METHODS AND SYSTEMS FOR TIME-GATED FLUORESCENT-BASED DETECTION

Abstract

The present disclosure provides methods, apparatus and systems for time-gated fluorescent-based detection. Time-based fluorescence analysis can be used in certain biochemical assays by measuring the emitted photon flux from fluorophores after an individual excitation pulse.

Claims

1.-30. (canceled)

31. A method for detecting a presence or absence of an analyte in a solution, comprising: directing the solution to a device comprising a reaction chamber configured to receive the solution comprising the analyte, and further comprising a temperature control system capable of changing the temperature of the solution in the reaction chamber, wherein the reaction chamber comprises a probe that binds to the analyte, and further wherein the probe is labeled with a long-lifetime fluorophore; applying a time-varying temperature profile to the reaction chamber; applying an optical excitation pulse to the analyte within a first time period; detecting periodically a fluorescent signal from the reaction chamber during a second time period subsequent to the first time period, wherein the optical excitation pulse is not applied during the second time period; and wherein the signal is indicative of the presence or absence of the analyte.

32. The method of claim 31, wherein the signal is indicative of binding of the analyte to the probe.

33. The method of claim 32, wherein the analyte is a nucleic acid.

34. The method of claim 33, comprising performing a polymerase chain reaction.

35. The method of claim 33, wherein the nucleic acid is an amplicon.

36. The method of claim 31, wherein the probe is a nucleic acid probe.

37. The method of claim 31, wherein the reaction chamber comprises a plurality of probes.

38. The method of claim 31, wherein the long-lifetime fluorophore is quenched by a quencher.

39. The method of claim 31, wherein the long-lifetime fluorophore has a lifetime longer than the first time period.

40. The method of claim 39, wherein the long-lifetime fluorophore has a lifetime of greater than 100 ns.

41. The method of claim 31, wherein the long-lifetime fluorophore is a lanthanide chelate-based fluorophore.

42. The method of claim 41, wherein the lanthanide is europium.

43. The method of claim 31, wherein the analyte comprises a quencher.

44. The method of claim 31, wherein the optical excitation pulse has a wavelength between 300 nm and 800 nm.

45. The method of claim 31, wherein the time-varying temperature profile comprises a rate of temperature change of at least 20 C./minute.

46. The method of claim 31, wherein the first time period is longer than 50 ns.

47. The method of claim 31, wherein the reaction chamber has a volume between 1 and 100 L.

48. The method of claim 31, wherein the signal is detected in the absence of passing through an optical filter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also figure and FIG. herein), of which:

[0032] FIG. 1 illustrates a timing diagram and waveforms of an example time-gated fluorescence detection.

[0033] FIG. 2 illustrates architecture and functional blocks of an example time-gated fluorescence (TGF) system.

[0034] FIG. 3 illustrates architecture and functional blocks of an example time-gated fluorescence (TGF) complementary metal-oxide semiconductor (CMOS) biochip.

[0035] FIG. 4 illustrates architecture of an example CMOS integrated circuit (IC) for the TGF CMOS biochip system.

[0036] FIG. 5 shows topology of an example TGF pixel with an electronic shutter and periodic charge integrating (PCI) capability.

[0037] FIG. 6 shows timing diagram of a conventional TGF system.

[0038] FIG. 7 shows timing diagram of an example periodic charge integrating (PCI) TGF pixel.

[0039] FIG. 8A shows a planar surface configuration of a biosensing layer structure for a TGF CMOS biochip in accordance with one of the embodiments of the present disclosure. FIG. 8B shows a planar surface configuration of a biosensing layer structure for a TGF CMOS biochip with a thin film barrier in accordance with one of the embodiments of the present disclosure. FIG. 8C shows a configuration of a 3D and permeable matrix coated on the surface of a biosensing layer for a TGF CMOS biochip in accordance with one of the embodiments of the present disclosure. FIG. 8D shows a configuration of a 3D and permeable matrix coated on the surface of a biosensing layer with a thin film barrier for a TGF CMOS biochip in accordance with one of the embodiments of the present disclosure. FIG. 8E shows a microwell configuration for a biosensing layer of a TGF CMOS biochip in accordance with one of the embodiments of the present disclosure. FIG. 8F shows a microwell configuration for a biosensing layer with a 3D and permeable matrix coating the microwell for a TGF CMOS biochip in accordance with one of the embodiments of the present disclosure. FIG. 8G shows a configuration of a combination of microwells and micro-beads with immobilized beads for a TGF CMOS biochip in accordance with one of the embodiments of the present disclosure.

[0040] FIG. 9 shows an example TGF CMOS biochip module and its TGF pixel structure in accordance with one of the embodiments of the present disclosure.

