Micro-integrated system and method for picoampere-level multi-channel fluorescence signal detection

Abstract

The present application relates to a picoampere-level multi-channel photoelectric signal detection micro-integrated system and method, which is used for nucleic acid molecule amplification fluorescence signal POCT detection; an electrical signal source is introduced in the primary of the amplification circuit to offset the bias current or voltage in the system, and this offset will also act on the secondary after amplification; when there is no fluorescence signal, the system can keep the light signal reading at a low level; when the nucleic acid molecule amplification generates a fluorescence signal, the detection system can detect the low-background photoelectric signal to obtain a larger signal-to-noise ratio. The picoampere-level multi-channel fluorescence signal detection micro-integrated system designed by the present invention can realize high-sensitivity, multiplex, and rapid nucleic acid molecule detection in the micro-integrated system, making molecular detection POCT a reality.

Description

Technical Field

[0001] The present disclosure relates to the field of medical electronic technology, and in particular to a picoampere-level multi-channel fluorescence signal detection micro-integrated system, a picoampere-level multi-channel fluorescence signal micro-integrated detection method, and a nucleic acid rapid detector. Background Art

[0002] Currently, molecular detection instruments, such as qPCR instruments, cost more than RMB 100,000 and are benchtop instruments used in laboratories. Patients need to go to the hospital for sampling, which is not only time-consuming but also prone to cross-infection. To solve these problems, point-of-care testing (POCT) has become a rapidly developing trend.

[0003] Common infectious diseases are mostly caused by pathogens such as viruses, mycoplasmas, bacteria or fungi; the genome of the pathogen can be composed of RNA or DNA. For RNA pathogens, the common detection method is to reverse transcribe RNA (reverse transcription or RT) into cDNA, and then perform exponential molecular amplification of cDNA, such as using polymerase chain reaction (polymerase chain reaction or PCR) to amplify cDNA molecules by 2 N Exponential molecular amplification; RT-PCR is to carry out the RT phase reaction first and then the PCR reaction in the same reaction body. When the genome of the pathogen is composed of DNA, PCR can be directly used to detect such pathogens. qPCR (quantitative PCR) is a type of PCR, which is characterized by that when there is a DNA target molecule in the reaction system, the fluorescent signal generated by exponential molecular amplification can be visualized as an S-shaped curve over time. According to the time and morphological characteristics of the S curve, it can be judged whether there are pathogens in the sample or the amount of pathogens.

[0004] In a pathogen detection reaction based on fluorescence signal detection, the initial light signal is the signal collected by a photosensor (such as a photodiode PD) in the instrument system before or after the start of the reaction and at a certain time point or time period before the reaction conditions stabilize; the background fluorescence signal is the fluorescence signal after the initial light signal and after the reaction conditions stabilize, but before the exponential fluorescence signal is generated.

[0005] POCT for molecular testing is still in its infancy, and the first issues to be addressed are instrument miniaturization, low cost, high sensitivity, and ease of use. Figure 1 The first generation amplifier circuit shown ("IN5" is the PD input terminal, and the R45 output is directly connected to the AD pin of the MCU) can amplify the pA-level photoelectric signal to the range that the MCU can detect. However, in actual application to the AirPOC (nucleic acid rapid detector) product, there are the following defects:

[0006] 1. Due to the low light intensity of the light source, the required amplification factor in the circuit is very large. In the actual reaction system, if the fluorescence signal is too strong or low, the amplification circuit will amplify the fluorescence signal collected by the photodiode (PD) to exceed 3.3V, or be lower than 0V, causing the device to malfunction;

[0007] 2. In the actual assembly of the instrument, due to some factors such as assembly method, individual differences of PDs and individual differences of operational amplifiers, the fluorescence signal may be exposed in the primary amplification circuit, or the background signals of multiple channels may not be at the same level, making the detection range inconsistent, resulting in the inability to see the amplification curve when doing positive samples;

[0008] 3. If the mass production standards need to be met, qualified PDs and op amps need to be selected, and there are also high requirements for structural parts such as filters in the equipment, which greatly increases the difficulty of mass production. Summary of the invention

[0009] In order to solve the above problems, the present application proposes a picoampere-level multi-channel fluorescence signal detection micro-integrated system, a picoampere-level multi-channel fluorescence signal micro-integrated detection method and a nucleic acid rapid detector.

