Gene sequencing device, gene sequencing method, and nucleic acid detection method

Abstract

The invention provides a gene sequencing device and a gene sequencing method. The gene sequencing device comprises: a biological sensing layer comprising one or more sample receiving sites and one or more photosensitive circuits in one-to-one correspondence with the one or more sample receiving sites, the sample receiving sites being configured to receive samples; the light sensing circuit is configured to receive fluorescence signals emitted by the samples on the corresponding sample receiving sites and generate analog electric signals according to the fluorescence signals, and the light sensing circuit comprises a quantum image sensor; the circuit structure layer comprises an analog-to-digital conversion circuit which is configured to convert an analog electric signal into a digital electric signal; and the logic processing layer is configured to determine the characteristics of the fluorescence signal according to the digital electric signal, and determine the type and / or sequence of the basic group contained in the sample according to the characteristics of the fluorescence signal.

Description

Gene sequencing device, gene sequencing method and nucleic acid detection method

[0001] The present disclosure relates to the field of biotechnology, and in particular to a gene sequencing device, a gene sequencing method and a nucleic acid detection method.

[0002] In the related technology, a high-throughput gene sequencer amplifies nucleic acid fragments and transfers them to a sequencing substrate or amplifies them directly on a sequencing substrate. Sequencing reagents are then added to the sequencing substrate to achieve biochemical reactions such as base synthesis and excision. The signal reporter group generates a light signal or an electrical signal that is acquired by the sequencing instrument. The internal computer of the sequencing instrument performs algorithm calculations to obtain the base sequence.

[0003] Sequencing substrates are also called sequencing chips, and the most common types are surface sequencing chips and semiconductor integrated circuits. Neither of them has the ability to directly calculate the base sequence. Since the signal of the fluorescent group on the label is very weak, the nucleic acid fragment needs to be amplified.

[0004] In single-molecule sequencing technology, common devices include nanopore sequencers, which identify single base molecules through the impedance change caused by nucleic acid fragments passing through the nanopore. In another technology, a total reflection layer of laser is formed on the semiconductor image sensor, and the evanescent field effect of the total reflection laser excites the fluorescent group. The signal-to-noise ratio is high enough, so single base molecules can also be detected.

[0005] Summary of the invention

[0006] According to one aspect of the present disclosure, a gene sequencing device is provided, comprising: a biosensing layer, comprising one or more sample receiving sites and one or more photosensitive circuits corresponding to the one or more sample receiving sites, the sample receiving sites being configured to receive samples; the photosensitive circuits being configured to receive fluorescence signals emitted by samples at corresponding sample receiving sites, and to generate analog electrical signals based on the fluorescence signals, the photosensitive circuits comprising quantum image sensors; a circuit structure layer, comprising: an analog-to-digital conversion circuit, configured to convert the analog electrical signals into digital electrical signals; and a logic processing layer, configured to determine the characteristics of the fluorescence signals based on the digital electrical signals, and to determine the type and / or sequence of bases contained in the sample based on the characteristics of the fluorescence signals.

[0007] In some embodiments, the quantum image sensor comprises a single photon avalanche diode.

[0008] In some embodiments, the photosensitive circuit also includes a resistor, wherein a first end of the resistor is electrically connected to a first voltage terminal, a second end of the resistor is electrically connected to an anode terminal of the single-photon avalanche diode, a cathode terminal of the single-photon avalanche diode is electrically connected to a second voltage terminal, and a voltage at the second voltage terminal is greater than a voltage at the first voltage terminal.

[0009] In some embodiments, the characteristics of the fluorescence signal include: at least one of: fluorescence intensity and fluorescence quenching time; and the digital electrical signal includes: a digital square wave signal.

[0010] In some embodiments, the logic processing layer is configured to determine the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal, determine at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal based on the number of photons, and determine the type and / or sequence of the bases contained in the sample based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0011] In some embodiments, the circuit structure layer further includes: a memory electrically connected to the analog-to-digital conversion circuit and the logic processing layer, and configured to store data.

[0012] In some embodiments, the gene sequencing device further includes: a substrate; wherein the logic processing layer, the circuit structure layer and the biological sensing layer are all located above the substrate.

[0013] In some embodiments, the logic processing layer is located above the substrate; the circuit structure layer is located on a side of the logic processing layer away from the substrate; and the biosensing layer is located on a side of the circuit structure layer away from the substrate.

[0014] According to another aspect of the present disclosure, a gene sequencing method is provided, comprising: performing gene detection using the gene sequencing device as described above.

[0015] In some embodiments, performing genetic testing using the gene sequencing device includes: placing a sample in the sample receiving site, wherein the sample emits a fluorescent signal; receiving the fluorescent signal emitted by the sample, and generating an analog electrical signal based on the fluorescent signal; converting the analog electrical signal into a digital electrical signal; and determining a characteristic of the fluorescent signal based on the digital electrical signal, and determining the type and / or sequence of bases contained in the sample based on the characteristic of the fluorescent signal.

[0016] In some embodiments, the characteristics of the fluorescence signal include: at least one of: fluorescence intensity and fluorescence quenching time; and the digital electrical signal includes: a digital square wave signal.

[0017] In some embodiments, determining the characteristics of the fluorescence signal according to the digital electrical signal, and determining the type and / or sequence of the bases contained in the sample according to the characteristics of the fluorescence signal includes: determining the number of photons of the received fluorescence signal according to the number of square waves in the digital square wave signal; determining at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal according to the number of photons; and determining the type and / or sequence of the bases contained in the sample according to at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0018] In some embodiments, the fluorescent signal is generated by a bioluminescent reaction.

[0019] In some embodiments, the bioluminescent reaction refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal.

[0020] In some embodiments, the gene sequencing device is used to perform sequencing based on bioluminescent reactions. Such sequencing methods are known in the art.

