Method and system for time-gated fluorescence-based detection

Abstract

The present disclosure provides methods, apparatus, and systems for time-gated fluorescence-based detection. Time-based fluorescence analysis can be used in certain biochemical assays by measuring the photon flux emitted from a fluorophore after each excitation pulse. A device for detecting the presence or absence of an analyte in a solution includes a chip including a sensor with an electronic shutter, the sensor configured to: (i) collect a signal from the solution generated in response to exposure of the solution to the excitation pulse within a first time period; (ii) remove, using the electronic shutter, a light-induced charge generated in the sensor by the excitation pulse within a second time period, the second time period being different from the first time period; and (iii) generate an output signal derived at least in part from the signal following removal of the light-induced charge, the output signal indicating the presence or absence of the analyte.

Description

Background Art

[0001] (Cross-reference) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 818,614, filed Mar. 14, 2019, which is hereby incorporated by reference in its entirety.

[0002] (Background) Continuous-wave (CW) fluorescence-based spectroscopy, which is employed in both heterogeneous and homogeneous biochemical assays, can be used in life science research as well as in vitro diagnostics. Endpoint fluorescence-based detection methods can be widely applied, for example, to detect and / or monitor capture probe and analyte binding in surface-based (solid-phase) biochemical assays. Generally, an analyte can contain a fluorophore construct that can emit light when excited by an optical excitation source. The emission can occur at a wavelength longer than that of the excitation source. When a capture probe is attached to specific and / or addressable coordinates on a surface, analyte capture can result in the generation of a localized fluorescence signal, i.e., a phenomenon that can be detected by an optical detection device. Exemplary optical detection devices can include a charge-coupled device (CCD) or a complementary metal-oxide semiconductor (CMOS) camera.

[0003] Time-gated fluorescence (TGF) analysis is a variant of fluorescence spectroscopy that can be used in certain biochemical assays. Unlike the CW fluorescence method, in TGF, the excitation light need not be continuous and can be applied only within a certain fraction of time, i.e., it can be time-gated.

Summary of the Invention

Means for Solving the Problems

[0004] (Abstract) In TGF, the response of the specimen to a series of finite-time optical excitation pulses is analyzed after each excitation pulse is turned off. In conventional TGF, the photon flux emitted from the phosphor can be measured for each individual excitation pulse. One way to measure the photon flux is to quantify the optically induced charge within a specific integration time interval of the detector (Figure 1). Such measured signals can be used to evaluate the presence, abundance, and possibly the characteristics of the target specimen when combined with a capture probe and / or a phosphor label of the specimen.

[0005] In certain applications, TGF can have advantages over CW fluorescence. For example, TGF can provide a much higher signal-to-background ratio when the phosphor copy number is relatively low. By using a sufficiently fast optical excitation switching source, during detection, the background from the excitation signal can be almost non-existent. Further, when phosphors with long lifetime times are used (e.g., lanthanide chelates), the short-lived autofluorescence background emission can be excluded from the surrounding materials and / or biomolecular structures. Examples of autofluorescence sources can include plastics, organic polymers, or cellular debris.

[0006] TGF can be advantageous, but its practical implementation can be very difficult. The first set of challenges can be related to speed, where pulsed excitation and detection can occur. In conventional TGF configurations, optical and electronic systems with a pulse frequency > 100 MHz can be required. The second set of challenges can arise from the small number of photons emitted per excitation pulse, and the total count can be less than or equal to the number of phosphors. Finally, TGF can provide a limited "multicolor" capability compared to CW fluorescence. Differentiating phosphors based on their lifetime times in TGF can require higher speed and lower noise performance for the optical and electronic systems.

[0007] In the present disclosure, apparatuses and methods are provided for creating a high-performance, highly integrated, and cost-effective TGF system using semiconductor biochip devices and technologies. The methods and apparatuses of the present disclosure may be used in genomics and proteomics, particularly in life sciences and molecular diagnostics in large-scale parallel DNA and protein analysis and DNA sequencing.

[0008] Aspects of the present disclosure provide a device for detecting the presence or absence of an analyte in a solution, the device comprising a chip comprising a sensor comprising an electronic shutter, the sensor configured to: (i) collect a signal from the solution generated in response to exposure of the solution to an excitation pulse during a first time period; (ii) use the electronic shutter to remove photoinduced charge generated within the sensor by the excitation pulse during a second time period, the second time period being different from the first time period; and (iii) generate an output signal at least partially derived from the signal subsequent to the removal of the photoinduced charge, the output signal being configured to indicate the presence or absence of the analyte.

[0009] In some embodiments, the second time period precedes the first time period. In some embodiments, the second time period exceeds the duration of the excitation pulse. In some embodiments, the chip comprises a plurality of individually addressable locations, the sensor comprising the electronic shutter is disposed on a first location of the plurality of individually addressable locations, and an additional sensor comprising an additional electronic shutter is disposed on an additional location of the plurality of individually addressable locations.

[0010] In some embodiments, the signal comprises an electrical signal, and the sensor further comprises at least one transducer configured to convert an optical signal from a solution into an electrical signal. In some embodiments, the electronic shutter comprises an electronic shutter switch operably coupled to the at least one transducer, and the electronic shutter switch is configured to facilitate removal of photoinduced charge from the at least one transducer in response to application of a voltage 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 integration switch disposed between and operably coupled to the at least one transducer and the at least one integrator, and the at least one integration switch is configured to transport 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, and the at least one additional transducer is configured to convert the electrical signal integrated by the at least one integrator into an output signal. In some embodiments, the signal comprises photoinduced charge and the output signal comprises a voltage. In some embodiments, the chip is included within a complementary metal oxide semiconductor (CMOS) integrated circuit (IC).

[0011] In some embodiments, the chip further comprises a biosensing layer adjacent to the sensor, the biosensing layer comprising at least one probe that specifically binds to the analyte. In some embodiments, the signal is derived at least in part from an optical signal generated by a label associated with the analyte in response to binding of the analyte to at least one probe. In some embodiments, the label is a phosphor. In some embodiments, the signal is derived at least in part from an optical signal or a change thereof from at least one probe or the analyte in response to binding of the analyte to at least one probe. In some embodiments, at least one probe comprises an energy donor and the analyte comprises an energy acceptor. In some embodiments, the energy donor is a phosphor and the energy acceptor is an additional phosphor or a quencher. In some embodiments, the biosensing layer comprises at least one control probe, and the sensor is configured to collect a control signal from the at least one control probe and use the control signal to normalize the collected signal. In some embodiments, the at least one control probe does not bind to 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 interfacially contact the solution and the chip, the interfacial contact comprising an interaction between the analyte and the biosensing layer of the chip. In some embodiments, the controllable fluid unit is configured to transport at least a portion of the solution in and out of the reaction chamber. In some embodiments, the digital unit is configured to receive or store an output signal from the chip. In some embodiments, the chip is configured to repeat (i)-(ii) a plurality of times prior to (iii). In some embodiments, the output signal is a single output.

[0012] Aspect of the present disclosure is a method for detecting the presence or absence of an analyte in a solution, comprising (a) Activating a chip comprising a sensor with an electronic shutter, wherein the sensor: (i) collects a signal generated in response to exposure of a solution to an excitation pulse during a first time period; (ii) uses the electronic shutter to remove photoinduced charges generated by the excitation pulse within the sensor during a second time period, the second time period being different from the first time period; and (iii) generates an output signal at least partially derived from the signal following removal of the photoinduced charges, the output signal being configured to indicate the presence or absence of an analyte. (b) Using an electronic shutter to remove photoinduced charges generated by an excitation pulse within the sensor during a second time period. (c) Collecting a signal generated in response to exposure of a solution to an excitation pulse during a first time period. (d) Generating an output signal at least partially derived from the signal following removal of the photoinduced charges, the output signal indicating the presence or absence of an analyte. A method is provided that includes the above.

[0013] In some embodiments, the sensor is a time-gated fluorescence (TGF) optical sensor. In some embodiments, the method further includes integrating the signal collected in (c) using the sensor. In some embodiments, the method further includes repeating (b)-(c) one or more times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 100 times.