[0010] On the one hand, the present application proposes a picoampere-level multi-channel fluorescence signal detection micro-integrated system, comprising:

[0011] A light-transmitting reaction container, used for nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is an enzymatic reaction;

[0012] At least one light source device, used to excite the fluorescein in the nucleic acid amplification reaction to generate at least one fluorescent signal, the light source device is no larger than 10 mm 3 ;

[0013] At least one photosensitive (PD) device, used to sense the fluorescent signal and output a corresponding PD signal, the volume of the photosensitive (PD) device is no larger than 100 mm 3 ;

[0014] An MCU control board, used for outputting digital-to-analog (DAC) signals and collecting analog-to-digital (ADC) signals, the MCU control board including an MCU microprocessing unit, used for implementing a peeling algorithm and a signal collection function, wherein the peeling algorithm is based on a preset MCU peeling control logic;

[0015] At least one set of DA buffer circuits, the buffer circuit comprising a first follower circuit and a second follower circuit, the first follower circuit outputs a reference voltage, the second follower circuit buffers the DAC signal and generates a digital-to-analog buffer signal after dividing the reference voltage: DA buffer signal;

[0016] At least one stage of amplification circuit, used for superimposing the PD signal and the DA buffer signal to obtain a peeled ADC signal;

[0017] The photosensitive (PD) device and the DA buffer circuit are respectively connected to the amplifier circuit on the PCB;

[0018] The average PCB area occupied by a single channel of the buffer circuit and amplifier circuit is no more than 500mm 2 ;

[0019] The DA buffer circuit and the amplifier circuit are electrically connected to the MCU control board respectively.

[0020] Preferably, the peeling control logic comprises:

[0021] a. Set a pico value in MCU;

[0022] b. A DAC value is provided by the MCU;

[0023] c. Based on the DAC value, the buffer circuit outputs a DA buffer value;

[0024] d. Output ADC value after passing through the amplification circuit according to the PD signal value and DA buffer value;

[0025] e. Determine whether the ADC value reaches the set value:

[0026] If yes, the DAC signal is locked and stably output, the peeling operation is completed, and the output ADC signal value is the detected fluorescence signal value;

[0027] If not, adjust the MCU to output the corrected DAC value, perform the peeling operation from step d to step e, until the ADC value reaches the set peeling value, then lock and stably output the corrected DAC value, complete the peeling operation, and the output ADC signal value is the detected fluorescence signal value;

[0028] and / or

[0029] f. If necessary, repeat the second stage peeling steps from steps a to e.

[0030] Preferably, the peeling algorithm comprises:

[0031] ,

[0032] in:

[0033] u(t) is the control output at time t, which is the DAC value output by the MCU control board;

[0034] K Pis a proportional gain, used to adjust the response strength of the MCU control board to the current error;

[0035] K i is the integral gain, used to eliminate steady-state errors;

[0036] K d is the differential gain, which is used to predict the error trend and reduce the overshoot and oscillation of the system;

[0037] is the error at time t, calculated as the difference between the set target pico value and the actual optical signal currently collected by the MCU control board: , where SP is the set value of the skin value, PV(t) is the process variable, that is, the current integrated term, which is the accumulation of error over time and is used to eliminate steady-state errors:

[0038] The integral term is the accumulation of error over time and is used to eliminate steady-state error:

[0039] ;

[0040] The derivative term is a prediction of the error rate of change and is used to predict the error trend:

[0041] ;

[0042] PV(T) is a process variable calculated by the MCU control board using the trimmed mean algorithm through multiple data collected within a certain period of time. The calculation is as follows:

[0043] Let X = {x1, x2, x3, ..., xn} be a data set of a certain original optical signal collected, which contains n values;

[0044] Find the m maximum values ​​max(X) and minimum values ​​min(X) in the original data set and remove them to get the data set Y;

[0045] Calculate the sum S of the remaining values:

[0046] ,in:

[0047] x Represents each element in the set Y;

[0048] Calculate the number of remaining values, k is the number of elements in the data set Y:

[0049] K=nm

[0050] Calculate the Y mean of the data set:

[0051] PV(t)=S / k.