[0021] According to another aspect of the present disclosure, a nucleic acid detection method is provided, comprising: marking a nucleic acid molecule at a sample receiving site with a fluorescent marker, wherein the fluorescent marker is used to identify a target nucleotide base in the nucleic acid molecule; irradiating the nucleic acid molecule marked with the fluorescent marker with excitation light, so that the nucleic acid molecule marked with the fluorescent marker absorbs the excitation light to generate a fluorescent signal, and the fluorescent signal is transmitted to a photosensitive circuit having a quantum image sensor, wherein the photosensitive circuit generates an analog electrical signal according to the fluorescent signal; performing digital-to-analog conversion on the analog electrical signal to obtain a corresponding digital electrical signal, determining at least one feature of the fluorescent signal received at the quantum image sensor based on the digital electrical signal; and determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the at least one feature.

[0022] In some embodiments, the quantum image sensor comprises a single photon avalanche diode.

[0023] In some embodiments, the at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0024] In some embodiments, the digital electrical signal includes a digital square wave signal; the determining of at least one feature of the fluorescence signal received at the quantum image sensor based on the digital electrical signal includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; and determining at least one of the fluorescence intensity and fluorescence quenching time of the fluorescence signal based on the number of photons.

[0025] In some embodiments, determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the at least one feature includes: determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0026] In some embodiments, the nucleic acid detection method is nucleic acid sequencing based on excitation light fluorescence technology.

[0027] According to another aspect of the present disclosure, a nucleic acid detection method is provided, comprising: marking a nucleic acid molecule at a sample receiving site with a biofluorescent substance, wherein the biofluorescent substance is used to identify a target nucleotide base in the nucleic acid molecule; changing the environment of the nucleic acid molecule or adding a reaction substrate so that the nucleic acid molecule marked with the biofluorescent substance emits a fluorescent signal, and the fluorescent signal is transmitted to a photosensitive circuit having a quantum image sensor, wherein the photosensitive circuit generates an analog electrical signal based on the fluorescent signal; performing digital-to-analog conversion on the analog electrical signal to obtain a corresponding digital electrical signal, determining at least one feature of the fluorescent signal received at the quantum image sensor based on the digital electrical signal; and determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the at least one feature.

[0028] In some embodiments, the quantum image sensor comprises a single photon avalanche diode.

[0029] In some embodiments, the at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0030] In some embodiments, the digital electrical signal includes a digital square wave signal; the determining of at least one feature of the fluorescence signal received at the quantum image sensor based on the digital electrical signal includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; and determining at least one of the fluorescence intensity and fluorescence quenching time of the fluorescence signal based on the number of photons.

[0031] In some embodiments, determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the at least one feature includes: determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0032] In some embodiments, the bioluminescent substance is luciferase.

[0033] In some embodiments, the nucleic acid detection method is nucleic acid sequencing based on bioluminescence technology. According to another aspect of the present disclosure, a method for in-situ detection of biological samples is provided, comprising: fixing the biological sample on a biosensing layer, the biological sample comprising a plurality of nucleic acid molecules, the biosensing layer comprising one or more sample receiving sites and one or more photosensitive circuits corresponding to the one or more sample receiving sites, the nucleic acid molecules are correspondingly fixed by the sample receiving sites, and the nucleic acid molecules are marked with fluorescent markers or bioluminescent substances; under predetermined conditions, the nucleic acid molecules marked with the fluorescent markers or the bioluminescent substances generate fluorescent signals, and the fluorescent signals are transmitted to a photosensitive circuit having a quantum image sensor, the photosensitive circuit generates an analog electrical signal according to the fluorescent signals; performing digital-to-analog conversion on the analog electrical signals to obtain corresponding digital electrical signals, determining at least one feature of the fluorescent signal received at the quantum image sensor based on the digital electrical signals; and determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecules at the corresponding positions in the biological sample based on the at least one feature.

[0034] In some embodiments, the nucleic acid molecule is coupled to one or more of a protein, a peptide, an antibody, or an affinity tag.

[0035] In some embodiments, the nucleic acid molecule is an oligonucleic acid tag sequence.

[0036] In some embodiments, the quantum image sensor comprises a single photon avalanche diode.

[0037] In some embodiments, the at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0038] In some embodiments, the digital electrical signal includes a digital square wave signal; the determining of at least one feature of the fluorescence signal received at the quantum image sensor based on the digital electrical signal includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; and determining at least one of the fluorescence intensity and fluorescence quenching time of the fluorescence signal based on the number of photons.

[0039] In some embodiments, determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at the corresponding position in the biological sample based on the at least one feature includes: determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at the corresponding position in the biological sample based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0040] In some embodiments, the biological sample is a tissue section.

[0041] In some embodiments, the biological sample in situ detection method is spatial transcriptome technology.

[0042] Other features and advantages of the present disclosure will become apparent from the following detailed description of exemplary embodiments of the present disclosure with reference to the accompanying drawings.

[0043] The accompanying drawings, which constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0044] The present disclosure may be more clearly understood from the following detailed description with reference to the accompanying drawings, in which:

[0045] FIG1 is a schematic diagram showing the structure of a gene sequencing device according to some embodiments of the present disclosure;

[0046] FIG2 is a schematic diagram showing the structure of a photosensitive circuit according to some embodiments of the present disclosure;

[0047] FIG3 is a schematic diagram showing the structure of a gene sequencing device according to other embodiments of the present disclosure;

[0048] FIG4 is a flow chart showing a gene sequencing method according to some embodiments of the present disclosure;

[0049] FIG5 is a schematic diagram illustrating a gene sequencing method according to some embodiments of the present disclosure;

[0050] FIG6 is a flow chart showing a nucleic acid detection method according to some embodiments of the present disclosure;

[0051] FIG7 is a flow chart showing a nucleic acid detection method according to other embodiments of the present disclosure;

[0052] FIG8 is a flow chart showing a method for in-situ detection of a biological sample according to some embodiments of the present disclosure.

[0053] It should be understood that the size of each part shown in the accompanying drawings is not necessarily drawn according to the actual proportional relationship. In addition, the same or similar reference numerals represent the same or similar components.

[0054] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. The description of the exemplary embodiments is merely illustrative and is in no way intended to limit the present disclosure and its application or use. The present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to make the present disclosure thorough and complete and to fully express the scope of the present disclosure to those skilled in the art. It should be noted that unless otherwise specifically stated, the relative arrangement of the components and steps, the composition of the materials, the numerical expressions and the numerical values ​​set forth in these embodiments should be interpreted as being merely exemplary and not as limiting.