[0041] FIG. 10A shows architecture of an example biochip in accordance with one of the embodiments of the present disclosure. FIG. 10B shows an example TGF pixel and its dedicated decimation cells in accordance with one of the embodiments of the present disclosure.

[0042] FIG. 11 shows photo-sensor pixel schematic and timing diagram of an example TGF mode in accordance with one of the embodiments of the present disclosure.

[0043] FIG. 12 shows measured primary electrical and optical performances of an example TGF pixel in accordance with one of the embodiments of the present disclosure.

[0044] FIG. 13 shows signal-to-background and kinetics measurements for CWF and TGF modes in accordance with one of the embodiments of the present disclosure.

[0045] FIG. 14 shows die micrograph of an example TGF CMOS biochip in accordance with one of the embodiments of the present disclosure.

[0046] FIG. 15A illustrates circuit schematic of an example six transistor (6T) PCI-TGF pixel in accordance with one of the embodiments of the present disclosure. FIG. 15B shows the layout of the pixel of FIG. 15A in sub-micron dimensions.

[0047] FIG. 16 shows timing diagram of an example PCI-TGF pixel in accordance with one of the embodiments of the present disclosure in which correlated double sampling (CDS) is implemented by reading V.sub.S in the reset cycle and after N PCI cycles.

DETAILED DESCRIPTION

[0048] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0049] The term analyte or target as used herein generally refers to a 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.

[0050] The term probe or capturing probe as used herein generally refers to a molecular species and/or other markers that can bind to a specific analyte or target. Probes can comprise molecules and can be bound to the substrate, molecules, or other solid surface, directly or via a linker. Non-limiting examples of linkers include amino acids, polypeptides, nucleotides, oligonucleotides, and chemical linkers. A plurality of probes can be immobilized to a substrate, molecule or other solid surface and can be referred to as a probe array. A plurality of probes of a probe array may be arranged uniformly, for example as an arrangement of spots, or non-uniformly.

[0051] The term about or nearly as used herein generally refers to within +/15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the designated amount.

[0052] The term label as used herein refers to a molecular structure that can be attached to a molecule (e.g., a target and/or a probe), to make the molecule detectable, distinguishable and/or traceable by providing a characteristic which may not be intrinsic to the target molecule. Examples of labels may include are luminescent molecules (e.g., fluorophores), reduction-oxidation (redox) species, or enzymes. In some cases, labels may comprise fluorophores with long lifetimes, such as, for example, lanthanide chelates and transition metal chelates, which are luminescent or phosphorescent.

[0053] The term nucleotide, as used herein, generally refers a molecule that can serve as the monomer, or subunit, of a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid RNA). A nucleotide can be a deoxynucleotide triphosphate (dNTP) or an analog thereof, e.g., a molecule having a plurality of phosphates in a phosphate chain, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphates. 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 strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof). A subunit can enable individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or uracil-counterparts thereof) to be resolved. A nucleotide may be labeled or unlabeled. A labeled nucleotide may yield a detectable signal, such as an optical, electrostatic or electrochemical signal.

[0054] As used herein, the terms polynucleotide, oligonucleotide, nucleotide, nucleic acid and nucleic acid molecule generally refer to a polymeric form of nucleotides (polynucleotides) of various lengths, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). Examples of nucleotide sequences are sequences corresponding to natural or synthetic RNA or DNA including genomic DNA and messenger RNA. The length of the sequence can be any length that can be amplified into nucleic acid amplification products, or amplicons, 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 nucleotides in length, 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 nucleotides in length.

[0055] As used herein, the terms peptide, polypeptide, and protein are used interchangeably, and generally refer to a compound comprised of amino acid residues covalently linked by peptide bonds. Polypeptides may include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. Examples of polypeptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptides and variants thereof, modified polypeptides, derivatives, analogs, fusion proteins, or combinations thereof. A polypeptide may be a natural peptide, a recombinant peptide, or a combination thereof.

[0056] As used herein, the singular forms a, an, and the include plural references unless the context clearly dictates otherwise.

[0057] The term detector as used herein generally refers to a device, generally including optical and/or electronic components that can detect signals.

TGF System Overview

[0058] 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 semiconductor-integrated. In some cases, the semiconductor platform and manufacturing process through which the system is created is complementary metal-oxide-semiconductor (CMOS).

[0059] 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 TGF transduction methods. The TGF CMOS biochip can be a monolithically-integrated biosensor array with addressable locations. See, e.g., U.S. Pat. Nos. 9,708,647, 9,499,861 and 10,174,367, each of which is entirely incorporated herein by reference. Each addressable location may comprise an independently operating TGF photo-sensor that detects TGF signals from its dedicated sensing area. The sensing/detection may be conducted in real-time and in the presence of an aqueous sample, or when such a sample is washed away. The TGF photo-sensor can adopt periodic charge integration (PCI) methods in which periodical signal accumulation is performed by applying multiple time-gated excitation pulses. The TGF CMOS biochip system can physically interface with the aqueous sample and apply physiochemical processes to the sample, including, for example, applying time-varying temperature profiles, biochemical reagents, or pulsed excitation photon fluxes to the sample.