[0052] Preferably, the DA buffer circuit includes an operational amplifier chip U6B, a resistor R40, a resistor R42, a resistor R44, a capacitor C27 and a resistor R45, wherein:

[0053] The positive input terminal MCU_DA_PD_Zero (DAC value output by MCU) of the operational amplifier chip U6B is connected to the MCU DA output pin of the MCU control board, the negative input terminal is connected to one end of the resistor R40, and the output terminal is connected to the other end of the resistor R40; the input terminal of the resistor R42 is connected to the output terminal of the operational amplifier chip U6B, and the output terminal is connected to the input terminal of the resistor R45; the output terminal of the resistor R45 is grounded; the input terminal of the resistor R44 is connected in parallel to the output terminal of the resistor R42, and the output terminal is connected to the reference power supply; the input terminal of the capacitor C27 is connected in parallel to the output terminal of the resistor R42, and the output terminal is grounded.

[0054] Preferably, the operational amplifier chip U6B is an operational amplifier chip with a single power supply of at least 3V and a gain bandwidth of at least 1Mhz, preferably LM358.

[0055] Preferably, the amplifier circuit includes a resistor R7, a capacitor C3, a resistor R3, a resistor R4, a resistor R1, an operational amplifier chip U1A, a resistor R13, a resistor R17, an operational amplifier chip U2A, a resistor R8, a resistor R9, a capacitor C1, a resistor R14 and a capacitor C7, wherein;

[0056] The current signal source DA_PD_Zero (DAC buffer value) acts on the negative input terminal of the operational amplifier chip U1A through the resistor R7, the positive input terminal of the operational amplifier chip U1A is grounded, and the output terminal of the operational amplifier chip U1A is connected to the input terminal of the resistor R13; the resistor R3 and the resistor R4 are connected in series, the input terminal of the resistor R3 is connected to the negative input terminal of the operational amplifier chip U1A, and the output terminal of the resistor R4 is connected to the output terminal of the resistor R13; the two ends of the capacitor C3 are respectively connected in parallel to the input terminal of the resistor R3 and the output terminal of the resistor R4; one end of the resistor R1 is connected to the output terminal of the resistor R3, and the other end is grounded;

[0057] The current after being operationally amplified by the operational amplifier chip U1A acts on the positive input terminal of the operational amplifier chip U2A; the input terminal of the resistor R17 is connected to the output terminal of the resistor R13, and the output terminal is connected to D-0.01V; the output terminal of the operational amplifier chip U2A is connected to the input terminal of the resistor R14, and the negative input terminal of the operational amplifier chip U2A is connected to the output terminal of the resistor R8; the input terminal of the resistor R8 is grounded, and the output terminal is connected to the input terminal of the resistor R9, and the output terminal of the resistor R9 is connected to the input terminal of the resistor R14; the two ends of the capacitor C1 are respectively connected in parallel to the output terminal and the input terminal of the resistor R9; the input terminal of the capacitor C7 is connected to the input terminal of the resistor R14, and the output terminal is grounded; the output terminal of the resistor R14 outputs the final acquisition signal MCU_AD_PD0.

[0058] Preferably, the operational amplifier chip U1A is an operational amplifier chip with a single power supply of at least 1.8V and a gain bandwidth of at least 10Mhz, preferably OPA2320A.

[0059] Preferably, the operational amplifier chip U2A is an operational amplifier chip with a single power supply of at least 1.8V and a gain bandwidth of at least 1.2Mhz, preferably GS85502.

[0060] Preferably, the positive input terminal of the power supply of the operational amplifier chip U1A is OPA2320A and connected to the positive pole of the power supply: D+3.3V, and the negative input terminal of the power supply is grounded.

[0061] On the one hand, the present application also proposes a picoampere-level multi-channel photoelectric signal detection method, which is implemented based on the above-mentioned picoampere-level multi-channel photoelectric signal detection micro-integrated system, and includes the following steps:

[0062] The MCU control board outputs DAC to the DA buffer circuit;

[0063] After buffering the initial DAC signal output by the MCU through the DA buffer circuit, a voltage signal source DA_PD_Zero (DAC buffer value) of -8mV to +550mV is provided for collecting the PD signal;

[0064] The fluorescent signal generated by the nucleic acid amplification reaction is sensed and detected by a photosensitive (PD) device and the corresponding PD signal is output to the amplification circuit, wherein the fluorescent signal includes an initial light signal and a background fluorescent signal generated in the RT stage and the PCR stage in sequence;

[0065] The PD signal and the initial DAC signal are amplified by an amplifying circuit to obtain and output a final acquisition signal MCU_AD_PD0 to the MCU control board;

[0066] Through the MCU control board, the peeling function is performed in the RT stage and the PCR stage respectively based on the preset MCU peeling control logic.