[0055] The words "first", "second" and similar words used in this disclosure do not indicate any order, quantity or importance, but are only used to distinguish different parts. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0056] In the present disclosure, when a specific device is described as being located between a first device and a second device, there may or may not be an intermediate device between the specific device and the first device or the second device. When a specific device is described as being connected to other devices, the specific device may be directly connected to the other device without an intermediate device, or may not be directly connected to the other device but have an intermediate device.

[0057] All terms (including technical terms or scientific terms) used in the present disclosure have the same meanings as those understood by ordinary technicians in the field to which the present disclosure belongs, unless otherwise specifically defined. It should also be understood that terms defined in general dictionaries, such as general dictionaries, should be interpreted as having meanings consistent with their meanings in the context of the relevant technology, and should not be interpreted in an idealized or extremely formal sense, unless explicitly defined herein.

[0058] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0059] Currently, most high-throughput gene sequencing systems use focused lasers to excite four-color or two-color fluorescent dyes, and then use a microscope system to collect the excitation light for further processing and analysis to obtain the base sequence. This type of sequencer often uses a high-power light source, which requires constant temperature control to ensure its stability. The instrument contains a high-precision, high-complexity optical system and a scientific-grade camera, which requires operations such as collimation, distortion elimination, focusing, spectroscopy, and filtering. The optical system is quite precise and complex, with high cost, large size, and high requirements for the laboratory environment. Therefore, it is not conducive to near-end field applications and applications in complex use environments.

[0060] For some semiconductor sequencing systems, the signal is generated by biological self-luminescence, and the image sensor semiconductor chip directly reads the signal into the internal circuit of the instrument, making the instrument "optical-free". There is no need for leveling and focusing operations when turning on the instrument after transportation, and the environmental requirements are greatly reduced.

[0061] Currently, signal amplification can be performed through bridge amplification technology on the chip surface; alternatively, template amplification can be completed in solution. Such technology can avoid error accumulation during the amplification process and effectively improve sequencing accuracy.

[0062] The inventors of the present disclosure have found that, regardless of excitation light or spontaneous light, amplification is required before sequencing due to the weak light signal, which leads to a complicated library construction process and a long library construction time. The short read length and amplification bias also limit the ability to interpret the complete genome.

[0063] In view of this, an embodiment of the present disclosure provides a gene sequencing device so as to realize gene detection under weak light signals and improve the sensitivity of gene detection.

[0064] FIG. 1 is a schematic diagram showing the structure of a gene sequencing device according to some embodiments of the present disclosure.

[0065] As shown in Fig. 1, the gene sequencing device includes a biosensing layer 110. The biosensing layer 110 includes one or more sample receiving sites 111 and one or more photosensitive circuits 112 corresponding to the one or more sample receiving sites.

[0066] It should be noted that although three sample receiving sites and three photosensitive circuits are shown in FIG1 , this is merely exemplary. The scope of the present disclosure is not limited to the number of sample receiving sites and photosensitive circuits. For example, the biosensing layer may include less than three (e.g., one or two) sample receiving sites and less than three (e.g., one or two) photosensitive circuits, or may include more than three (e.g., eight or ten, etc.) sample receiving sites and more than three (e.g., eight or ten, etc.) photosensitive circuits.

[0067] The sample receiving site 111 is configured to receive a sample. For example, the sample receiving site 111 may adopt a physical structure (e.g., a groove) to receive the sample; or, the sample receiving site 111 may receive the sample through a magnetic ball, a chemical bond, avidin, or an antigen-antibody, for example, to achieve a link between a chip surface group and ssDNA (single stranded DNA).

[0068] The photosensitive circuit 112 is configured to receive the fluorescent signal emitted by the sample at the corresponding sample receiving site and generate an analog electrical signal according to the fluorescent signal. The photosensitive circuit 112 includes a quantum image sensor (Quanta Image Sensor, referred to as QIS). The photosensitive circuit 112 is configured to transmit the analog electrical signal to the circuit structure layer 120.

[0069] Here, the photosensitive circuit includes a quantum image sensor. Since the quantum image sensor has the characteristic of high sensitivity when performing photon detection, the gene sequencing device can realize gene detection under weak light signals, thereby improving the sensitivity of gene detection.

[0070] As shown in FIG1 , the gene sequencing device further includes a circuit structure layer 120. The circuit structure layer 120 includes an analog-to-digital conversion circuit 121. The analog-to-digital conversion circuit 121 is configured to convert an analog electrical signal into a digital electrical signal. For example, the digital electrical signal includes a digital square wave signal. For example, the analog-to-digital conversion circuit includes an analog-to-digital converter.

[0071] As shown in FIG1 , the gene sequencing device further includes a logic processing layer 130. The logic processing layer 130 is configured to determine the characteristics of the fluorescence signal according to the digital electrical signal, and determine the type and / or sequence of bases contained in the sample according to the characteristics of the fluorescence signal. For example, the logic processing layer 130 includes a logic processing circuit. The logic processing circuit can adopt a known circuit structure, which will not be described in detail here.

[0072] For example, the characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0073] In some embodiments, the logic processing layer 130 is configured to determine the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal, determine at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal based on the number of photons, and determine the type and / or sequence of bases contained in the sample based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0074] For example, a photon can cause a square wave to be generated, that is, the number of square waves in the digital square wave signal is equal to the number of photons in the fluorescence signal. Therefore, the number of photons of the received fluorescence signal is determined according to the number of square waves in the digital square wave signal, and the number of photons can reflect the fluorescence intensity and / or fluorescence quenching time of the fluorescence signal. Therefore, the fluorescence intensity and / or fluorescence quenching time of the fluorescence signal can be determined according to the relationship between the number of photons and the fluorescence intensity and / or fluorescence quenching time of the fluorescence signal known in advance. For example, a certain number of photons corresponds to a certain intensity of fluorescence intensity and a certain fluorescence quenching time. Here, the relationship between the number of photons and the fluorescence intensity and / or fluorescence quenching time of the fluorescence signal is a relationship that can be known in advance after multiple experiments, and will not be described in detail here.