[0060] The TGF CMOS biochip system, as illustrated in FIG. 2, can comprise of components including, but not limited to: [0061] 1. TGF CMOS biochip which can identify and detect analytes interfaces to its top surface through TGF transductions methods in a 2D-array format; [0062] 2. Reaction Chamber which can interface the sample fluid (e.g., a fluidic aqueous sample that includes the analytes) with the TGF CMOS biochip; [0063] 3. Excitation Source which can introduce wavelength-specific photon flux into the reaction chamber and/or TGF CMOS biochip surface in a controlled fashion and synchronized with the TGF CMOS biochip operation; [0064] 4. Controllable Fluidic System configured to move into and/or, remove and/or, hold the reagents and/or sample from, and into, the reaction chamber in a controlled fashion and synchronized with the TGF CMOS biochip operation; [0065] 5. Temperature Controller which can set the temperature of the fluidic within the reaction chamber in a controlled fashion and synchronized with the TGF CMOS biochip operation; and [0066] 6. TGF Reagents and Reporter Molecular Constructs which can enable the detection of the analytes and targets by the TGF CMOS biochip within the reaction chamber and according to a specific assay methodology. [0067] 7. Digital System which can coordinate the operation of one or more components comprised in the system, collect the data and/or communicate the data to a processing and/or data analysis unit.

TGF CMOS Biochip

[0068] As shown in FIG. 3, The TGF CMOS biochip can comprise components including, but not limited to: [0069] A. CMOS Integrated Circuit (IC), which can include the following functional blocks embedded within its monolithically-integrated semiconductor substrate: [0070] i. TGF photo-sensor array comprising a plurality of detectors in a 2D array format. The individual detectors (e.g., a biosensing element or pixel) can measure the emitted photon flux from the fluorophores (F.sub.e) at their addressable location, in parallel, simultaneously, and independently. The detectors can also adopt periodic charge integration (PCI) TGF methods; [0071] ii. Readout circuity which may acquire data from individual TGF pixels and communicate them sequentially, in parallel, or a combination thereof, to an off-chip unit (external destination); and [0072] iii. On-chip passive resistive heater and temperature sensor. [0073] B. Biosensing Layer, which can be located on a surface of the CMOS IC and can utilize TGF methods to create analyte-specific, localized TGF signal coupled with the TGF pixels. The biosensing layer may comprise a plurality of probes at independently (and/or individually) addressable locations on a solid surface. Each pixel can comprise a plurality of identical or different probes molecules that can specifically bind to or interact with a specific target/analyte or reagents in the reaction chamber;

CMOS Integrated Circuits (IC)

[0074] The architecture of the integrated CMOS IC for the TGF biochip is illustrated in FIG. 4. The CMOS die include a 2D photo-sensor array, with a similar general readout circuitry architecture to other biosensor arrays. See, e.g., U.S. Pat. Nos. 9,708,647, 9,499,861 and 10,174,367, each of which is entirely incorporated herein by reference. The photo-sensor array, where identical CMOS embedded TGF pixels are placed may be read sequentially (i.e., one pixel at a time) using a row and column decoder. The output of the chip, sent to off-chip through an output buffer, can be either analog or digital.

[0075] The chip may also include a resistive heater and a temperature sensor to accommodate the temperature control of the reaction chamber (e.g., Hassibi, A. et al. A fully integrated CMOS fluorescence biochip for DNA and RNA testing, IEEE Journal of Solid-State Circuits, 52(11):2857-2870, 2017). In addition, the CMOS IC can also include a control block to be programmed and accessed off-chip by the user to set the functionality of the chip and manage the data acquisition.

[0076] The general topology of an example TGF pixel is shown in FIG. 5. The TGF receive both F.sub.e and F.sub.x from the addressable location on its biosensing layer and the photons may be converted into electrical charge by using a photon-charge transducer (PCT). Examples of PCT in CMOS processes include lateral photodiodes (e.g., Cauwenberghs, G., et al. Which photodiode to use: A comparison of CMOS-compatible structures, IEEE sensors journal, 9(7):752-760, 2009), or pinned photodiode devices (e.g., Hondongwa, 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 comprise two switches connected to it. The first may be an electronic shutter switch (S.sub.S) which removes the charge completely out of the PCT through connecting it to the electronic shutter voltage source (V.sub.S). The second may be an integration switch (S.sub.I) which transfer the created charge into a charge integrator element (CIE). The CIE device may be continually connected to a charge-to-voltage transducer (CVT) to produce a TGF pixel output. In addition, the CIE may have a reset switch (S.sub.R) to remove the integrated charge at any time and basically reset the CIE output value to V.sub.R.