[0014] Another aspect of the present disclosure provides a device for detecting a signal, the device comprising: a chip comprising a sensor and an electronic shutter, wherein the sensor is configured to: (i) detect a signal during a given time period; and (ii) provide data indicative of charges generated by the signal; and the electronic shutter is configured to remove photoinduced charges comprising charges generated by an excitation pulse during a time period prior to the given time period; and a readout circuitry operably coupled to the sensor and configured to transmit data from the sensor to a memory.

[0015] In some embodiments, the readout circuitry is part of the chip. In some embodiments, the memory is external to the readout circuitry. In some embodiments, the signal is a fluorescence signal. In some embodiments, the chip comprises a sensor array having a plurality of individually addressable locations, the sensor and the electronic shutter are disposed on a first one of the plurality of individually addressable locations, and a second sensor and a second electronic shutter are disposed on a second one of the plurality of individually addressable locations. In some embodiments, the sensor is further configured to integrate charge generated by the signal. In some embodiments, the sensor comprises an integration switch. In some embodiments, the sensor comprises at least one photo / charge transducer and at least one charge integrator, and at least one integration switch is positioned between the at least one photo / charge transducer and the at least one charge integrator. In some embodiments, the chip is included within a complementary metal oxide semiconductor (CMOS) integrated circuit (IC). In some embodiments, the CMOS IC further comprises a heater and a temperature control system. In some embodiments, the heater and the temperature control system control the temperature at a plurality of individually addressable locations.

[0016] In some embodiments, the chip further comprises a biosensing layer adjacent to the sensor, the biosensing layer comprising a surface with a plurality of probes. In some embodiments, the probes of the plurality of probes are the same. In some embodiments, the sensor receives fluorescent light from a fluorescent source associated with the biosensing layer. In some embodiments, the fluorescent source is a phosphor. In some embodiments, the phosphor is attached to at least one probe 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 the target molecule. In some embodiments, each probe of the plurality of probes specifically binds to or interacts with the target molecule. In some embodiments, the target molecule comprises a target molecule label. In some embodiments, the target molecule label comprises a target phosphor. In some embodiments, each probe of the plurality of probes further comprises a molecular label. In some embodiments, the molecular label comprises a phosphor. In some embodiments, the specific binding or interaction between the probe and the target molecule changes the fluorescence emitted from the phosphor. 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 interfaces the sample and the biochip, the interfacing comprising an interaction between the sample and the biosensing layer of the chip. In some embodiments, the controllable fluid system transports at least one reagent into and / or out of the reaction chamber. In some embodiments, the at least one reagent comprises the sample. In some embodiments, the temperature control system sets a first temperature in the reaction chamber at a first point in time. 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 further receives data from the chip.

[0017] Yet another aspect of the present disclosure is a method for detecting a signal, comprising: (a) activating a chip comprising a sensor and an electronic shutter, wherein the sensor is configured to (i) detect a signal within a given time period and (ii) provide data indicative of the charge generated by the signal, and the electronic shutter is configured to remove photoinduced charge comprising charge generated by an excitation pulse within a time period prior to the given time period; (b) using the electronic shutter to remove photoinduced charge within a time period prior to the given time period; (c) using the sensor to detect a signal within the given time period and provide data indicative of the charge generated by the signal; and (d) transmitting the data to a memory.

[0018] In some embodiments, the sensor is a time-gated fluorescence (TGF) optical sensor. In some embodiments, (c) further includes using the sensor to integrate the charge generated by the signal. In some embodiments, the method further includes repeating (a)-(c) one or more times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 10 times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 50 times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 100 times. In some embodiments, the method further includes using the chip to generate an output signal. In some embodiments, the output signal is a single output signal. In some embodiments, the chip comprises a plurality of independently addressable locations. In some embodiments, the chip further comprises an additional sensor and an additional electronic shutter, the sensor and the electronic shutter are disposed on a first location of the plurality of independently addressable locations, and the additional sensor and the additional electronic shutter are disposed on a second location of the independently addressable locations. In some embodiments, the first location is different from the second location. In some embodiments, the method further includes using the additional electronic shutter to remove additional photoinduced charge within a time period prior to a given time period. In some embodiments, the method further includes using the additional sensor to detect additional charge generated by an additional signal within a given time period and providing additional data indicative of the additional charge generated by the additional signal. In some embodiments, the method further includes using the sensor to integrate the additional charge. In some embodiments, the plurality of independently addressable locations comprises a number greater than or equal to about 100 locations. In some embodiments, the plurality of independently addressable locations comprises a number greater than or equal to about 1,000 locations. In some embodiments, the plurality of independently addressable locations comprises a number 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 locations, which are pixels.

[0019] Another aspect of the present disclosure is a method for operating time-gated fluorescence (TGF) detection, comprising: (a) activating a chip comprising a surface and an integrated circuit (IC) comprising at least one optical sensor, the IC comprising an electronic shutter; (b) directing a pulse of excitation light from an excitation light source onto the surface; (c) using the electronic shutter to remove a first optically induced charge from the optical sensor during a first time period, the first optically induced charge comprising the charge generated by the pulse of excitation light during the first time period; (d) measuring a second optically induced charge generated within the optical sensor during a second time period following the first time period, the surface not being exposed to the excitation pulse during the second time period; and (e) integrating the second optically induced charge measured in (d) during the second time period.

[0020] In some embodiments, the excitation pulse is generated by a laser. In some embodiments, integrating is performed by using a sub-circuit provided within the chip. In some embodiments, the method further includes repeating (a)-(e) one or more times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 10 times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 50 times. In some embodiments, the number of one or more times comprises a number greater than or equal to about 100 times. In some embodiments, the method further includes generating an output signal. In some embodiments, the output signal is a single output. In some embodiments, the method further includes resetting the sub-circuit once. In some embodiments, the sub-circuit is not reset during the repetition. In some embodiments, the sub-circuit is not reset during each of the repetitions. In some embodiments, the method further includes resetting the sub-circuit prior to (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 phosphor. In some embodiments, the phosphor emits a fluorescence signal when excited by excitation light. In some embodiments, the surface comprises a target molecule. In some embodiments, the at least one target molecule comprises a phosphor. In some embodiments, the phosphor emits a fluorescence signal when excited by excitation light. In some embodiments, the at least one probe specifically binds to or interacts with the target molecule, thereby modulating the fluorescence signal emitted from the phosphor provided within the at least one probe. In some embodiments, integrating includes integrating a photocurrent. In some embodiments, the method further includes converting the integrated photocurrent from an analog format to a digital format.

[0021] Another aspect of the present disclosure provides a device comprising a chip operably coupled to a light source, the chip comprising a sensor configured to: (a) periodically detect one or more signals from a sample associated with the surface of the chip, the one or more signals being generated during or subsequent to exposing the sample 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.

[0022] 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 comprise an optical 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 an individually addressable location of the sensor array. In some embodiments, the analyte comprises a phosphor. In some embodiments, the output signal is used to measure the lifetime of the phosphor. In some embodiments, the analyte is immobilized on a surface. In some embodiments, the analyte is part of a molecule that is immobilized on a surface. In some embodiments, the analyte is immobilized on a surface via a linker. In some embodiments, one or more signals comprise fluorescent photons. In some embodiments, the sensor comprises a transducer configured to convert fluorescent photons into an electrical signal. In some embodiments, the sensor comprises a transducer configured to convert fluorescent photons into charge. In some embodiments, the sensor further comprises an integrator configured to integrate 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 transports 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 an output signal comprising an 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.

[0023] Another aspect of the present disclosure is a method comprising: (a) (i) periodically detecting one or more signals from a sample associated with a surface of a chip, the one or more signals being generated during or subsequent to exposing the sample to a light source, (ii) integrating at least a subset of the one or more signals detected in (i) to generate an integrated signal, and (iii) activating a chip comprising a sensor configured to generate an output signal based on the integrated signal; (b) directing a light source at the chip to generate one or more signals; (c) periodically detecting one or more signals from the sample during or subsequent to exposing the sample to the light source; (d) integrating at least a subset of the one or more signals to generate an integrated signal; and (e) generating an output signal based on the integrated signal.