[0075] Next, the type and / or sequence of the bases contained in the sample is determined based on the relationship between at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal and the type and / or sequence of the bases. For example, a certain fluorescence intensity and / or a certain fluorescence quenching time of the fluorescence signal corresponds to a certain type of base and / or a certain sequence of bases. Here, the relationship between at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal and the type and / or sequence of the base is a relationship that can be known in advance after multiple experiments and is not described in detail here.

[0076] So far, a gene sequencing device according to some embodiments of the present disclosure is provided. The gene sequencing device includes: a biosensing layer, including one or more sample receiving sites and one or more photosensitive circuits corresponding to the one or more sample receiving sites, the sample receiving sites are configured to receive samples; the photosensitive circuit is configured to receive the fluorescent signal emitted by the sample on the corresponding sample receiving site, and generate an analog electrical signal according to the fluorescent signal, and the photosensitive circuit includes a quantum image sensor; a circuit structure layer, including: an analog-to-digital conversion circuit, configured to convert the analog electrical signal into a digital electrical signal; and a logic processing layer, configured to determine the characteristics of the fluorescent signal according to the digital electrical signal, and determine the type and / or sequence of the bases contained in the sample according to the characteristics of the fluorescent signal. The gene sequencing device can realize gene detection under weak light signals, thereby improving the sensitivity of gene detection.

[0077] In addition, the above gene sequencing device can sequence multiple gene molecules at the same time, thereby increasing the sequencing speed.

[0078] In the above embodiment, the signal detection of a single base molecule on the sequencing biochemical group is realized by a gene sequencing device, thereby realizing the single molecule sequencing technology. Since the gene sequence does not need to be amplified before sequencing, the complexity of the library construction process and the library construction time are greatly reduced. Since the spatial conformation of the single molecule group is simpler than the cluster / ball structure after amplification, the read length of the synthetic sequencing method can be extended and the sequencing time can be reduced.

[0079] In some embodiments, the quantum image sensor includes a single photon avalanche diode (SPAD). Of course, those skilled in the art will appreciate that the quantum image sensor may also use other types of quantum image sensors, and the scope of the present disclosure is not limited thereto.

[0080] FIG. 2 is a schematic diagram showing the structure of a photosensitive circuit according to some embodiments of the present disclosure.

[0081] As shown in Fig. 2, the photosensitive circuit includes a single photon avalanche diode 210. The single photon avalanche diode 210 is used as a quantum image sensor.

[0082] In some embodiments, as shown in FIG. 2 , the photosensitive circuit further includes a resistor 220. A first end 221 of the resistor 220 is electrically connected to a first voltage end 231, and a second end 222 of the resistor 220 is electrically connected to an anode end 211 of the single-photon avalanche diode 210. A cathode end 212 of the single-photon avalanche diode 210 is electrically connected to a second voltage end 232. The voltage of the second voltage end 232 is greater than the voltage of the first voltage end 231. For example, the first voltage end 231 is a ground end, and the second voltage end 232 is a power supply voltage end. After receiving a photon, the photosensitive circuit can generate an analog electrical signal S A .

[0083] In this embodiment, by setting a resistor in the photosensitive circuit, a current limiting function can be achieved, thereby preventing as much as possible the problem of burning out the single-photon avalanche diode due to excessive current of the generated analog electrical signal, thereby protecting the single-photon avalanche diode.

[0084] For example, the resistance value of the resistor 220 ranges from 1000 ohms to 9000 ohms. Of course, those skilled in the art will appreciate that the range of the resistance value of the resistor is only exemplary, and the scope of the present disclosure is not limited thereto. The resistance value of the resistor can be set according to actual needs.

[0085] The inventors of the present disclosure have discovered that, on a semiconductor sequencing platform, after amplifying the gene molecule 200 times, the signal intensity and signal-to-noise ratio can achieve high-accuracy gene sequencing, and its read length and error rate are basically the same as those of the mainstream excitation light sequencing principle.

[0086] At room temperature, quantum image sensors (QIS) are capable of photon number resolution and high-speed readout. Thanks to the photon number resolution capability of QIS devices, low-light imaging performance is greatly enhanced despite the small pixel size, and its dynamic range is improved thanks to the extended sensitivity in low light.

[0087] For example, the semiconductorized QIS sensor is a SPAD device, which is a device in which the PN junction operates in Geiger mode. In this mode, the reverse bias voltage of the PN junction is above the breakdown voltage (Break Down). When a photon is absorbed by the depletion region of the semiconductor, it generates an electron-hole pair, which splits and accelerates under the action of the electric field. If the energy of the electron or hole is high enough, impact ionization will produce more electron-hole pairs, triggering a self-sustaining avalanche. When the electric field strength is higher than the critical field of the material, this avalanche phenomenon occurs due to the impact ionization of the carriers. For example, in silicon, the critical field Ecr≈3×10 5 V / cm.

[0088] In some cases, when an avalanche begins, a large current of the order of milliamperes may flow through the device, which may destroy the device. Therefore, a resistor 220 may be used in series with the SPAD 210 to prevent burnout. The resistor may be in the order of kilo-ohms. When the anode voltage is biased toward the excess bias voltage V EB When it increases, it suppresses (quenching) the avalanche by reducing the current to less than 100μA (microamps). After suppression is completed, the charging process begins, allowing the photosensitive circuit to return to idle mode and enter the next cycle.

[0089] In Geiger mode, the optical gain of the photodiode is almost infinite, and only the number of carriers in the avalanche increases. Therefore, quantum efficiencies of more than 400,000 e- / Lux-sec (electrons / lux-second) can be easily achieved through the avalanche-suppression process.

[0090] As mentioned above, after the gene molecule is amplified 200 times, the sequencing process can be realized on an ordinary image sensor. Then, after the quantum efficiency is increased by 200 times, the signal generated by a single molecule can be sufficient to realize the gene sequencing process, that is, single-molecule gene sequencing.