[0077] TGF pixel of the present disclosure may be different from conventional detectors for TGF or time-resolved fluorescence. One difference is the absence of the S.sub.S and S.sub.I and the capability of selectively discarding or integrating the generated charge of the PCT. In FIG. 6, an example timing diagram for the operation for a conventional TGF system is shown. As FIG. 6 shows, F.sub.e is measured after every N individual F.sub.x pulse by quantifying the photo-induced charge during integration time intervals. The N outputs (X.sub.OUT[1] to X.sub.OUT[N]) are then averaged to estimate, F.sub.e. Multiple challenges and non-idealities may exist with this system. For example: [0078] It may be needed to take N consecutive measurements (reads) to estimate F.sub.e or every pixel. Since F.sub.e may be low, extensive averaging may be required and, for example, values of N>100 may be needed in such TGF systems. [0079] Due to the low level of signal (e.g., 10 total electrons per F.sub.e pulse), CVT may require very high gain (e.g., >20 V/e) with an analog-to-digital quantization noise of equivalent to less than a few electrons per read. [0080] When large biosensor arrays may be implemented with number of pixels M>1000, the number of reads per frame becomes NM which can become quickly overwhelming. For example, if a fluorophore used in TGF has a lifetime of .sub.L=100 ns, it is possible to create the F.sub.x pulse sequence with period 1 ms=10 .sub.L. If N=100 and M=1000, then the readout speed will be 10.sup.5 reads/ms or 100 million sample/s. Given the noise requirements of the system, this may require very complex readout circuitry and call for a significant amount of power. As a result, one may consider reducing the pulse sequence frequency and essentially slowing down TGF measurements.

[0081] In the present disclosure, by using the topology shown in FIG. 4, the above-mentioned challenges may be addressed. FIG. 7, depicts a timing diagram of the TGF pixel of the present disclosure, which adopts an in-pixel periodic charge integration (PCI) scheme to improve both the speed and performance of the TGF measurements. As FIG. 7 shows, by using S.sub.S and S.sub.I and applying an electronic shutter, responses of N pulses of the PCT may be integrated into the CIE which may create a single output. This may enable a readout of output once every N pulses with an amplitude N times larger than conventional TGF. Additional advantages to this approach may include, but are not limited to: [0082] One read in PCI-TGF may be equal to N reads in conventional TGF. [0083] The accumulated charge and the output amplitude signal of PCI-TGF may be N times of conventional TGF and can be read N times slower. Therefore, PCI-TGF can use a much more relaxed the readout circuitry with lower speed and signal higher chain quantization noise. [0084] When large biosensor arrays, with number of pixels, M>1000 elements are used, the required readout and pixel scanning speed requirement may be N times less than conventional TGF. Therefore, it may become quite feasible to create arrays with M>10.sup.6, a number that may be necessary for the adoption of for massively parallel arrays used in life-science research.

[0085] The challenges in the implementation of PCI-TGF may revolve around the circuit and device implementation of the switches, efficient approaches of transferring charge in time intervals compatible with TGF, and CIE.

Biosensing Layer

[0086] The biosensing layer as provided herein may include an organic layer that may be created on top of a CMOS IC and interfacing the reaction chamber to: (a) form addressable location(s) for probes on top of the pixels; and (b) enable TGF transduction by first capturing targets and subsequently creating TGF signals as a function of the probe target interactions and/or structure of the captured target.

[0087] Biosensing layers may be created by various methods. For example, specific probe structures may be physically printed, immobilized, or spotted or chemically synthesized on a surface. In some cases, probes are first randomly distributed within the array 2D surface and then identified prior to detecting the targets by alternative approaches that are known in the field. In some cases, the surface of the IC (typically made of SiO.sub.2 or Si.sub.3N.sub.4) may be chemically modified with linkers and/or thin film structures to become compatible with probe attachment.

[0088] FIGS. 8A-8G shows examples of biosensing structures that are compatible with CMOS ICs and TGF transduction methods, including PCI-TGF. In FIG. 8A and FIG. 8B, a planar surface may be implemented to immobilize probes and an addressable array may be created with and without a thin film barrier, respectively. In FIG. 8C and FIG. 8D, a 3D and permeable matrix may be coated on the surface to allow for probe immobilization at the intimate proximity of the surface) In FIG. 8E and FIG. 8F, microwells may be used to better isolate the immobilized probes and isolate the TGF pixels. In FIG. 8G, a combination of microwells and micro-beads with immobilized beads may be used to create an addressable array.