[0024] In some embodiments, the light source is a pulsed light source. In some embodiments, (c) is performed periodically at a given time interval. In some embodiments, (c) occurs during or after each time the 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 in the absence of passage through an optical filter.

[0025] Additional aspects and advantages of the present disclosure will be readily apparent to those of ordinary skill in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be recognized by those of skill in the art, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the spirit and scope of the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive. The present invention provides, for example, the following. (Item 1) A device for detecting the presence or absence of an analyte in a solution, comprising a chip having a sensor with an electronic shutter, the sensor configured to: (i) collect a signal from the solution generated in response to exposure of the solution to an excitation pulse during a first time period; (ii) use the electronic shutter to remove photoinduced charge generated within the sensor by the excitation pulse during a second time period, the second time period being different from the first time period; and (iii) generate an output signal at least partially derived from the signal following removal of the photoinduced charge, the output signal indicating the presence or absence of the analyte. (Item 2) The device according to item 1, wherein the second time period precedes the first time period. (Item 3) The device according to item 1, wherein the second time period exceeds the duration of the excitation pulse. (Item 4) The chip comprises a plurality of individually addressable locations, the sensor with the electronic shutter is disposed on a first location of the plurality of individually addressable locations, and an additional sensor with an additional electronic shutter is disposed on an additional location of the plurality of individually addressable locations. (Item 5) The device according to item 1, wherein the signal comprises an electrical signal, and the sensor further comprises at least one transducer configured to convert an optical signal from the solution into the electrical signal. (Item 6) The device according to item 5, wherein the electronic shutter comprises an electronic shutter switch operably coupled to the at least one transducer, the electronic shutter switch configured to facilitate removal of the photoinduced charge from the at least one transducer in response to application of a voltage to the electronic shutter switch. (Item 7) The device according to item 5, wherein the sensor further comprises at least one integrator configured to integrate the electrical signal. (Item 8) The sensor further comprises at least one integration switch disposed between and operatively coupled to the at least one transducer and the at least one integrator, the at least one integration switch being configured to transport the electrical signal from the at least one transducer to the at least one integrator, the device of item 7. (Item 9) The sensor further comprises at least one additional transducer operatively 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, the device of item 7. (Item 10) The signal comprises photoinduced charge and the output signal comprises a voltage, the device of item 1. (Item 11) The chip is included within a complementary metal oxide semiconductor (CMOS) integrated circuit (IC), the device of item 1. (Item 12) The chip further comprises a biosensing layer adjacent to the sensor, the biosensing layer comprising at least one probe that specifically binds to the analyte, the device of item 1. (Item 13) The signal is at least partially derived from an optical signal generated by a label associated with the analyte in response to binding of the analyte and the at least one probe, the device of item 12. (Item 14) The label is a phosphor, the device of item 13. (Item 15) The signal is at least partially derived from an optical signal or a change thereof from the at least one probe or the analyte in response to binding of the analyte and the at least one probe, the device of item 12. (Item 16) The at least one probe comprises an energy donor and the analyte comprises an energy acceptor, the device of item 15. (Item 17) The energy donor is a phosphor and the energy acceptor is an additional phosphor or a quencher, the device of item 16. (Item 18) The biosensing layer comprises at least one control probe, and the sensor is configured to collect a control signal from the at least one control probe and use the control signal to normalize the collected signal, the device according to item 12. (Item 19) The at least one control probe does not bind to or interact with the analyte, the device according to item 18. (Item 20) The device according to item 12, further comprising a reaction chamber, a controllable fluid unit, a temperature control unit, and a digital unit. (Item 21) The reaction chamber is configured to bring the solution and the chip into interfacial contact, the interfacial contact comprising an interaction between the analyte and the biosensing layer of the chip, the device according to item 20. (Item 22) The controllable fluid unit is configured to transport at least a portion of the solution in and out of the reaction chamber, the device according to item 20. (Item 23) The digital unit is configured to receive or store the output signal from the chip, the device according to item 21. (Item 24) The chip is configured to repeat (i)-(ii) a plurality of times prior to (iii), the device according to item 1. (Item 25) The output signal is a single output, the device according to item 24. (Item 26) A method for detecting the presence or absence of an analyte in a solution, comprising: (a) activating a chip comprising a sensor with an electronic shutter, the sensor being configured to (i) collect a signal generated in response to exposure of the solution to an excitation pulse within a first time period, (ii) use the electronic shutter to remove photoinduced charges generated within the sensor by the excitation pulse within a second time period, the second time period being different from the first time period, and (iii) generate an output signal at least partially derived from the signal following removal of the photoinduced charges, the output signal indicating the presence or absence of the analyte; (b) using the electronic shutter to remove photoinduced charges generated within the sensor by the excitation pulse within the second time period; (c) collecting a signal generated in response to exposure of the solution to the excitation pulse within the first time period; (d) generating an output signal at least partially derived from the signal following removal of the photoinduced charge, the output signal indicating the presence or absence of the analyte; A method comprising. (Item 27) The method according to item 26, wherein the sensor is a time-gated fluorescence (TGF) optical sensor. (Item 28) The method according to item 26, further comprising integrating the signal collected in (c) using the sensor. (Item 29) The method according to item 26, further comprising repeating (b)-(c) one or more times. (Item 30) The method according to item 29, wherein the one or more times comprises a number greater than or equal to about 100 times. (Item 31) A device for detecting a signal, A chip comprising a sensor and an electronic shutter, the sensor being configured to (i) detect the signal within a given time period and (ii) provide data indicative of the charge generated by the signal, the electronic shutter being configured to remove photoinduced charge comprising charge generated by an excitation pulse within a time period prior to the given time period; A readout circuitry operably coupled to the sensor, the readout circuitry being configured to transmit data from the sensor to a memory; A device comprising. (Item 32) The device according to item 31, wherein the readout circuitry is part of the chip. (Item 33) The device according to item 31 or 32, wherein the memory is external to the readout circuitry. (Item 34) The chip comprises a sensor array having a plurality of individually addressable locations, The sensor and the electronic shutter are disposed on a first location of the plurality of individually addressable locations, The device according to any one of items 31-33, wherein a second sensor and a second electronic shutter are disposed on a second location of the plurality of individually addressable locations. (Item 35) The device according to any one of items 31-34, wherein the sensor is further configured to integrate the charge generated by the signal. (Item 36) The device according to any one of items 31-35, wherein the sensor comprises an integration switch. (Item 37) The sensor includes at least one photo / charge transducer and at least one charge integrator, and the at least one integration switch is located between the at least one photon / charge transducer and the at least one charge integrator, the device according to item 36. (Item 38) The chip is included within a complementary metal oxide semiconductor (CMOS) integrated circuit (IC), the device according to any one of items 31-37. (Item 39) The chip further includes a biosensing layer adjacent to the sensor, and the biosensing layer includes a surface with a plurality of probes, the device according to any one of items 31-38. (Item 40) The phosphor is attached to at least one of the plurality of probes, the device according to item 39. (Item 41) The plurality of probes includes at least one control probe, the device according to any one of items 39-40. (Item 42) The device further includes a reaction chamber, a controllable fluid system, a temperature control system, and a digital system, according to any one of items 39-41. (Item 43) The reaction chamber interfaces the sample and the biochip, and the interfacing includes an interaction between the sample and the biosensing layer of the chip, the device according to item 42. (Item 44) The controllable fluid system transports at least one reagent into and / or out of the reaction chamber, the device according to item 42 or 43. (Item 45) A method for detecting a signal, (a) activating a chip including a sensor and an electronic shutter, where the sensor is configured to (i) detect the signal within a given time period and (ii) provide data indicative of the charge generated by the signal, and the electronic shutter is configured to remove photoinduced charge including charge generated by an excitation pulse within a time period prior to the given time period, (b) using the electronic shutter to remove the photoinduced charge within the time period prior to the given time period, (c) using the sensor to detect the signal within the given time period and provide data indicative of the charge generated by the signal, (d) transmitting the data to a memory and including, the method. (Item 46) The method according to item 45, wherein the sensor is a time-gated fluorescence (TGF) optical sensor. (Item 47) (c) The method according to item 45 or 46, further comprising integrating the charge generated by the signal using the sensor. (Item 48) The method according to any one of items 45-47, further comprising repeating (a)-(c) one or more times. (Item 49) The method according to item 48, wherein the number of times of the one or more times is equal to or greater than about 10 times, 50 times, or 100 times. (Item 50) The method according to any one of items 48-49, further comprising generating an output signal using the chip. (Item 51) The method according to item 50, wherein the output signal is a single output signal. (Item 52) The method according to any one of items 45-51, wherein the chip comprises a plurality of independently addressable locations. (Item 53) The method according to item 52, wherein the chip further comprises an additional sensor and an additional electronic shutter, the sensor and the electronic shutter are disposed on a first location of the plurality of independently addressable locations, and the additional sensor and the additional electronic shutter are disposed on a second location of the independently addressable locations. (Item 54) The method according to item 53, further comprising removing additional optically induced charge within the time period prior to the given time period using the additional electronic shutter. (Item 55) The method according to item 54, further comprising detecting additional charge generated by an additional signal within the given time period using the additional sensor, and providing additional data indicating the additional charge generated by the additional signal. (Item 56) The method according to item 55, further comprising integrating the additional charge using the sensor. (Item 57) The method according to any one of items 52-56, wherein the plurality of independently addressable locations comprises a location equal to or greater than about 100 locations, 1,000 locations, or 100,000 locations. (Item 58) The method according to any one of items 52-57, wherein the plurality of independently addressable locations comprises a location equal to or greater than about 100 locations and is a pixel. (Item 59) A method for operating time-gated fluorescence (TGF) detection, comprising: (a) Activating a chip comprising a surface and an integrated circuit (IC) having at least one optical sensor, wherein the IC comprises an electronic shutter; (b) Directing a pulse of excitation light from an excitation light source onto the surface; (c) Using the electronic shutter to remove first optically induced charge from the optical sensor during a first time period, the first optically induced charge comprising charge generated by the pulse of excitation light during the first time period; (d) Measuring second optically induced charge generated within the optical sensor during a second time period following the first time period, wherein the surface is not exposed to the excitation pulse during the second time period; (e) Integrating the second optically induced charge measured in (d) during the second time period; A method comprising the above steps. (Item 60) The method according to item 59, wherein the integrating is performed by using a sub-circuit provided within the chip. (Item 61) The method according to any one of items 59 - 60, further comprising repeating (a) - (e) one or more times. (Item 62) The method according to item 61, wherein the number of times of the one or more times is about 10 times, 50 times, or more than or equal to 100 times. (Item 63) The method according to any one of items 61 - 62, further comprising generating an output signal. (Item 64) The method according to item 63, wherein the output signal is a single output. (Item 65) The method according to any one of items 60 - 64, further comprising resetting the sub-circuit once. (Item 66) The method according to item 65, wherein the sub-circuit is not reset during the repetition. (Item 67) The method according to item 65, wherein the sub-circuit is not reset during each of the repetitions. (Item 68) (b) The method according to item 65, further comprising resetting the sub-circuit prior to (b). (Item 69) The method according to any one of items 59 - 68, wherein there is a gap between the first time period and the second time period. (Item 70) The method according to any one of items 59 - 69, wherein the integrating comprises integrating a photocurrent. (Item 71) The method according to item 70, further comprising converting the integrated photocurrent from an analog format to a digital format. (Item 72) A device, comprising a chip operably coupled to a light source, said chip comprising a sensor, said sensor being configured to (a) periodically detect one or more signals from a specimen associated with a surface of said chip, said one or more signals being generated during or subsequent to exposing said specimen to said light source, (b) integrate at least a subset of said one or more signals detected in (a) to generate an integrated signal, and (c) generate an output signal based on said integrated signal, a device. (Item 73) The device according to item 72, wherein said chip comprises an integrated complementary metal oxide semiconductor (CMOS) chip. (Item 74) The device according to item 72 or 73, wherein said output signal is a single output signal. (Item 75) The device according to any one of items 72-74, wherein said sensor is a time-gated fluorescence (TGF) sensor. (Item 76) The device according to any one of items 72-75, wherein said device does not comprise an optical filter disposed adjacent to said chip. (Item 77) The device according to any one of items 72-76, wherein said chip comprises a sensor array comprising a plurality of sensors. (Item 78) The device according to item 77, wherein each of said plurality of sensors is disposed at an individually addressable location of said sensor array. (Item 79) The device according to any one of items 72-77, wherein said specimen comprises a phosphor. (Item 80) The device according to item 79, wherein said output signal is used to measure the lifetime of said phosphor. (Item 81) The device according to any one of items 72-80, wherein said specimen is immobilized on said surface. (Item 82) The device according to any one of items 72-81, wherein said one or more signals comprise fluorescent photons. (Item 83) The device according to item 82, wherein said sensor comprises a transducer configured to convert said fluorescent photons into an electrical signal. (Item 84) The device according to item 82 or item 83, wherein said sensor comprises a transducer configured to convert said fluorescent photons into charge. (Item 85) The device according to any one of items 72-84, wherein said sensor further comprises an integrator configured to integrate said one or more signals. (Item 86) The sensor is the device according to item 85, comprising a switch operably coupled to the transducer and the integrator. (Item 87) The switch is the device according to item 86, transporting the charge from the transducer to the integrator. (Item 88) The integrator is the device according to any one of items 84 - 87, operably coupled to an additional transducer. (Item 89) The additional transducer is the device according to item 88, converting the charge into an electrical signal, thereby generating the output signal comprising the electrical signal. (Item 90) The electrical signal is the device according to item 89, comprising a voltage. (Item 91) The light source is the device according to any one of items 72 - 90, which is a pulsed light source. (Item 92) A method, comprising: (a) Activating a chip comprising a sensor, wherein the sensor is configured to: (i) periodically detect one or more signals from a sample associated with the surface of the chip, the one or more signals being generated during or subsequent to exposing the sample to a 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; (b) Directing the light source towards the chip to generate the one or more signals; (c) Periodically detecting the one or more signals from the sample during or subsequent to exposing the sample to the light source; (d) Integrating at least the subset of the one or more signals to generate the integrated signal; (e) Generating an output signal based on the integrated signal. A method including the above steps. (Item 93) The light source is the pulsed light source according to item 92. (Item 94) (c) is performed periodically at a given time interval according to any one of items 92 - 93. (Item 95) (c) occurs during or after each time the pulsed light source is turned off according to any one of items 93 - 94. (Item 96) (d) is performed using an integrator according to any one of items 92 - 95. (Item 97) (c) or (e) is performed using a transducer according to any one of items 92 - 96. (Item 98) The method according to any one of Items 92-97, wherein the output signal is an electrical signal. (Item 99) The method according to any one of Items 92-98, wherein the one or more signals are detected by the sensor in the absence of passage through an optical filter.