[0067] Preferably, it also includes:

[0068] A charging and discharging module, used to provide charging and discharging services;

[0069] At least 2 heating and temperature control modules to provide heating services for nucleic acid testing;

[0070] Multi-channel LED control circuit, used to provide different types of LED response services;

[0071] The communication interface Type-C is used to communicate with the detection terminal, including: transmitting the sampling value of the fluorescent signal generated by the nucleic acid detection molecule detection to the detection terminal;

[0072] The charging and discharging module, the heating and temperature control module, the multi-channel LED control circuit and the communication interface Type-C are respectively connected to the MCU control board.

[0073] Technical effects of the present invention:

[0074] This application introduces a current signal source in the primary of the amplifier circuit to offset the photocurrent, bias current, etc. This offset will also act on the secondary after amplification, so that when no fluorescence signal is generated, the light signal reading can be maintained at a lower level, so that after the fluorescence signal is generated, a complete amplification curve can be seen. Specifically, the current signal source comes from the DA module of the MCU. The DA module can convert the digital signal into an analog signal of 0 to 3.3V. After the operational amplifier buffer and voltage division, this analog signal is converted into a voltage signal of -8mV to +550mV. Then this voltage signal is connected to the signal input terminal of the primary operational amplifier U1A through a 100MΩ resistor, so that a -8pA to +550pA offset current can be generated, which can release the primary skin value voltage of the operational amplifier, so that the voltage of the secondary output will also be reduced, so that the AD finally collected by the MCU will also be reduced to an acceptable level. In this way, normal data can be collected, and the initial AD value collected on the circuit can be reduced to an acceptable level. When doing positive samples, the amplification curve can be seen.

[0075] In addition, the picoampere-level multi-channel fluorescence signal detection micro-integrated system designed by the present invention can use a low-cost optical detection solution on AirPOC (multi-channel fluorescence signal detector) to achieve rapid mass production, reduce the difficulty of mass production, and obtain reliable multi-channel data. Through optoelectronic integration and innovative control logic, low-cost devices (cost <500 yuan) are used to achieve high-sensitivity (pA level, or single molecule), multiple (multiple light signal sources or multiple pathogens), and rapid (<30min) nucleic acid molecule detection on a handheld instrument, making molecular detection POCT a reality, thereby solving the problem of excessive background signals or even exceeding the limit, and collecting reliable multi-channel optical signal data.

[0076] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0078] Figure 1 Shown is a schematic diagram of the first generation amplification circuit of a molecular detection POCT device;

[0079] Figure 2 Shown is a schematic diagram of a control circuit of a micro-integrated system of the present invention;

[0080] Figure 3 Shown is the MUC peeling control logic diagram of the present invention;

[0081] Figure 4 Shown is a DA buffer circuit diagram of the present invention;

[0082] Figure 5 Shown is an amplification circuit diagram of the present invention;

[0083] Figure 6 Shown is a secondary peeling timing diagram of the present invention;

[0084] Figure 7 Shown is the first peeling control logic diagram of the present invention;

[0085] Figure 8 Shown is the second peeling control logic diagram of the present invention;

[0086] Fig. 9 It is shown as a schematic diagram of 4-way detection of the present invention;

[0087] Fig.10 Shown is a comparison diagram of the detection data after peeling of the present invention;

[0088] Fig.11Shown is a structural diagram of the control system of the rapid nucleic acid detector of the present invention. DETAILED DESCRIPTION

[0089] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0090] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0091] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present disclosure.

[0092] In this embodiment, the values ​​of each electronic device in the circuit of the attached figure only represent an example of this embodiment. For other schemes, it can be decided by the user. As long as the nucleic acid molecule detection system is implemented according to the technical principles of the present invention, it is within the protection scope of the present invention. Example 1

[0093] like Figure 2 As shown, on one hand, the present application proposes a picoampere-level multi-channel fluorescence signal detection micro-integrated system, comprising:

[0094] A light-transmitting reaction container, used for nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is an enzymatic reaction;

[0095] At least one light source device, used to excite the fluorescein in the nucleic acid amplification reaction to generate at least one fluorescent signal, the light source device is no larger than 10 mm 3 ;