Reaction Chamber

[0089] The reaction chamber as provided herein may be a fluidic chamber that interfaces with the CMOS TGF biochip and contains the fluidic sample with analytes, targets, and other biochemical reagents that are required for the execution of the TGF assay.

[0090] The volume of this reaction chamber can be between about 0.1 L to 10,000 L, e.g., between about 1 L to 100 L.

[0091] The reaction chamber may comprise a plurality of inlets and outlets to accommodate the interfacing with the controllable fluidic system to insert or remove fluids.

[0092] To accommodate TGF, the fluidic system can provide a transparent optical travel path for the pulse F.sub.x to go through the fluidic and reach the biosensing layer. The transmittance in the wavelengths of F.sub.x can be from 1% to 99.9%, but typically is from 5% to 80%.

[0093] The reaction chamber can be built using a variety of materials such as polymers, glass, semiconductor, crystals, or ceramics materials, or a combination of them.

Excitation Source

[0094] The excitation source as provided herein may comprise an optical light source that can create a wavelength selective photon flux (F.sub.x) with a controllable and time-varying amplitude. The light source may illuminate the biosensing layer of the system and the coordinates in which TGF transduction takes place.

[0095] The excitation source center wavelength can be anywhere between about 200 nm to 1500 nm, e.g., between about 300 nm to 800 nm.

[0096] The excitation source spectral span (bandwidth) may be from about 1 nm to 500 nm, e.g., from about 10 nm to 100 nm.

[0097] The excitation source photon flux may be directional and may be optically collimated.

[0098] The excitation source peak output power may be from about 10 mW to 100 W, e.g., from about 100 mW to 10 W.

[0099] The excitation source power may be controllable and modulated with bandwidth of up to about 1 GHz, e.g., up to about 1 MHz

[0100] The excitation source turns off and on times may be as fast as about 0.1 nanosecond (ns), e.g., as fast as about 1 microsecond (s).

Controllable Fluidic System

[0101] The controllable fluidic system introduces into, and/or removes from, and/or confines within the reaction chamber aqueous media that can include samples and assay reagents, and/or TGF transduction reagents in a controlled fashion by the user. The workflow and sequence of each fluidic operation may be defined by the assaying method and can be, for example, flow-through and mono-directional, or closed-tube.

[0102] The controllable fluidic system may use fluidic components such as pumps, valves, and tubing to perform the workflow.

Temperature Controller

[0103] The temperature controller system can establish a specific temperature for the fluidic of the reaction chamber, and/or create a temperature profile that requires heating and/or cooling. A temperature controller can include a feedback control system that measures the temperature, using temperature sensors within the CMOS biochip IC and/or sensor devices coupled with the reaction chamber (such as a thermistor or a thermocouple), and, based on the measured temperature, add or remove heat from the reaction chamber using CMOS biochip IC heaters and/or thermal devices (such as Peltier devices or resistive heaters). Temperature controllers can comprise heat sinks for removing heat. Temperature controllers can have components within the CMOS IC, including resistive heaters and/or temperature sensors.

[0104] Temperature controllers can change the temperature of a substrate, reaction chamber, or array pixel. The rate of temperature change can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C./minute. The rate of temperature change can be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C./minute. The rate of temperature change can be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C./minute. Temperature controllers can change temperature at a linear rate (e.g., 5 C./second). Alternatively, temperature controllers can change temperature at a non-linear rate. Temperature controllers can increase or decrease temperature.

Digital System

[0105] The digital system is essentially a computing and controlling digital hardware And embedded software than can control and coordinate the functionality of the components of the system.

TGF Reagents and Reporter Molecular Constructs

[0106] To enable TGF transduction, we can use molecular structures and constructs that exhibit fluorescence activity. Such molecular structures are sometimes referred to as fluorophores (or fluorochromes, similarly to chromophores) which are chemical compound that can re-emit light upon light excitation with life-times from about 10 ps to 10 ms, e.g., from about 1 ns to 100 ns.

[0107] As provided herein, various types of fluorophores can be adopted by a TGF system. In some cases, fluorophores that have longer life-times, e.g., greater than about 100 ns, may be used. In cases where fluorophores with longer lifetime are used: [0108] Longer life-time fluorophores may require lower speed PCI-TGF systems in the CMOS Biochip IC; [0109] The excitation source switching speed can be more manageable, and more cost-efficient light sources can be used; and [0110] The negative effects of background autofluorescence from the biological sample and/or materials in the fluidic chamber and/or biolayer may be mitigated, if the they have shorter life-time compared to the adopted TGF fluorophore.

[0111] TGF systems may not need an excitation and emission filter set, or other filters of wavelength to transmit a desired signal for an analyte and/or remove background fluorescence from signals of the analyte. In some cases, the emission filter may filter out violet, blue, green, yellow, orange, and red light, or any combination thereof.