[0026] (Incorporation by reference) All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated and incorporated by reference.

Brief Description of the Drawings

[0027] The novel features of the invention are set forth in the appended claims. A more complete understanding of the features and advantages of the invention will be obtained by reference to the following detailed description of the invention, which describes illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures" and "FIGs").

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[0044] (Detailed Description) Although various detailed embodiments are illustrated and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, adaptations, and substitutions can be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments described herein may be employed.

[0045] 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 acid (PNA), polypeptides such as proteins, peptides, antibodies, antigens, enzymes, and receptors, as well as tissues, organelles, and other receptor probes.

[0046] As used herein, the term "probe" or "capture probe" generally refers to a molecular species and / or other marker that can specifically bind to an analyte or target. A probe can comprise a molecule and can be bound directly or via a linker to a substrate, molecule, or other solid surface. Non-limiting examples of linkers include amino acids, polypeptides, nucleotides, oligonucleotides, and chemical linkers. A plurality of probes can be immobilized on a substrate, molecule, or other solid surface and can be referred to as a probe array. The plurality of probes in a probe array can be arranged uniformly, for example, as an array of spots, or non-uniformly.

[0047] As used herein, the term "about" or "approximately" generally refers to within + / - 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the specified amount.

[0048] 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) and that can render the molecule detectable, distinguishable, and / or traceable by providing a property that cannot be inherent to the target molecule. Examples of labels can include luminescent molecules (e.g., phosphors), redox (reduction-oxidation) species, or enzymes. In some cases, the label may comprise a phosphor with a long lifetime, such as lanthanide chelates and transition metal chelates, which are, for example, luminescent or phosphorescent.