[0096] At least one photosensitive (PD) device, used to sense the fluorescent signal and output a corresponding PD signal, the volume of the photosensitive (PD) device is no larger than 100 mm 3 ;

[0097] An MCU control board, used for outputting digital-to-analog (DAC) signals and collecting analog-to-digital (ADC) signals, the MCU control board including an MCU microprocessing unit, used for implementing a peeling algorithm and a signal collection function, wherein the peeling algorithm is based on a preset MCU peeling control logic;

[0098] At least one set of DA buffer circuits, the buffer circuit comprising a first follower circuit and a second follower circuit, the first follower circuit outputs a reference voltage, the second follower circuit buffers the DAC signal and generates a digital-to-analog buffer signal after dividing the reference voltage: DA buffer signal;

[0099] At least one stage (optical signal) amplification circuit, used for superimposing the PD signal and the DA buffer signal to obtain a peeled ADC signal;

[0100] The photosensitive (PD) device and the DA buffer circuit are respectively connected to the amplifier circuit on the PCB;

[0101] The average PCB area occupied by a single channel of the buffer circuit and amplifier circuit is no more than 500mm 2 ;

[0102] The DA buffer circuit and the amplifier circuit are electrically connected to the MCU control board respectively.

[0103] The light-transmitting reaction container and the light source device can be selected from reaction containers of corresponding light-transmitting materials (such as quartz experimental cups) according to the analysis and detection requirements, and the light source device excites the fluorescent element in the nucleic acid amplification reaction, such as:

[0104] Halogen tungsten light source: can emit a continuous spectrum, suitable for a variety of PCR applications, and can be equipped with multi-color filters to achieve different excitation wavelengths;

[0105] Argon ion laser: high brightness, good monochromaticity, often used in PCR experiments that require highly precise excitation wavelengths, with high energy density and stability;

[0106] Light-emitting diode (LED) light source: low price, low energy consumption, long life, especially suitable for large-scale, long-running PCR experiments. Monochromatic LED light source requires different LEDs to achieve different excitation wavelengths.

[0107] The photosensitive (PD) device can use a PD photoelectrode tube.

[0108] The present invention requires a photosensitive (PD) device to collect multi-channel photoelectric signals (specifically, multiple photosensitive (PD) devices are arranged around the reaction tube to collect fluorescence signals of several channels, i.e., photoelectric signals). The multi-channel photoelectric signals are corresponding electrical signals generated by detecting multiple interval light signals within the wavelength range of 320nm to 1200nm.

[0109] The MCU control board is a computing and control unit based on an MCU chip (such as an MCU microprocessor unit such as a single-chip microcomputer chip of the STM32 series), which can perform signal analysis and processing. The MCU control board includes an MCU microprocessor unit (program block, etc.) for implementing a peeling algorithm and signal acquisition functions, wherein the peeling algorithm is based on a preset MCU peeling control logic; Figure 3 As shown, the peeling control logic includes:

[0110] a. Set a pico value in MCU;

[0111] b. A DAC value is provided by the MCU;

[0112] c. Based on the DAC value, the buffer circuit outputs a DA buffer value;

[0113] d. Output ADC value after passing through the amplification circuit according to the PD signal value and DA buffer value;

[0114] e. Determine whether the ADC value reaches the set value:

[0115] If yes, the DAC signal is locked and stably output, the peeling operation is completed, and the output ADC signal value is the detected fluorescence signal value;

[0116] If not, adjust the MCU to output the corrected DAC value, perform the peeling operation from step d to step e, until the ADC value reaches the set peeling value, then lock and stably output the corrected DAC value, complete the peeling operation, and the output ADC signal value is the detected fluorescence signal value;

[0117] and / or

[0118] f. If necessary, repeat the second stage peeling steps from steps a to e.

[0119] The logic of the second stage peeling operation can be referred to Figure 7 and Figure 8 Secondary tare logic shown.

[0120] If you want to perform secondary peeling, please refer to the peeling node of the corresponding signal. Figure 6 shown.