[0112] Various types of fluorophores may permit multi-color capabilities. In TGF, differentiating fluorophores may be determined by the differences in their fluorescence lifetimes after excitation. In some cases, these fluorophores can be reactive and/or conjugated dyes, nucleic acid dyes, fluorescent proteins, and cell function dyes. Once emission light is pulsed in the direction of a substrate containing the fluorophore species, a shutter may close off the detection apparatus from the emission light and the reflected emission light. The shutter may be removed to let in the desired fluorescent light. A first fluorophore with a shorter lifetime can be detected among the detected signals if the shutter opens shortly after the emission is stopped. A second fluorophore 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 fluorophore (longer lifetime) may be detected with little or no interference of the first fluorophore (shorter lifetime). In addition, readings of the signals corresponding to the first fluorophore (shorter lifetime) in the presence of the second fluorophore (longer lifetime) can be estimated or calculated by calibration of the detected signals using information about the second fluorophore (longer lifetime) detected and/or determined later. Other experimental designs for the multiplex detection of multiple fluorophores are possible with or without the use of emission and excitation filter sets. Accordingly, multiple fluorophores can be detected in a single experiment by the opening and closing of the shutter following the emission in the absence of excitation and emission filter sets. Individual species of fluorophores can be detected based on the differences in their decay rates by varying the delay profiles of the shutter and the time during which the shutter is opened. The multi-color capability may be limited by shutter speed and the overlap between fluorescence decay rates of the fluorophores to be detected.

[0113] For example, in some examples, metal chelate, such as Lanthanide chelates can be used as TGF fluorophores. In some cases, TGF fluorophores may predominantly act as molecular reporters in TGF assays either as a standalone reporter or an element (donor or acceptor) in a fluorescence energy transfer moiety. Examples include, but are not limited to, Frster Resonance Energy Transfer (FRET) technologies. 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 TGF fluorophores may include facilitating the generation of a specific TGF signal that may be correlated to the presence or absence of a molecular reaction or presence or absence of a specific target molecule.

[0114] TGF fluorophores can be used as labels for specific target analytes, in applications where the targets can be chemically modified to incorporate a TGF fluorophore. Examples includes, but are not limited to, Northern blots, Southern blots, DNA microarrays, quantitative Polymerase Chain Reaction (PCR), digital PCR, and diagnostic assays. [0115] In microarrays and Northern blots, the mRNA target analyte may be converted into a fluorophore-labelled complementary DNA (cDNA), for example, through reverse transcription. [0116] In Southern blots, a fluorophore-labeled cDNA may be used to identify a target sequence. [0117] In quantitative polymerase chain reaction (PCR) and digital PCR (dPCR), the fluorophore may be incorporated into an amplified nucleic acid sequence or a primer sequence to demonstrate the accumulation of a target sequence (See, e.g., Y. Wong et al., Applications of digital PCR in precision medicine, Expert Review of Precision Medicine and Drug Development 2(3):177-186, 2017). [0118] In a diagnostic assay, a device may be used to sequester target nucleic acids, and a fluorophore-labelled cDNA may be used for direct detection.

[0119] TGF fluorophores can also be used as labels for the detection of probes in sandwich assays. Examples include, but are not limited to, Western Blots, Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme-Linked Immuno SPOT (ELISPOT) including FluoroSpot (See, e.g., 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. [0120] In these methods, the TGF fluorophores may be used as a direct method for detection, in which the fluorophore is conjugated to the primary detection antibody. [0121] In these methods, the TGF fluorophore may also be used as an indirect method for detection, in which the fluorophore is conjugated to a secondary antibody. [0122] ELISPOT is a type of assay that quantitatively measures the frequency of cytokine secretion for a single cell. The ELISPOT Assay is also a form of immunostaining that uses antibodies to detect an analyte, including but not limited to, any biological or chemical substance being identified or measured, such as, for example, protein analyte. [0123] The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses fluorescence to analyze multiple analytes. It can detect the secretion of more than one type of protein or other analytes.

[0124] TGF fluorophores can be used as labels in cell sorting, counting, and detecting methods. An example may be flow cytometry, in which cells are labeled with a fluorophore. [0125] In this method, the cells may be sorted and counted by their fluorescence profiles. [0126] In this method, the specific cellular characteristics and/or functions may be identified by their fluorescence profiles.