[0049] As used herein, the term "nucleotide" generally refers to a molecule that can serve as a monomer or subunit of a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleotide can be a deoxyribonucleoside triphosphate (dNTP) or an analog thereof, for example, a molecule having multiple phosphates, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphates, in a phosphate chain. A nucleotide can generally include adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid chain. Such a subunit can be A, C, G, T, or U, or one or more complementary A, C, G, T, or U, or any other subunit that is specific to A, C, G, T, or U, or is complementary to a purine (i.e., A or G or a variant thereof) or a pyrimidine (i.e., C, T, or U or a variant thereof). The subunit can be capable of allowing individual nucleobases or base groups (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or the uracil counterparts thereof) to be cleaved. Nucleotides can be labeled or not. Labeled nucleotides can provide a detectable signal, such as an optical, electrostatic, or electrochemical signal.

[0050] As used herein, the terms "polynucleotide", "oligonucleotide", "nucleotide", "nucleic acid", and "nucleic acid molecule" generally refer to polymeric forms of nucleotides (polynucleotides) of any length, 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 of any length that can be amplified in 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 than 10,000 nucleotide lengths, or at least about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000, or 10,000 nucleotide lengths.

[0051] As used herein, the terms "peptide", "polypeptide", and "protein" are used synonymously 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 joined to each other by peptide bonds. Examples of polypeptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptides, and their isoforms, modified polypeptides, derivatives, analogs, fusion proteins, or combinations thereof. A polypeptide may be a natural peptide, a recombinant peptide, or a combination thereof.

[0052] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0053] As used herein, the term "detector" generally refers to a device that can detect signals and generally includes optical and / or electronic components. TGF System Overview

[0054] The present disclosure provides methods, devices, reagents, and systems based on time-gated fluorescence (TGF). The system may comprise a TGF-based biochip. The TGF biochip may be semi-conductor integrated. In some cases, the semiconductor platform and manufacturing process through which the system is created is complementary metal oxide semiconductor (CMOS).

[0055] The methods and systems of the present disclosure may be used to detect, analyze, and / or quantify a plurality of analytes present in an aqueous sample through a TGF transduction method. The TGF CMOS biochip can be a monolithically 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 hereby incorporated by reference in its entirety. Each addressable location may comprise an independently operating TGF optical sensor that detects a TGF signal from its dedicated sensing area. Sensing / detection may be performed in real time and in the presence of an aqueous sample or when such a sample is washed. The TGF optical sensor can employ a periodic charge integration (PCI) method in which periodic signal accumulation is performed by applying a plurality of gate excitation pulses. The TGF CMOS biochip system can apply physiochemical processes to the aqueous sample, including physically interfacing with the aqueous sample and applying, for example, a time-varying temperature profile, biochemical reagents, or a pulsed excitation photon beam to the sample.

[0056] The TGF CMOS biochip system as illustrated in FIG. 2 can consist of components including, but not limited to, the following. 1. TGF CMOS Biochip: In a 2D array format, the analyte interface with its top surface can be identified and detected through the TGF transduction method. 2. Reaction Chamber: The sample fluid (e.g., a fluid aqueous sample containing the analyte) can be interfaced with the TGF CMOS biochip. 3. Excitation Source: In a controlled manner and synchronized with the TGF CMOS biochip operation, a photon beam specific to the wavelength can be introduced into the reaction chamber and / or onto the TGF CMOS biochip surface. 4. Controllable Fluid System: In a controlled manner and synchronized with the TGF CMOS biochip operation, it is configured to move reagents and / or samples into, and / or remove from, and / or hold in the reaction chamber. 5. Temperature Controller: In a controlled manner and synchronized with the TGF CMOS biochip operation, the temperature of the fluid in the reaction chamber can be set. 6. TGF Reagents and Reporter Molecule Constructs: Detection of analytes and targets can be enabled by the TGF CMOS biochip in the reaction chamber and according to a specific assay methodology. 7. Digital System: It can coordinate the operations of one or more components provided within the system, collect data, and / or process data and / or communicate with a data analysis unit. TGF CMOS Biochip

[0057] As shown in Figure 3, the TGF CMOS biochip can comprise components including, but not limited to, the following. A. CMOS Integrated Circuit (IC): It can include the following functional blocks embedded within its monolithically integrated semiconductor substrate. i. TGF optical sensor array: Comprises a plurality of detectors in a 2D array format. Individual detectors (e.g., "biosensing elements" or "pixels") can measure, in parallel, simultaneously, and independently, the photon flux emitted from a phosphor (F e ) at their addressable locations. The detectors can also employ the periodic charge integration (PCI) TGF method. ii. Readout circuitry: Can obtain data from individual TGF pixels and communicate them, serially, in parallel, or in combinations thereof, to an off-chip unit (external destination). iii. On-chip passive resistor heater and temperature sensor. B. Biosensing layer: Can be located on the surface of a CMOS IC and create a sample-specific localized TGF signal that is coupled to the TGF pixels using the TGF method. The biosensing layer can comprise a plurality of probes at independently (and / or individually) addressable locations on a solid surface. Each pixel can comprise a plurality of the same or different probe molecules that specifically bind to or interact with a specific target / sample or reagent within the reaction chamber. CMOS integrated circuit (IC)

[0058] The architecture of an integrated CMOS IC for a TGF biochip is illustrated in FIG. 4. The CMOS die includes a 2D optical sensor array and has a general readout circuitry architecture similar to other biosensor arrays. 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 hereby incorporated by reference in its entirety. The optical sensor array where the same CMOS-embedded TGF pixels are installed can be read serially (i.e., one pixel at a time) using row and column decoders. The output of the chip, which is sent off-chip through an output buffer, can be either analog or digital.

[0059] The chip may also include a resistive heater and a temperature sensor and be adapted for 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). Additionally, the CMOS IC may also include a control block that is programmed and accessed off-chip by the user to configure the functionality of the chip and manage data acquisition.

[0060] The general topology of an exemplary TGF pixel is shown in FIG. 5. The TGF receives both F e and F X from addressable locations on its biosensing layer, and photons can be converted to electrical charge by using a photon-charge transducer (PCT). Examples of PCTs in a CMOS process 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 buried photodiode devices (e.g., Hondongwa, D. B. et al. “A review of the pinned photodiode for CCD and CMOS image sensors,” IEEE J. Electron Devices Soc., 2(3):33-43, 2014). The PCT device may be provided with two switches connected thereto. The first one may be an electronic shutter switch (S S ), which completely removes the charge from the PCT by connecting it to an electronic shutter voltage source (V S ). The second one is an integration switch (S I) may also be, which transports the created charge into the charge integrator element (CIE). The CIE device may be continuously connected to a charge / voltage transducer (CVT) and generate a TGF pixel output. In addition, the CIE has a reset switch (S R ) and can remove the integrated charge at any time, essentially "resetting" the CIE output value to V R .

[0061] The TGF pixels of the present disclosure may differ from conventional detectors for TGF or time-resolved fluorescence. One difference is the absence of S S and S I and the ability to selectively discard or integrate the generated charge of PCT. In FIG. 6, an exemplary timing diagram regarding the operation for a conventional TGF system is shown. As shown in FIG. 6, F e is measured for each of the N individual F X pulses by quantifying the photoinduced charge during the integration time interval. The N outputs (X OUT [1] to X OUT [N]) are then averaged to estimate F e . Multiple problems and non-idealities may exist in this system. For example, the following are. · It may be necessary to obtain N consecutive measurements (readings) and estimate F e or all pixels. Since F e can be low, a wide range of averaging may be required. For example, values of N > 100 may be required in such TGF systems. · Due to low-level signals (e.g., 10 total electrons per F e pulse), the CVT may require a very high gain (e.g., > 20 μV / e) with analog / digital quantization noise comparable to less than a few electrons per reading. · When a large biosensor array can be implemented with a pixel count M > 1,000, the number of readings per frame is N × M, which can quickly become a compressed state. For example, if the phosphor used in TGF has a lifetime of τ L = 100 ns, the period 1 ms = 10τL accompanied by an F X pulse sequence can be created. When N = 100 and M = 1,000, the readout speed will be 10 5 readings / ms or 100 million samples / second. Assuming the noise requirements of the system, this requires a very complex readout circuitry and can require a significant amount of power. As a result, reducing the pulse sequence frequency and essentially slowing down the TGF measurement can be considered.