[0121] The peeling algorithm includes:

[0122] ,

[0123] in:

[0124] u(t) is the control output at time t, which is the DAC value output by the MCU control board;

[0125] K Pis a proportional gain, used to adjust the response strength of the MCU control board to the current error;

[0126] K i is the integral gain, used to eliminate steady-state errors;

[0127] K d is the differential gain, which is used to predict the error trend and reduce the overshoot and oscillation of the system;

[0128] is the error at time t, calculated as the difference between the set target pico value and the actual optical signal currently collected by the MCU control board: , where SP is the set value of the skin value, PV(t) is the process variable, that is, the current integrated term, which is the accumulation of error over time and is used to eliminate steady-state errors:

[0129] The integral term is the accumulation of error over time and is used to eliminate steady-state error:

[0130] ;

[0131] The derivative term is a prediction of the error rate of change and is used to predict the error trend:

[0132] ;

[0133] PV(T) is a process variable calculated by the MCU control board using the trimmed mean algorithm through multiple data collected within a certain period of time. The calculation is as follows:

[0134] Let X = {x1, x2, x3, ..., xn} be a data set of a certain original optical signal collected, which contains n values;

[0135] Find the m maximum values ​​max(X) and minimum values ​​min(X) in the original data set and remove them to get the data set Y;

[0136] Calculate the sum S of the remaining values:

[0137] ,in:

[0138] x Represents each element in the set Y;

[0139] Calculate the number of remaining values, k is the number of elements in the data set Y:

[0140] K=nm

[0141] Calculate the Y mean of the data set:

[0142] PV(t)=S / k.

[0143] The above algorithm is implemented through an MCU chip embedded program, etc., to realize the calculation process of the corresponding peeling control logic.

[0144] like Figure 3 The following is the execution control logic of the MCU (chip embedded program in the control board) of this system.

[0145] This current signal source comes from a DA module that comes with the MCU. This DA module can convert digital signals into analog signals of 0 to 3.3V. After being buffered and divided by the op amp, this analog signal is converted into a voltage signal of -8mV to +550mV. Then this voltage signal is connected to the signal input terminal of the primary op amp through a 100MΩ resistor, so that a -8pA to +550pA offset current can be generated, which can release the primary skin value voltage of the op amp, so that the voltage of the secondary output will also be reduced, so that the AD finally collected by the MCU will also be reduced to an acceptable level, so that normal data can be collected. After the design of the present invention is used in the circuit, the collected initial AD value can be reduced to an acceptable level, and the amplification curve can be seen when a positive sample is made.

[0146] In traditional technology, in the actual assembly of instruments, due to some factors such as assembly methods, individual differences in PDs, and individual differences in op amp devices, the fluorescence signal may be exposed in the primary amplification circuit, or the background signals of multiple channels may not be at the same level, or the detection range may be inconsistent, resulting in the inability to see the amplification curve when doing positive samples.

[0147] Therefore, the present invention adopts a peeling function similar to that of an electronic scale to remove the background current signal in the optical circuit, thereby increasing the signal dynamic range, normalizing multi-channel background signals, and improving detection sensitivity, thereby laying a foundation for miniaturization, low cost, and high sensitivity of the instrument.

[0148] In view of the shortcomings of the prior art, the present invention modifies the original circuit and introduces a current signal source (buffered voltage signal output by the DA buffer circuit: DA_PD_Zero) at the primary stage of the amplifier circuit to offset the photocurrent, bias current, etc. This offset will also act on the secondary stage after amplification, so that when no fluorescence signal is generated, the light signal reading can be maintained at a lower level, so that after the fluorescence signal is generated, a complete amplification curve can be seen.

[0149] In this embodiment, the type of MCU chip is not limited and can be a single chip microcomputer or the like.

[0150] The following is a description of the buffer and operational amplifier circuit of the present invention.

[0151] The fluorescence signal DA buffer circuit is composed of two voltage followers (also called buffers). Voltage follower 1 (buffer) outputs the 10mv voltage gain obtained by voltage division; voltage follower 2 (buffer) outputs the DAC signal output by the MCU after gain. The main purpose is to increase the input impedance and reduce the output impedance, and to isolate the front-stage signal source from the back-stage.

[0152] Voltage follower function:

[0153] 1.Signal Isolation:

[0154] In the signal chain, the voltage follower can isolate the previous and subsequent circuits to prevent the subsequent circuit from affecting the previous circuit.

[0155] 2. Enhanced driving capability:

[0156] The voltage follower can enhance the driving capability of the signal, enabling it to drive more loads or transmit over long distances.

[0157] 3. Buffering noise

[0158] Voltage followers can be used to buffer noise in input signals and improve signal stability.