[0127] TGF fluorophores can be used in applications where solid-phase and immobilized probes are labeled. Examples are inverse fluorophore assays (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)

[0128] TGF fluorophores can be used in assays in which the chemical reactions are monitored while a target molecule is introduced to a reacting reagent. The target molecule and/or the reacting reagent may include TGF fluorophores. Examples are Sanger sequencing, Next Generation Sequencing (NGS) assays such as sequence-by-synthesis (SBS) (See, Ansorge; Metzker; and Pareek et al., Sequencing technologies and genome sequencing, J. Appl. Genet., 52(4):413-435, 2011), sequence-by-hybridization (SBH) (See, Qin, Schneider and Brenner, Sequencing by Hybridization of Long Targets, PLOS One., 7 (5): e35819, 2012), and pyrosequencing. [0129] In this method, Single Molecule Real Time (SMRT) sequencing and Illumina sequencing can use TGF fluorophore-labeled nucleotides to determine the sequence of a nucleic acid

[0130] Sequence information of nucleic acids may be used to improve people's lives. (See, e.g., Ansorge, W., Next-generation DNA sequencing techniques, New Biotech. 25(4):195-203, 2009). Several DNA sequencing platforms have been commercially available. The availability of parallel NGS technologies may enable the comprehensive analysis for biological targets, including but not limited to genomes, transcriptomes and interactomes. (See, e.g., Shendure, J. and Ji, H., Next-generation DNA sequencing, Nature Biotech. 26:1135-45, 2008). However, although NGS technologies may produce comprehensive results, their turnaround time may be too slow to address the rapid progression of an infectious process in critically ill patients. In addition, while multiplexing a large number of target amplification reactions (e.g., multiplexed PCR) may be possible, but it is not straightforward to detect multiple amplicons simultaneously.

[0131] Commercially available NGS sequencing platforms may include the Illumina Genome Analyzer, the Roche (454) Genome Sequencer, the Life Technologies SOLID platform, and real-time sequencers such as those from Pacific Biosciences. These platforms may require the construction of a set of DNA fragments from a biological sample. In most cases, the DNA fragments are flanked by platform-specific adapters.

EXAMPLE 1

[0132] In this example, a fully-integrated TGF CMOS biochip is presented that is specifically designed for DNA and protein addressable arrays of biotechnology. As shown in FIG. 9, a CMOS IC is assembled on a printed circuit board (PCB) substrate and then integrated with the fluidic module to create the biochip consumable. The biochip IC includes an array of 1024 biosensors pixels with an optical density (OD)5.8 integrated emission filter and addressable (unique) immobilized probes (DNA) on every pixel. Pixel-level photo-sensors with Nwell-Psub photodiodes (acting as the PCT elements) are designed to be shot-noise-limited and offer >130 dB detection dynamic range (DDR). A temperature control and cycling system is also integrated in this biochip to accommodate thermal control. For that reason, a bandgap temperature sensor and a resistive heater are integrated that together can achieve heating/cooling rates of +/10 C./s with an overall accuracy of 0.25 C. within 25 C. to 100 C. range.

[0133] The architecture of the chip and 120 m-pitch biosensing pixels and decimation cells are shown in FIG. 10A and FIG. 10B. The TGF pixels within the 3232 array include a current detector that takes the photocurrent, I.sub.ph, as its input and produces a 1-bit digital output stream that is transferred into the on-chip decimation array. The photo-sensor circuitry (FIG. 10B) includes a current integrator (acting as the CIE+CVT), a clocked comparator (ADC) and a programmable current source (DAC).

[0134] In the CWF mode (i.e., no pulsed excitation source or electronic shuttering), the current detector operates continuously with frequency of f.sub.c while the decimation cell implements a sinc.sup.2 filter, by performing a two-stage 32-bit accumulation followed by down-sampling and readout with frequency of f.sub.s. In the TGF mode, similar operation is done, but with the exception of periodic activation of an electronic shutter capable of diverting I.sub.ph from the integrator. This operation blocks the optical excitation pulses and reduces the natural autofluorescence background from biological media that typically have lifetimes <50 ns. The chip then accumulates and measures the fluorescence emissions at pre-programmed time intervals.

[0135] In this chip, the TGF pixels, the decimation arrays, bandgap temperature sensor, and reference voltage DACs are all operated and read by a single digital core block operating at 50 MHz and is accessible through a serial peripheral interface (SPI) port (FIG. 10A). The single resistive heater can provide up to 20 W using an external source, has a serpentine structure, and is uniformly distributed in the top metal layer. This chip can be fully operated using 14 pins (and bond wires) aggregated on one side of the die to facilitate efficient fluidic assembly and consumable manufacturing (FIG. 9).