[0062] In the present disclosure, by using the topology shown in FIG. 4, the aforementioned problems can be solved. FIG. 7 depicts a timing diagram of the TGF pixel of the present disclosure that employs an in-pixel periodic charge integration (PCI) scheme to improve both the speed and performance of TGF measurements. As shown in FIG. 7, S S and S I are used and by applying an electronic shutter, the responses of the N pulses of PCT can be integrated into the CIE, which can create a single output. This can enable the reading of one output per N pulses with an amplitude N times larger than that of conventional TGF. Additional advantages of this approach can include, but are not limited to, the following. · One reading in PCI-TGF can be equal to N readings in conventional TGF. · The accumulated charge and output amplitude signals of PCI-TGF can be N times that of conventional TGF and can be read at 1 / N of the low frequency. Thus, PCI-TGF can use a much less restrictive readout circuitry with slower speed and higher serial quantization noise of the signal. · When a large biosensor array with the number of pixels M > 1,000 elements is used, the required readout and pixel scanning speed requirements can be 1 / N of that of conventional TGF. Thus, it can be very feasible to create an array with a number of M > 10 6 which may be necessary for the adoption of large-scale parallel arrays used in life science research.

[0063] The challenges in the implementation of PCI-TGF can be addressed by the switch circuitry and device implementation, an efficient approach to transport charge over time intervals compatible with TGF, and the CIE. Biosensing layer

[0064] A biosensing layer as provided herein is fabricated on top of a CMOS IC, interfacing with a reaction chamber, and may include an organic layer that (a) forms an addressable location for probes on top of the pixels, and (b) enables TGF transduction to first capture a target and subsequently create a TGF signal as a function of the probe-target interaction and / or structure of the captured target.

[0065] The biosensing layer may be fabricated in a variety of ways. For example, specific probe structures may be physically printed, immobilized, or spot welded onto the surface, or chemically synthesized. In some cases, the probes are first randomly dispersed within the 2D surface of the array and then identified prior to detecting the target by alternative approaches known in the art. In some cases, the surface of the IC (typically made from SiO 2 or Si 3 N 4 may be chemically modified with linkers and / or thin film structures to be compatible with probe attachment.

[0066] Figure 8 shows an embodiment of a biosensing structure that is compatible with a TGF transduction method, including a CMOS IC and a PCI-TGF. In FIGS. 8A and 8B, the planar surface may be implemented to immobilize the probes, and the addressable arrays may be created with or without a thin film barrier, respectively. In FIGS. 8C and 8D, a 3D and permeable matrix may be coated on the surface to enable probe immobilization in close proximity to the surface. In FIGS. 8E and 8F, microwells may be used to better isolate the immobilized probes and isolate TGF pixels. In FIG. 8G, a combination of microwells and microbeads with immobilized beads may be used to create an addressable array. Reaction chamber

[0067] A reaction chamber as provided herein may be a fluid chamber that interfaces with a CMOS TGF biochip and contains a fluid sample with a specimen, a target, and other biochemical reagents required for the execution of a TGF assay.

[0068] The volume of the reaction chamber can be from about 0.1 μL to 10,000 μL, for example, from about 1 μL to 100 μL.

[0069] The reaction chamber may comprise a plurality of inlets and outlets and be adapted for interfacing with a controllable fluid system to insert or remove fluid.

[0070] To be adapted for TGF, the fluid system can provide a transparent optical propagation path for the pulse F X to pass through the fluid and reach the biosensing layer. The transmittance of the wavelength of F X can be from 1% to 99.9%, but typically can be from 5% to 80%.

[0071] The reaction chamber can be constructed using various materials such as polymers, glass, semiconductors, crystals, or ceramic materials, or combinations thereof. Excitation source

[0072] An excitation source as provided herein may comprise an optical light source capable of creating a wavelength - selective photon beam (F X ) with controllable and time - varying amplitude. The light source may illuminate the biosensing layer of the system and the coordinates where TGF transduction occurs.

[0073] The excitation source center wavelength can be anywhere from about 200 nm to 1500 nm, for example, from about 300 nm to 800 nm.

[0074] The excitation source spectral span (bandwidth) may be from about 1 nm to 500 nm, for example, from about 10 nm to 100 nm.

[0075] The excitation source photon beam may be directional and may be optically collimated.

[0076] The excitation source peak output power may be from about 10 mW to 100 W, for example, from about 100 mW to 10 W.

[0077] The excitation source power is controllable and may be modulated using a bandwidth of up to about 1 GHz, for example, up to about 1 MHz.

[0078] The excitation source off and on times may be as fast as about 0.1 nanoseconds (ns), for example, as fast as about 1 microsecond (μs). Controllable fluid system

[0079] A controllable fluid system may introduce, and / or remove, and / or confine, in a controlled manner, an aqueous medium containing a sample and assay reagents and / or TGF transduction reagents into the reaction chamber by the user. The workflow and sequence of each fluid operation may be defined by the assay method and can be, for example, flow - through and unidirectional, or a closed - tube.

[0080] A controllable fluid system may perform a workflow using fluid components such as pumps, valves, and pipes. Temperature controller

[0081] A temperature controller system can establish a specific temperature for the fluid in a reaction chamber and / or create a temperature profile that requires heating and / or cooling. The temperature controller can measure the temperature using a temperature sensor (such as a thermistor or thermocouple) within a CMOS biochip IC and / or sensor device that is coupled to the reaction chamber, and based on the measured temperature, use a CMOS biochip IC heater and / or a heat device (such as a Peltier device or a resistive heater) to add heat to or remove heat from the reaction chamber, and can include a feedback control system. The temperature controller can include a heat sink for removing heat. The temperature controller can have components that include a resistive heater and / or a temperature sensor within the CMOS IC.

[0082] The temperature controller can change the temperature of the substrate, reaction chamber, or array pixel. The rate of change of temperature 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 / min. The rate of change of temperature 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 / min. The rate of change of temperature 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 / min. The temperature controller can change the temperature at a linear rate (e.g., 5 °C / sec). Alternatively, the temperature controller can change the temperature at a non-linear rate. The temperature controller can increase or decrease the temperature. Digital system

[0083] A digital system is essentially computing and control digital hardware and embedded software that can control and coordinate the functionality of the components of the system. TGF reagent and reporter molecule construct

[0084] To enable TGF transduction, molecular structures and constructs that exhibit fluorescence activity can be used. Such molecular structures are sometimes also referred to as phosphors (or fluorescent dyes, similar to chromophores), which are chemical compounds that re-emit light in response to photoexcitation, with a lifetime of about 10 ps to 10 ms, e.g., about 1 ns to 100 ns.

[0085] As provided herein, various types of phosphors can be employed by the TGF system. In some cases, for example, phosphors having a longer lifetime, greater than about 100 ns, may be used. When phosphors with longer lifetimes are used, the following occurs. · Phosphors with longer lifetimes may require a slower PCI-TGF system within the CMOS biochip IC. · The excitation source switching speed can be more manageable, and more cost-effective light sources can be used. · The adverse effects of background autofluorescence from biological samples and / or materials within the fluid chamber and / or biological layer can be mitigated if they have a shorter lifetime compared to the TGF phosphor employed.

[0086] The TGF system may not require an excitation and emission filter set or other wavelength filters to transmit the desired signal regarding the analyte and / or remove background fluorescence from the analyte signal. In some cases, the emission filter may filter out violet, blue, green, yellow, orange, and red light, or any combination thereof.