[0159] The amplifying circuit mainly performs superposition processing on the PD signal and the DA buffer signal to obtain a peeled ADC signal and output it to the MCU control board (after ADC sampling).

[0160] like Figure 4 As shown, preferably, the DA buffer circuit includes an operational amplifier chip U6B, a resistor R40, a resistor R42, a resistor R44, a capacitor C27 and a resistor R45, wherein:

[0161] The positive input terminal MCU_DA_PD_Zero (DAC value output by MCU) of the operational amplifier chip U6B is connected to the MCU DA output pin of the MCU control board, the negative input terminal is connected to one end of the resistor R40, and the output terminal is connected to the other end of the resistor R40; the input terminal of the resistor R42 is connected to the output terminal of the operational amplifier chip U6B, and the output terminal is connected to the input terminal of the resistor R45; the output terminal of the resistor R45 is grounded; the input terminal of the resistor R44 is connected in parallel to the output terminal of the resistor R42, and the output terminal is connected to the reference power supply; the input terminal of the capacitor C27 is connected in parallel to the output terminal of the resistor R42, and the output terminal is grounded.

[0162] Preferably, the operational amplifier chip U6B is an operational amplifier chip with a single power supply of at least 3V and a gain bandwidth of at least 1Mhz, preferably LM358.

[0163] In the DA buffer circuit, "MCU_DA_PD_Zero" is connected to the MCU DA output pin, and "DA_PD_Zero" is a converted voltage signal between -8mV and +550mV. Therefore, the MCU output voltage can be buffered and stepped down to -8mV to +550mV (DA_PD_Zero), providing the operating voltage for the (optical signal) amplifier circuit.

[0164] like Figure 5 As shown, preferably, the amplifier circuit includes a resistor R7, a capacitor C3, a resistor R3, a resistor R4, a resistor R1, an operational amplifier chip U1A, a resistor R13, a resistor R17, an operational amplifier chip U2A, a resistor R8, a resistor R9, a capacitor C1, a resistor R14 and a capacitor C7, wherein;

[0165] The current signal source DA_PD_Zero (DAC buffer value) acts on the negative input terminal of the operational amplifier chip U1A through the resistor R7, the positive input terminal of the operational amplifier chip U1A is grounded, and the output terminal of the operational amplifier chip U1A is connected to the input terminal of the resistor R13; the resistor R3 and the resistor R4 are connected in series, the input terminal of the resistor R3 is connected to the negative input terminal of the operational amplifier chip U1A, and the output terminal of the resistor R4 is connected to the output terminal of the resistor R13; the two ends of the capacitor C3 are respectively connected in parallel to the input terminal of the resistor R3 and the output terminal of the resistor R4; one end of the resistor R1 is connected to the output terminal of the resistor R3, and the other end is grounded;

[0166] The current after being operationally amplified by the operational amplifier chip U1A acts on the positive input terminal of the operational amplifier chip U2A; the input terminal of the resistor R17 is connected to the output terminal of the resistor R13, and the output terminal is connected to D-0.01V; the output terminal of the operational amplifier chip U2A is connected to the input terminal of the resistor R14, and the negative input terminal of the operational amplifier chip U2A is connected to the output terminal of the resistor R8; the input terminal of the resistor R8 is grounded, and the output terminal is connected to the input terminal of the resistor R9, and the output terminal of the resistor R9 is connected to the input terminal of the resistor R14; the two ends of the capacitor C1 are respectively connected in parallel to the output terminal and the input terminal of the resistor R9; the input terminal of the capacitor C7 is connected to the input terminal of the resistor R14, and the output terminal is grounded; the output terminal of the resistor R14 outputs the final acquisition signal MCU_AD_PD0.

[0167] Preferably, the operational amplifier chip U1A is an operational amplifier chip with a single power supply of at least 1.8V and a gain bandwidth of at least 10Mhz, preferably OPA2320A.

[0168] Preferably, the operational amplifier chip U2A is an operational amplifier chip with a single power supply of at least 1.8V and a gain bandwidth of at least 1.2Mhz, preferably GS85502.

[0169] Preferably, the positive input terminal of the power supply of the operational amplifier chip U1A is OPA2320A and connected to the positive pole of the power supply: D+3.3V, and the negative input terminal of the power supply is grounded.