[0091] The SPAD array is built using CMOS (Complementary Metal Oxide Semiconductor) technology. This change in design and production platform allows for the reliable production of high-pixel-count SPAD detectors, as well as new pixel circuits that integrate quenching and recharging, time-stamping, and photon counting functions. In terms of data acquisition, this device supports both single SPAD pulse output and fully digital signal processing. In addition to SPAD devices, other QIS devices can achieve high-pixel-count (e.g., 165 million pixel) arrays on a single semiconductor chip.

[0092] FIG3 is a schematic diagram showing the structure of a gene sequencing device according to other embodiments of the present disclosure.

[0093] Similar to the gene sequencing device shown in Fig. 1, the gene sequencing device shown in Fig. 3 includes a biosensing layer 110, a circuit structure layer 120 and a logic processing layer 130. Here, the same or similar structures will not be described in detail.

[0094] In some embodiments, as shown in FIG3 , the circuit structure layer 120 includes a memory 122 in addition to the analog-to-digital conversion circuit. The memory 122 is electrically connected to the analog-to-digital conversion circuit 121 and the logic processing layer 130. The memory 122 is configured to store data. For example, the memory 122 can store comparison data or logic processing data, etc. For another example, the memory can store the relationship between the number of photons and the fluorescence intensity and / or fluorescence quenching time of the fluorescence signal, and / or the relationship between at least one of the fluorescence intensity and fluorescence quenching time of the fluorescence signal and the type and / or sequence of the base, etc.

[0095] For example, the memory may be a SRAM (Static Random-Access Memory). For example, the SRAM is a high-speed SRAM.

[0096] In some embodiments, as shown in Fig. 3, the gene sequencing device further includes a substrate 140. The logic processing layer 130, the circuit structure layer 120 and the biosensing layer 110 are all located above the substrate 140. For example, the substrate may be a silicon substrate or the like.

[0097] In some embodiments, as shown in FIG3 , the logic processing layer 130 is located above the substrate 140. The circuit structure layer 120 is located on the side of the logic processing layer 130 away from the substrate 140. The biosensing layer 110 is located on the side of the circuit structure layer 120 away from the substrate 140. In this way, the integration of related structural layers is achieved, which is convenient for the implementation of gene sequencing.

[0098] The above gene sequencing device is used in the field of gene sequencing, which can reduce costs, realize gene detection under weak light signals, and improve the sensitivity of gene detection. The gene sequencing device can realize basic single-molecule sequencing, high-speed sequencing (for example, high-speed sequencing of hundreds of bases per second) and long read length sequencing (for example, ultra-long read length sequencing of more than 10k).

[0099] The above gene sequencing device can be used in synthetic sequencing. Since the time resolution of the SPAD device is very high, which can reach the ms (millisecond) level, it can also be applied to the sequencing method that extends to the excision base to generate signals. The above gene sequencing device can achieve ultra-high-speed (for example, 960 frames / second) dynamic image acquisition.

[0100] In the above embodiment, the signal detection of a single base molecule on the sequencing biochemical group is realized by an array-type gene sequencing device, thereby realizing the single molecule sequencing technology. Since the gene sequence does not need to be amplified before sequencing, the complexity of the library construction process and the library construction time are greatly reduced. Since the spatial conformation of the single molecule group is simpler than the cluster / ball structure after amplification, the read length of the synthetic sequencing method can be extended and the sequencing time can be reduced.

[0101] In some embodiments of the present disclosure, a gene sequencing method is also provided, comprising: performing gene detection using the gene sequencing device as described above.

[0102] FIG. 4 is a flow chart showing a gene sequencing method according to some embodiments of the present disclosure. FIG. 5 is a schematic diagram showing a gene sequencing method according to some embodiments of the present disclosure. The gene sequencing method according to some embodiments of the present disclosure is described in detail below in conjunction with FIG. 4 and FIG. 5. The gene sequencing method includes steps S402 to S408. That is, the process of performing gene detection using a gene sequencing device includes steps S402 to S408.

[0103] In step S402, a sample is placed in a sample receiving site, wherein the sample emits a fluorescent signal. For example, as shown in FIG5, a sample 510 is placed in the sample receiving site 111, wherein the sample 510 can emit a fluorescent signal.

[0104] For example, DNA fragments can be interrupted in the sample solution, and the linker sequence can be added to denature it; then the capture particles / molecular groups / base sequences / protein sequences, etc. can be mixed into the solution; the sample can be transferred to the chip surface; the sample can be labeled with a dual luciferase-substrate labeling system; and then the sample can be placed in the sample receiving site for detection. Here, by capturing particles / molecular groups / sequences, one pixel on the array chip can correspond to one ssDNA.

[0105] In step S404, the fluorescent signal emitted by the sample is received, and an analog electrical signal is generated according to the fluorescent signal. For example, a photosensitive circuit is used to receive the fluorescent signal emitted by the sample at the corresponding sample receiving site, and an analog electrical signal is generated according to the fluorescent signal.

[0106] In step S406, the analog electrical signal is converted into a digital electrical signal. For example, an analog-to-digital conversion circuit is used to convert the analog electrical signal into a digital electrical signal. For example, the digital electrical signal includes a digital square wave signal.

[0107] In step S408, the characteristics of the fluorescent signal are determined according to the digital electrical signal, and the type and / or sequence of the bases (i.e., nucleotide bases) contained in the sample are determined according to the characteristics of the fluorescent signal. For example, the characteristics of the fluorescent signal are determined according to the digital electrical signal by the logic processing layer, and the type and / or sequence of the bases contained in the sample are determined according to the characteristics of the fluorescent signal.

[0108] In some embodiments, the characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0109] In some embodiments, step S408 includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; determining at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal based on the number of photons; and determining the type and / or sequence of bases contained in the sample based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0110] So far, a gene sequencing method according to some embodiments of the present disclosure is provided. The gene sequencing method includes: placing a sample in a sample receiving site, wherein the sample emits a fluorescent signal; receiving the fluorescent signal emitted by the sample, and generating an analog electrical signal according to the fluorescent signal; converting the analog electrical signal into a digital electrical signal; and determining the characteristics of the fluorescent signal according to the digital electrical signal, and determining the type and / or sequence of bases contained in the sample according to the characteristics of the fluorescent signal. The gene sequencing method can realize gene detection under weak light signals, thereby improving the sensitivity of gene detection.