[0087] Various types of phosphors can enable multi - color capabilities. In TGF, differentiating phosphors can be determined by differences in their fluorescence lifetimes after excitation. In some cases, these phosphors can be reactive and / or conjugated dyes, nucleic acid dyes, fluorescent proteins, as well as cell - function dyes. Once the emitted light is pulsed in the direction of the substrate containing the phosphor species, the shutter can block the detection device from the emitted light and the reflected emitted light. The shutter can be removed to allow the desired fluorescent light to pass through. A first phosphor with a shorter lifetime can be detected between the detected signals if the shutter is opened immediately after the emission is stopped. A second phosphor with a longer lifetime can be detected if the shutter is opened after waiting for a longer time after the emission is stopped. In this scenario, the second phosphor (longer lifetime) can be detected with little or no interference to the first phosphor (shorter lifetime). Additionally, the read - out value of the signal corresponding to the first phosphor (shorter lifetime) in the presence of the second phosphor (longer lifetime) can be estimated or calculated by calibration of the detected signal using information about the second phosphor (longer lifetime) that is detected and / or determined later. Other experimental designs for multiplex detection of multiple phosphors are also conceivable, with or without the use of emission and excitation filter sets. Thus, multiple phosphors can be detected within a single experiment by opening and closing the shutter following emission, in the absence of emission and excitation filter sets. Individual species of phosphors can be detected based on differences in their decay rates by varying the shutter delay profile and the time at which the shutter is opened. The multi - color capability can be limited by the overlap between the shutter speed and the fluorescence decay rate of the phosphors to be detected.

[0088] For example, in some embodiments, metal chelates such as lanthanide chelates can be used as TGF phosphors. In some cases, the TGF phosphor can act as a molecular reporter in a TGF assay, primarily as either an independent reporter or element (donor or acceptor) within the fluorescence energy transport portion. Embodiments include, but are not limited to, Forster 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 role of the TGF phosphor can include promoting the generation of a specific TGF signal that can correlate with the presence or absence of a molecular reaction or a specific target molecule.

[0089] The TGF phosphor can be used as a label for a specific target analyte in applications where the target can be chemically modified to incorporate the TGF phosphor. Embodiments include, but are not limited to, Northern blot, Southern blot, DNA microarray, quantitative polymerase chain reaction (PCR), digital PCR, and diagnostic assays. · In microarrays and Northern blots, mRNA target analytes can be converted to phosphor-labeled complementary DNA (cDNA), for example, through reverse transcription. · In Southern blots, phosphor-labeled cDNA can be used to identify target sequences. ·In quantitative polymerase chain reaction (PCR) and digital PCR (dPCR), the phosphor is incorporated into the amplified nucleic acid sequence or primer sequence and can demonstrate the accumulation of the target sequence (see, for example, Y. Wong et al., “Applications of digital PCR in precision medicine,” Expert Review of Precision Medicine and Drug Development 2(3):177-186, 2017). ·In diagnostic assays, a device can be used to isolate the target nucleic acid, and a phosphor-labeled cDNA can be used for direct detection.

[0090] The TGF phosphor can also be used as a label for the detection of probes in sandwich assays. Examples include, but are not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT) including FluoroSpot assay (see, for example, G. Kesa et al., “Comparison of ELISpot and FluoroSpot in the Analysis of Swine Flu-Specific IgG and IgA Secretion by in Vivo Activated Human B Cells,” Cells 1(2):27-34, 2012), and protein arrays. ·In these methods, the TGF phosphor can be used as a direct method for detection where the phosphor is conjugated to the primary detection antibody. ·In these methods, the TGF phosphor can also be used as an indirect method for detection where the phosphor is conjugated to the secondary antibody. ·ELISPOT is an assay type that quantitatively measures the frequency of cytokine secretion for single cells. The ELISPOT assay is also a form of immunostaining that uses antibodies to detect a specimen, including, but not limited to, any biological or chemical substance that has been identified or measured, such as a protein specimen. ·The FluoroSpot assay is a variant of the ELISPOT assay. The FluoroSpot assay uses fluorescence to analyze multiple specimens. It can detect the secretion of more than one type of protein or other specimen.

[0091] The TGF fluorophore can be used as a label in cell sorting, counting, and detection methods. An example can be flow cytometry where cells are labeled with the fluorophore. ·In this method, cells can be sorted and counted by their fluorescence profile. ·In this method, specific cell characteristics and / or functions can be identified by their fluorescence profile.

[0092] The TGF fluorophore can be used in applications where solid phases and immobilized probes are labeled. An example is the 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).

[0093] The TGF phosphor can be used in assays where a chemical reaction is monitored while a target molecule is introduced to a reaction reagent. The target molecule and / or the reaction reagent may contain the TGF phosphor. 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 pyrosequencing. · In this method, single molecule real time (SMRT) sequencing and Illumina sequencing can determine the sequence of nucleic acids using TGF phosphor-labeled nucleotides.

[0094] Nucleic acid sequence information can be used to improve people's lives (see, for example, Ansorge, W., “Next-generation DNA sequencing techniques,” New Biotech. 25(4):195-203, 2009). Several DNA sequencing platforms are commercially available. The availability of parallel NGS technologies can enable comprehensive analysis for biological targets, including but not limited to genomes, transcriptomes, and interactomes (see, for example, Shendure, J. and Ji, H., “Next-generation DNA sequencing,” Nature Biotech. 26:1135-45, 2008). However, while NGS technologies can generate comprehensive results, their response time may be too slow to cope with the rapid progression of infectious processes in critically ill patients. In addition, multiplexing a large number of target amplification reactions (e.g., multiplexed PCR) may be considered possible, but it is not easy to detect multiple amplicons simultaneously.

[0095] Commercially available NGS sequencing platforms may include Illumina's Genome Analyzer, Roche (454)'s Genome Sequencing System, Life Technologies' SOLiD platform, and real-time sequencing devices such as those from Pacific Biosciences. These platforms may require constructs of sets of DNA fragments from biological samples. In most cases, the DNA fragments are adjacent to platform-specific adapters.

[0096] (Example 1) In this embodiment, a fully integrated TGF CMOS biochip specifically designed for addressable arrays of DNA and proteins in biotechnology is presented. As shown in FIG. 9, a CMOS IC is assembled on a printed circuit board (PCB) and then integrated with a fluid module to create a biochip consumable. The biochip IC includes an array of 1,024 biosensor pixels with an integrated emission filter of optical density (OD) of approximately 5.8, and addressable (unique) immobilized probes (DNA) for each pixel. Pixel-level optical sensors with Nwell-Psub photodiodes (acting as PCT elements) are designed to be shot-noise limited and provide a detection dynamic range (DDR) of >130 dB. A temperature control and circulation system is also integrated within the biochip to adapt to thermal control. For this reason, both a bandgap temperature sensor and a resistive heater are integrated to achieve a heating / cooling rate of + / -10 °C / second with an overall accuracy of ±0.25 °C within the range of 25 °C to 100 °C.

[0097] The chip architecture and 120 μm pitch biosensing pixels and decimation cells are shown in FIGS. 10A and 10B. The TGF pixels in the 32×32 array include a ΔΣ current detector that takes the photocurrent I ph as its input and generates a 1-bit digital output stream that is transported into an on-chip decimation array. The optical sensor circuitry (FIG. 10B) includes a current integrator (acting as CIE+CVT), a clocked comparator (ADC), and a programmable current source (DAC).

[0098] In CWF mode (i.e., without a pulsed excitation source or electronic shutter), the ΔΣ current detector operates continuously with a frequency of f C while the decimation cell performs a two-stage 32-bit accumulation and then continues downsampling and readout with a frequency of f S such that sinc 2Implement a filter. In TGF mode, similar operations are performed, except for the periodic activation of an electronic shutter that can bypass I ph This operation blocks the optical excitation pulse and typically reduces the natural autofluorescence background from biological media having a lifetime < 50 ns. The chip then accumulates and measures the fluorescence emission at pre-programmed time intervals.

[0099] In this chip, all TGF pixels, decimation array, bandgap temperature sensor, and reference voltage DAC are operated, read, and accessible through a Serial Peripheral Interface (SPI) port (Figure 10A) by a single digital core block operating at 50 MHz. A single resistive heater can provide up to 20 W using an external source, has a meandering structure, and is uniformly distributed within the upper metal layer. This chip can be fully operated and promote efficient fluid assembly and consumable manufacturing (Figure 9) using 14 pins (and bonding wires) integrated on one side of the die.