[0136] In FIG. 11, the schematic and timing diagram of the photo sensing pixel in the TGF mode is depicted. A capacitive trans-impedance amplifier (CTIA) is used as the CIE+CVT and a clocked comparator creates the pixel output, D.sub.out. The DAC is implemented by using a current source that can be used to apply a current pulse into the CTIA input with two adjustable durations (.sub.1 and .sub.2). The electronic shutter uses SH.sub.1, SH.sub.2, and SH.sub.3 to temporarily remove C.sub.f, the feedback capacitor of the CTIA, out of the circuit and simultaneously shorting I.sub.ph to V.sub.d using the op-amp. Due to transistor mismatch, a small quantity of charge is injected into C.sub.f at every shutter operation that manifests itself as a pixel-dependent electronic shuttering offset current, I.sub.s. This current when added to dark current I.sub.dc, forms the random offset current of the pixel I.sub.o=I.sub.s+I.sub.dc, which, in both CWF and TGF modes, is measured and extracted to estimate I.sub.ph. This is done using a CDS approach in which one frame with excitation light and one without are taken, and then the measurements are subtracted from one another.

[0137] The decimation array has a dedicated bit cell for every pixel. The bit cell consists of a 32-bit incrementor, followed by a 32-bit adder, forming the two-stage accumulation unit (FIG. 10B). At intervals of T=1/f.sub.s, the output of the adder is loaded onto the 32-bit shift register. The data from the shift registers are then passed into the digital unit in a serial scan chain fashion.

[0138] The electrical and optical measurements for this biochip are reported in FIG. 12. The measured signal-to-noise ratio (SNR) from pixels is demonstrate that the added sensor noise is 30% of the shot-noise when the quantization noise is not limiting within the 100 fA to 1 nA input current region. The total dual-depletion region (DDR) is 137 dB (1.33 fA10 nA) for f.sub.s=1.667 Hz. The photodiode external quantum efficiency (QE), with and without the integrated emission filter, show the pass-band and stop-band QE of 0.4 and 3.6910.sup.7 (OD5.8), respectively. The measured distribution of I.sub.dc and I.sub.o validate the expected randomness with maximum amplitude of 100 pA (<1% of the full scale. The output of the temperature sensor as a function of temperature are also reported in FIG. 12, which shows that with 2-point calibration accuracy of 0.25 C. is achievable across the 25 C.-100 C. temperature range.

[0139] In FIG. 13, the results from two (2) biosensing experiments are reported and compared, to demonstrate the different modes of operation. In all experiments, identical surface functionalization and array-based DNA hybridization or ligand-receptor bindings are performed. However, distinct molecular labels are attached to the targets, for CWF and TGF, respectively. In CWF, using a R-phycoerythrin fluorophore, the signal-to-background (S/B) shows the lowest value. This may be due to the non-ideal blocking of the excitation light. The S/B is increased significantly when using TGF and DTBTA-Eu3+, which is a Europium (Lanthanide) chelate-based long lifetime fluorophore. As evident, the background photon emission from the pulsed light-emitting diode (LED) excitation source decays significantly within 100 s and the background becomes much smaller than compared to CWF mode.

[0140] In FIG. 14, the micrograph of the implemented TGF biochip is shown.

EXAMPLE 2

[0141] This example shows how PCI-TGF pixels can be designed in applications where high-density biosensor pixels arrays are required, such as DNA SBS and DNA SBH systems. The example also shows how miniaturized PCI-TGF pixels can be incorporated into standard high-density image sensor arrays. As the example shows, PCI can be added into the circuitry of multi-million pixel CMOS image sensors that can have sub-micron pixel dimensions.

[0142] FIG. 15A depicts the circuit diagram example of a six transistor (6T) pixel topology which includes a pinned photodiode (PPD) as the PCT, and two (2) charge transfer gates; one to transfer charge to the sense node (TX) acting as an integrating switch, and one to act as an electronic shutter (SH). The charge is integrated on the floating diffusion (acting as the CIE+CVT) and the generated voltage V.sub.s is read using the source follower gate. In this depiction, we assume that the pixel is located at the (i,j) coordinate within a photo-sensor array and V.sub.s can be accessed by the column signal (COL[j]) by activating the row select signal (SEL[i]). The charge is the floating diffusion can be reset using RST[i].

[0143] In FIG. 15B, the layout of this pixel is shown that can be scaled down to sub-micron dimensions similar to equivalent CMOS image sensor pixels.

[0144] In FIG. 16, the diagram of the PCI-TGF pixel is shown. As FIG. 16 shows, correlated double sampling (CDS) can be implemented by reading V.sub.s in the reset cycle and after N PCI cycles. As shown in the reset cycle, the output of the pixel is V.sub.DDV.sub.nV.sub.th, where V.sub.n and V.sub.th are the offset and threshold voltages of the source follower transistor, respectively. Now, at the end of the N.sup.th integration cycle, V.sub.s=V.sub.DDV.sub.nV.sub.thNQ/C, where Q is the charge collected by the emission from an individual excitation pulse and C is the floating diffusion effective capacitance. Therefore, by subtracting these two values (i.e., CDS) we can have a value that follows the PCI schemes while is independent of the offset of the source follower that may vary from pixel to pixel within the array.

[0145] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.