[0111] In the above gene sequencing method, the signal detection of a single base molecule on the sequencing biochemical group is realized through an array-type gene sequencing device, thereby realizing the single molecule sequencing technology. Since the gene sequence does not need to be amplified before sequencing, the complexity of the library construction process and the time of library construction are greatly reduced. Since the spatial conformation of the single molecule group is simpler than the cluster / ball structure after amplification, the read length of the synthetic sequencing method can be extended and the sequencing time can be reduced.

[0112] Figure 6 is a flow chart showing a nucleic acid detection method according to some embodiments of the present disclosure. As shown in Figure 6, the nucleic acid detection method includes steps S602 to S608.

[0113] In step S602, the nucleic acid molecule at the sample receiving site is labeled with a fluorescent marker, and the fluorescent marker is used to identify the target nucleotide base in the nucleic acid molecule. Here, the fluorescent marker can generate fluorescence under the action of excitation light, and the fluorescent marker is used to identify the nucleotide base at a specific position in the nucleic acid molecule (i.e., the target nucleotide base). For example, the nucleic acid molecule is a nucleic acid macromolecule.

[0114] In step S604, the nucleic acid molecules marked with fluorescent markers are irradiated with excitation light, so that the nucleic acid molecules marked with fluorescent markers absorb the excitation light to generate fluorescent signals, and the fluorescent signals are transmitted to a photosensitive circuit with a quantum image sensor, and the photosensitive circuit generates an analog electrical signal according to the fluorescent signals.

[0115] In some embodiments, the quantum image sensor includes a single photon avalanche diode.

[0116] In step S606 , the analog electrical signal is converted into a digital-to-analog signal to obtain a corresponding digital electrical signal, and at least one feature of the fluorescent signal received at the quantum image sensor is determined based on the digital electrical signal.

[0117] In some embodiments, the at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0118] In some embodiments, the digital electrical signal comprises a digital square wave signal.

[0119] In some embodiments, determining at least one characteristic of a fluorescence signal received at a quantum image sensor based on a digital electrical signal includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; and determining at least one of a fluorescence intensity and a fluorescence quenching time of the fluorescence signal based on the number of photons.

[0120] In step S608, the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule is determined based on the at least one feature.

[0121] In some embodiments, step S608 includes: determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule according to at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0122] In some embodiments, at least one feature of the fluorescence signal can be associated with a fluorescent marker, so that the fluorescent marker can be determined based on at least one feature of the fluorescence signal, and the fluorescent marker is associated with the target nucleotide base in the nucleic acid molecule at the sample receiving site, thereby determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the fluorescent marker.

[0123] So far, a nucleic acid detection method according to some embodiments of the present disclosure is provided. The nucleic acid detection method can be nucleic acid sequencing based on excitation light fluorescence technology. The nucleic acid detection method can realize nucleic acid detection under weak light signals, thereby improving the sensitivity of nucleic acid detection. The above-mentioned nucleic acid detection method can realize signal detection of single base molecules, reduce the complexity and library construction time of the library construction process, and reduce the time of nucleic acid detection.

[0124] Fig. 7 is a flow chart showing a nucleic acid detection method according to other embodiments of the present disclosure. As shown in Fig. 7, the nucleic acid detection method includes steps S702 to S708.

[0125] In step S702, the nucleic acid molecules at the sample receiving site are labeled with a bioluminescent substance, which is used to identify the target nucleotide base in the nucleic acid molecule. That is, the bioluminescent substance is associated with the target nucleotide base in the nucleic acid molecule at the sample receiving site. Here, the bioluminescent substance refers to a substance that can produce bioluminescence without external excitation light irradiation, and the bioluminescent substance is used to identify the nucleotide base at a specific position in the nucleic acid molecule (i.e., the target nucleotide base). For example, the bioluminescent substance is luciferase. For example, the nucleic acid molecule is a nucleic acid macromolecule.

[0126] In step S704, the environment of the nucleic acid molecule is changed or a reaction substrate is added so that the nucleic acid molecule labeled with the bioluminescent substance emits a fluorescent signal, and the fluorescent signal is transmitted to a photosensitive circuit with a quantum image sensor, and the photosensitive circuit generates an analog electrical signal according to the fluorescent signal.

[0127] In some embodiments, the quantum image sensor includes a single photon avalanche diode.

[0128] In step S706 , the analog electrical signal is converted into a digital-to-analog signal to obtain a corresponding digital electrical signal, and at least one feature of the fluorescent signal received at the quantum image sensor is determined based on the digital electrical signal.

[0129] In some embodiments, the at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0130] In some embodiments, the digital electrical signal comprises a digital square wave signal.

[0131] In some embodiments, determining at least one characteristic of a fluorescence signal received at a quantum image sensor based on a digital electrical signal includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; and determining at least one of a fluorescence intensity and a fluorescence quenching time of the fluorescence signal based on the number of photons.

[0132] In step S708, the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule is determined based on the at least one feature.

[0133] In some embodiments, step S708 includes: determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule according to at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0134] In some embodiments, the at least one feature of the fluorescence signal can be associated with a biofluorescent substance, so that the biofluorescent substance can be determined based on the at least one feature of the fluorescence signal, and the biofluorescent substance is associated with the target nucleotide base in the nucleic acid molecule at the sample receiving site, and thus, the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule is determined based on the biofluorescent substance.

[0135] So far, a nucleic acid detection method according to some embodiments of the present disclosure is provided. The nucleic acid detection method can be nucleic acid sequencing based on bioluminescence technology. The nucleic acid detection method can realize nucleic acid detection under weak light signals, thereby improving the sensitivity of nucleic acid detection. The above-mentioned nucleic acid detection method can realize signal detection of single base molecules, reduce the complexity and library construction time of the library construction process, and reduce the time of nucleic acid detection.

