecting the one or more signals from the analyte during or after subjecting the analyte to the light source; (d) integrating the at least one subset of the one or more signals to generate the integrated signal; as well as (e) generating an output signal based on the integrated signal.

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