[0100] In Figure 11, a schematic and timing diagram of the light-sensing pixel in TGF mode are depicted. A capacitive transimpedance amplifier (CTIA) is used as CIE+CVT, and a clocked comparator creates the pixel output D out The DAC is implemented by using a current source that can be used to apply a current pulse into the CTIA input using two adjustable durations (Φ 1 and Φ 2 ). The electronic shutter uses SH 1 , SH 2 , and SH 3 to temporarily remove C f , the feedback capacitor of the CTIA, from the circuit and at the same time uses an operational amplifier to short I ph to V d . Due to transistor mismatch, a small amount of charge is added to C fis injected into this, which is exposed as the pixel-dependent electronic shutter offset current I S This current, when added to the dark current I dc forms the pixel random offset current I O = I S + I dc which is measured and extracted in both CWF and TGF modes to estimate I ph This is done using a CDS approach where one frame with excitation light and one frame without it are required and then the measurements are subtracted from each other.

[0101] The decimation array has a dedicated bit cell for each pixel. The bit cell consists of a 32-bit incrementer followed by a 32-bit adder, forming a two-stage accumulation unit (Figure 10B). At a time interval of T = 1 / f S the output of the adder is loaded onto a 32-bit shift register. The data from the shift register then passes into a digital unit in a serial scan chain fashion.

[0102] The electrical and optical measurements regarding this biochip are reported in Figure 12. The signal-to-noise ratio (SNR) measured from the pixel demonstrates that the additional sensor noise is about 30% of the shot noise when the quantization noise is not limited within the 100 fA - 1 nA input current region. The total dual depletion region (DDR) is 137 dB (1.33 fA - 10 nA) for f S = 1.667 Hz. The photodiode external quantum efficiency (QE) shows a passband and stopband QE of 0.4 and 3.69×10 -7 (OD ~ 5.8) respectively, regardless of the presence of the integrated emission filter. I dc and I OThe measured distribution verifies the expected randomness with a maximum amplitude of 100 pA (<1% of full scale). The output of the temperature sensor as a function of temperature is also reported in FIG. 12, which shows that an accuracy of ±0.25 °C can be achieved across the 25 °C to 100 °C temperature range using a two-point calibration.

[0103] In FIG. 13, results from two biosensing experiments are reported and compared, demonstrating different modes of operation. In all experiments, the same surface functionalization and array-based DNA hybridization or ligand-receptor binding is performed. However, distinctly different molecular labels are attached to the targets for CWF and TGF, respectively. For CWF, an R-phycoerythrin fluorophore is used and the signal-to-background ratio (S / B) shows a minimum value. This may be due to non-ideal blocking of the excitation light. The S / B is significantly increased when using TGF with DTBTA-Eu3+, a long-lived phosphor based on a europium (lanthanoid) chelate. As is evident, the background photon emission from a pulsed light-emitting diode (LED) excitation source decays significantly within 100 μs and the background becomes much smaller compared to the CWF mode.

[0104] In FIG. 14, a micrograph of the implemented TGF biochip is shown.

[0105] (Example 2) This example shows how PCI-TGF pixels can be designed for 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 a standard high-density image sensor array. As the example shows, PCI can be added into the circuitry of a CMOS image sensor of millions of pixels that can have sub-micron pixel dimensions.

[0106] Figure 15A depicts a circuit diagram embodiment of a 6-transistor (6T) pixel topology, which includes an embedded photodiode (PPD) as a PCT and two charge transport gates (one transports charge to a sensing node (TX) acting as an integration switch and one acts as an electronic shutter (SH)). The charge is integrated onto a floating diffusion layer (acting as CIE + CVT), and the generated voltage V S is read using a source follower gate. In this depiction, the pixel is located at the (i,j) coordinates within the photosensor array, and V S is assumed to be accessible by a column signal (COL[j]) by activating a row selection signal (SEL[i]). The charge in the floating diffusion layer can be reset using RST[i].

[0107] In Figure 15B, the layout of this pixel is shown, which can be scaled to sub-micron dimensions similar to equivalent CMOS image sensor pixels.

[0108] In Figure 16, a schematic of a PCI-TGF pixel is shown. As shown in Figure 16, correlated double sampling (CDS) can be implemented by reading V S during the reset cycle and after N PCI cycles. 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 voltage of the source follower transistor, respectively. Here, at the end of the Nth integration cycle, V S =V DD -ΔV n -V th -NΔQ / C, where ΔQ is the charge collected by the emission from individual excitation pulses and C is the floating diffusion layer effective capacitance. Therefore, by subtracting these two values (i.e., CDS), a value can be obtained that follows the PCI scheme while being independent of the offset of the source follower, which can vary for each pixel in the array.

[0109] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. The present invention has been described with reference to the foregoing specification, but the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. Further, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. Accordingly, the present invention is also considered to cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered.

Claims

1. A device for detecting the presence or absence of an analyte in a solution, comprising: a reaction chamber configured to receive and hold the solution; a biochip in interfacial contact with the reaction chamber, the biochip comprising: a biosensing layer comprising at least one probe that specifically binds to the analyte; a sensor that accumulates charge through fluorescence generated by an excitation pulse, the accumulated charge being resettable, the sensor comprising a photon - charge transducer (PCT), a charge integrator element (CIE), a charge / voltage transducer (CVT), and an electronic shutter configured to remove charge from the PCT to the outside, the sensor being configured to: (i) collect a signal from the solution generated in response to exposure of the solution to the excitation pulse during a first time period; (ii) use the PCT to convert the signal into a first photo - induced charge; (iii) selectively discard the first photo - induced charge during the first time period using the electronic shutter; (iv) during a second time period when the solution is not exposed to the excitation pulse, use the PCT to collect a fluorescence signal from the solution and convert it into a second photo - induced charge, the second time period following the first time period; (v) repeat steps (i) - (iv) a plurality of times and use the CIE to integrate the second photo - induced charge; (vi) generate an output signal at least partially derived from the integrated second photo - induced charge generated in (v) by the CVT; and a biochip, the output signal indicating the presence or absence of the analyte. A device comprising the above.

2. The biochip comprises a plurality of sensors arranged in a two - dimensional array at a plurality of individually addressable locations, each of the plurality of sensors being a pixel, a first sensor of the plurality of sensors being arranged on a first location of the plurality of individually addressable locations, a second sensor of the plurality of sensors being arranged on an additional location of the plurality of individually addressable locations. The device according to Claim 1.

3. The electronic shutter includes an electronic shutter switch operably coupled to the PCT, and the electronic shutter switch is configured to discard the first optically induced charge from the PCT in response to the application of a voltage to the electronic shutter switch. The device according to claim 1.

4. The sensor further includes an integration switch, the integration switch is disposed between the PCT and the CIE, and is operably coupled to the PCT and the CIE. The integration switch is configured to transport the second optically induced charge from the PCT to the CIE. The device according to claim 1.

5. The CVT is operably coupled to the CIE, and the CVT is configured to generate the output signal. The device according to claim 1.

6. The biochip is included within a complementary metal oxide semiconductor (CMOS) integrated circuit (IC). The device according to claim 1.

7. The fluorescent signal is generated by a label associated with the analyte in response to the binding of the analyte and the at least one probe. The device according to claim 1.

8. The label is a phosphor. The device according to claim 7.

9. The fluorescent signal is generated from the at least one probe or the analyte in response to the binding of the analyte and the at least one probe. The device according to claim 1.

10. The at least one probe includes an energy donor, and the analyte includes an energy acceptor. The device according to claim 9.

11. The energy donor is a phosphor, and the energy acceptor is an additional phosphor or a quencher. The device according to claim 10.

12. The device according to claim 1 further includes a controllable fluid unit, a temperature control unit, and a digital unit.

13. The reaction chamber is configured to interfacially contact the solution and the biochip, and the interfacial contact includes an interaction between the analyte and the biosensing layer of the biochip. The device according to claim 12.

14. The controllable fluid unit is configured to transport at least a portion of the solution in and out of the reaction chamber. The device according to claim 12.

15. The digital unit is configured to receive or store the output signal from the biochip, the device according to claim 13. **Claim 16** The output signal is a single output, the device according to claim 1.

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