[0136] Fig. 8 is a flow chart showing a biological sample in-situ detection method according to some embodiments of the present disclosure. As shown in Fig. 8, the biological sample in-situ detection method includes steps S802 to S808.

[0137] In step S802, the biological sample is fixed to the biosensing layer, the biological sample contains multiple nucleic acid molecules, the biosensing layer includes one or more sample receiving sites and one or more photosensitive circuits corresponding to the one or more sample receiving sites, the nucleic acid molecules are fixed by the sample receiving sites, and the nucleic acid molecules are marked with fluorescent markers or bioluminescent substances. Here, the fluorescent markers and bioluminescent substances have been described above and will not be repeated here. Furthermore, the biological sample can be fixed to the biosensing layer using known techniques.

[0138] In some embodiments, the nucleic acid molecule is coupled to one or more of a protein, a peptide, an antibody, or an affinity tag.

[0139] In some embodiments, the nucleic acid molecule is an oligonucleic acid tag sequence.

[0140] In some embodiments, the biological sample is a tissue section.

[0141] In step S804, under predetermined conditions, the nucleic acid molecules marked with fluorescent markers or bioluminescent substances generate fluorescent signals, and the fluorescent signals are transmitted to a photosensitive circuit having a quantum image sensor, and the photosensitive circuit generates an analog electrical signal according to the fluorescent signals.

[0142] For example, the predetermined condition may be a condition that causes a nucleic acid molecule labeled with a fluorescent marker to emit light using excitation light or a condition that causes a nucleic acid molecule labeled with a bioluminescent substance to emit light spontaneously.

[0143] In some embodiments, the quantum image sensor includes a single photon avalanche diode.

[0144] In step S806 , the analog electrical signal is converted into a digital-to-analog signal to obtain a corresponding digital electrical signal, and at least one feature of the fluorescent signal received at the quantum image sensor is determined based on the digital electrical signal.

[0145] In some embodiments, the at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time.

[0146] In some embodiments, the digital electrical signal comprises a digital square wave signal.

[0147] In some embodiments, determining at least one characteristic of a fluorescence signal received at a quantum image sensor based on a digital electrical signal includes: determining the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal; and determining at least one of a fluorescence intensity and a fluorescence quenching time of the fluorescence signal based on the number of photons.

[0148] In step S806, the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at the corresponding position in the biological sample is determined based on the at least one feature.

[0149] In some embodiments, step S806 includes: determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at the corresponding position in the biological sample according to at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

[0150] So far, a biological sample in situ detection method according to some embodiments of the present disclosure is provided. The biological sample in situ detection method can be a spatial transcriptome technology. The biological sample in situ detection method can realize nucleic acid detection under weak light signals, thereby improving the sensitivity of nucleic acid detection. The above-mentioned biological sample in situ detection method can realize signal detection of single base molecules, reduce the complexity and library construction time of the library construction process, and reduce the time of nucleic acid detection.

[0151] So far, various embodiments of the present disclosure have been described in detail. In order to avoid obscuring the concept of the present disclosure, some details known in the art are not described. Based on the above description, those skilled in the art can fully understand how to implement the technical solution disclosed here.

[0152] Although some specific embodiments of the present disclosure have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present disclosure. It should be understood by those skilled in the art that the above embodiments may be modified or some technical features may be replaced by equivalents without departing from the scope and spirit of the present disclosure. The scope of the present disclosure is defined by the appended claims.

Claims

A gene sequencing device, comprising: A biosensing layer, comprising one or more sample receiving sites and one or more photosensitive circuits corresponding to the one or more sample receiving sites, wherein the sample receiving sites are configured to receive a sample; The photosensitive circuit is configured to receive a fluorescent signal emitted by a sample at a corresponding sample receiving site and generate an analog electrical signal according to the fluorescent signal, and the photosensitive circuit includes a quantum image sensor; The circuit structure layer includes: an analog-to-digital conversion circuit configured to convert the analog electrical signal into a digital electrical signal; and a logic processing layer configured to determine the characteristics of the fluorescent signal according to the digital electrical signal, and to determine the type and / or sequence of the bases contained in the sample according to the characteristics of the fluorescent signal. The gene sequencing device according to claim 1, wherein: The quantum image sensor includes a single photon avalanche diode. The gene sequencing device according to claim 2, wherein: The photosensitive circuit also includes a resistor, wherein a first end of the resistor is electrically connected to a first voltage end, a second end of the resistor is electrically connected to an anode end of the single-photon avalanche diode, a cathode end of the single-photon avalanche diode is electrically connected to a second voltage end, and a voltage at the second voltage end is greater than a voltage at the first voltage end. The gene sequencing device according to claim 1, wherein: The characteristics of the fluorescence signal include: at least one of fluorescence intensity and fluorescence quenching time; the digital electrical signal includes: a digital square wave signal. The gene sequencing device according to claim 4, wherein: The logic processing layer is configured to determine the number of photons of the received fluorescence signal based on the number of square waves in the digital square wave signal, determine at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal based on the number of photons, and determine the type and / or sequence of the bases contained in the sample based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal. The gene sequencing device according to claim 1, wherein: The circuit structure layer further includes: a memory electrically connected to the analog-to-digital conversion circuit and the logic processing layer and configured to store data. The gene sequencing device according to claim 1, further comprising: Substrate; wherein the logic processing layer, the circuit structure layer and the biological sensing layer are all located above the substrate. The gene sequencing device according to claim 7, wherein: The logic processing layer is located above the substrate; the circuit structure layer is located on a side of the logic processing layer away from the substrate; and the biosensing layer is located on a side of the circuit structure layer away from the substrate. A gene sequencing method comprising: Gene testing is performed using the gene sequencing device as described in any one of claims 1 to 8. The gene sequencing method according to claim 9, wherein: Performing gene testing using the gene sequencing device includes: placing a sample in the sample receiving site, wherein the sample emits a fluorescent signal; receiving the fluorescent signal emitted by the sample, and generating an analog electrical signal based on the fluorescent signal; converting the analog electrical signal into a digital electrical signal; and determining the characteristics of the fluorescent signal based on the digital electrical signal, and determining the type and / or sequence of bases contained in the sample based on the characteristics of the fluorescent signal. The gene sequencing method according to claim 10, wherein: The characteristics of the fluorescence signal include: at least one of fluorescence intensity and fluorescence quenching time; the digital electrical signal includes: a digital square wave signal. The gene sequencing method according to claim 11, wherein: Determining the characteristics of the fluorescence signal according to the digital electrical signal, and determining the type and / or sequence of the bases contained in the sample according to the characteristics of the fluorescence signal includes: determining the number of photons of the received fluorescence signal according to the number of square waves in the digital square wave signal; determining at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal according to the number of photons; and determining the type and / or sequence of the bases contained in the sample according to at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal. The gene sequencing method according to claim 10, wherein: The fluorescent signal is generated by a bioluminescent reaction. The gene sequencing method according to claim 13, wherein the bioluminescence reaction refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal. A nucleic acid detection method, comprising: labeling the nucleic acid molecule at the sample receiving site with a fluorescent label, wherein the fluorescent label is used to identify a target nucleotide base in the nucleic acid molecule; The nucleic acid molecules marked with the fluorescent marker are irradiated with excitation light, so that the nucleic acid molecules marked with the fluorescent marker absorb the excitation light to generate a fluorescent signal, and the fluorescent signal is transmitted to a photosensitive circuit having a quantum image sensor, and the photosensitive circuit generates an analog electrical signal according to the fluorescent signal; the analog electrical signal is converted into a digital-to-analog signal to obtain a corresponding digital electrical signal, and at least one feature of the fluorescent signal received at the quantum image sensor is determined based on the digital electrical signal; and the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule is determined based on the at least one feature. The nucleic acid detection method according to claim 15, wherein The quantum image sensor includes a single photon avalanche diode. The nucleic acid detection method according to claim 15, wherein The at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time. The nucleic acid detection method according to claim 17, wherein: The digital electrical signal comprises a digital square wave signal; and determining at least one feature of the fluorescence signal received at the quantum image sensor based on the digital electrical signal comprises: determining the number of photons of the received fluorescence signal according to the number of square waves in the digital square wave signal; and determining at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal according to the number of photons. The nucleic acid detection method according to claim 18, wherein Determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the at least one feature includes: determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule according to at least one of the fluorescence intensity and fluorescence quenching time of the fluorescence signal. A nucleic acid detection method, comprising: labeling the nucleic acid molecule at the sample receiving site with a bioluminescent substance, wherein the bioluminescent substance is used to identify a target nucleotide base in the nucleic acid molecule; Changing the environment of the nucleic acid molecule or adding a reaction substrate so that the nucleic acid molecule labeled with the bioluminescent substance emits a fluorescent signal, and transmitting the fluorescent signal to a photosensitive circuit having a quantum image sensor, wherein the photosensitive circuit generates an analog electrical signal according to the fluorescent signal; performing digital-to-analog conversion on the analog electrical signal to obtain a corresponding digital electrical signal, and determining at least one feature of the fluorescent signal received at the quantum image sensor based on the digital electrical signal; and determining the type and / or sequence of target nucleotide bases contained in the nucleic acid molecule based on the at least one feature. The nucleic acid detection method according to claim 20, wherein The quantum image sensor includes a single photon avalanche diode. The nucleic acid detection method according to claim 20, wherein The at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time. The nucleic acid detection method according to claim 22, wherein: The digital electrical signal comprises a digital square wave signal; and determining at least one feature of the fluorescence signal received at the quantum image sensor based on the digital electrical signal comprises: determining the number of photons of the received fluorescence signal according to the number of square waves in the digital square wave signal; and determining at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal according to the number of photons. The nucleic acid detection method according to claim 23, wherein Determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule based on the at least one feature includes: determining the type and / or sequence of the target nucleotide base contained in the nucleic acid molecule according to at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal. The nucleic acid detection method according to claim 20, wherein The bioluminescent substance is luciferase. A biological sample in situ detection method, comprising: Fixing a biological sample on a biosensing layer, wherein the biological sample contains a plurality of nucleic acid molecules, the biosensing layer includes one or more sample receiving sites and one or more photosensitive circuits corresponding to the one or more sample receiving sites, the nucleic acid molecules are correspondingly fixed by the sample receiving sites, and the nucleic acid molecules are marked with fluorescent markers or bioluminescent substances; under predetermined conditions, the nucleic acid molecules marked with the fluorescent markers or the bioluminescent substances generate fluorescent signals, and the fluorescent signals are transmitted to a photosensitive circuit having a quantum image sensor, the photosensitive circuit generates an analog electrical signal according to the fluorescent signals; performing digital-to-analog conversion on the analog electrical signals to obtain corresponding digital electrical signals, and determining at least one feature of the fluorescent signal received at the quantum image sensor based on the digital electrical signals; and determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at a corresponding position in the biological sample based on the at least one feature. The biological sample in situ detection method according to claim 26, wherein: The nucleic acid molecule is coupled to one or more molecules selected from proteins, peptides, antibodies or affinity tags. The biological sample in situ detection method according to claim 27, wherein: The nucleic acid molecule is an oligonucleic acid tag sequence. The biological sample in situ detection method according to claim 26, wherein: The quantum image sensor includes a single photon avalanche diode. The biological sample in situ detection method according to claim 26, wherein: The at least one characteristic of the fluorescence signal includes at least one of fluorescence intensity and fluorescence quenching time. The biological sample in situ detection method according to claim 26, wherein: The digital electrical signal comprises a digital square wave signal; and determining at least one feature of the fluorescence signal received at the quantum image sensor based on the digital electrical signal comprises: determining the number of photons of the received fluorescence signal according to the number of square waves in the digital square wave signal; and determining at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal according to the number of photons. The biological sample in situ detection method according to claim 31, wherein: Determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at the corresponding position in the biological sample based on the at least one feature includes: determining the type and / or sequence of nucleotide bases contained in the nucleic acid molecule at the corresponding position in the biological sample based on at least one of the fluorescence intensity and the fluorescence quenching time of the fluorescence signal.

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