[0170] In the amplifier circuit, "PD_Cathode_0" is connected to the cathode of the photodiode (PD), the anode of PD is connected to AGND (that is, the sampling port), U1A is the primary op amp, U2A is the secondary op amp, and MCU_AD_PD0 is the output end of the final collected signal. Figure 4 "DA_PD_Zero" in Figure 5 It is introduced in and acts on pin 2 of U1A through a 100MΩ resistor to offset all the photocurrent, dark current, bias current, etc.

[0171] In this way, the analog signal is converted into a voltage signal of -8mV to +550mV after being buffered and divided by the op amp. Then this voltage signal is connected to the signal input terminal of the primary op amp U1A through a 100MΩ resistor, which can generate a -8pA to +550pA offset current, which can release the primary skin value voltage of the op amp, thereby reducing the voltage of the secondary output, and making the AD finally collected by the MCU drop to an acceptable level, so that normal data can be collected. The collected initial AD value can be reduced to an acceptable level, and the amplification curve can be seen when doing positive samples.

[0172] In the application of the system of the present invention, the number of various elements / components etc. can be configured according to the requirements, and multiple channels can be formed for detection.

[0173] like Fig. 9 As shown, the present invention can collect multiple groups of fluorescence signals, so a sampling circuit consisting of multiple photosensitive (PD) devices and amplifier circuits can be set. Fig.11 The architecture shown is a main architecture of DA peeling of the present invention, where PD0 to PD3 are photosensitive (PD) devices where the channels of four different light colors are located in the AirPOC device. Theoretically, one group of PD corresponds to one group of amplification circuits. Since the four PDs belong to the same device, the four groups of operational amplifiers can use one group of DA buffer circuits for peeling in design. The specific number of groups required can be set according to the detection requirements.

[0174] like Fig.10As shown in the figure, in a PCR amplification test, the corresponding amplification curve is drawn according to the test results (MCU samples and feeds back the sampling results to the test terminal such as PC). The results show that there are four curves in the figure, representing the curves formed by the voltage values ​​collected by the four PDs after passing through the amplification circuit. Before peeling, the AD value is already very high; the AD value is peeled by adjusting the DA, which greatly allows the subsequent collected light intensity to pass through and improves the sampling clarity.

[0175] The above operational amplifier chip is only an example of this embodiment.

[0176] Obviously, those skilled in the art should understand that all or part of the processes in the above embodiments can be implemented by instructing the relevant hardware through a computer program, and the program can be stored in a computer-readable storage medium, and when the program is executed, it can include the processes of the above-mentioned control embodiments. Among them, the storage medium can be a disk, an optical disk, a read-only memory (ROM), a random access memory (RAM), a flash memory, a hard disk drive (HDD) or a solid-state drive (SSD), etc.; the storage medium can also include a combination of the above-mentioned types of memory. Example 2

[0177] Based on the implementation principle of Example 1, on one hand, this application proposes a picoampere-level multi-channel photoelectric signal detection method, which is implemented based on the above-mentioned picoampere-level multi-channel photoelectric signal detection micro-integrated system, including the following steps:

[0178] The MCU control board outputs DAC to the DA buffer circuit;

[0179] After buffering the initial DAC signal output by the MCU through the DA buffer circuit, a voltage signal source DA_PD_Zero of -8mV to +550mV is provided for collecting the PD signal;

[0180] The fluorescent signal generated by the nucleic acid amplification reaction is sensed and detected by a photosensitive (PD) device and the corresponding PD signal is output to the amplification circuit, wherein the fluorescent signal includes an initial light signal and a background fluorescent signal generated in the RT stage and the PCR stage in sequence;

[0181] The PD signal and the initial DAC signal are amplified by an amplifying circuit to obtain and output a final acquisition signal MCU_AD_PD0 to the MCU control board;

[0182] Through the MCU control board, based on the preset MCU peeling control logic, the peeling function is performed in the RT stage and the PCR stage respectively (please refer to the peeling logic and algorithm description of Example 1 for details).

[0183] Please understand the details in conjunction with the corresponding steps in Example 1, and understand and implement the peeling algorithm in conjunction with the description in Example 1, which will not be described in detail in this example.

[0184] Please understand and implement the above steps in conjunction with Example 1, and this example will not be repeated.

[0185] The modules or steps of the present invention described above can be implemented by a general-purpose computing system, they can be concentrated on a single computing system, or distributed on a network composed of multiple computing systems, and optionally, they can be implemented by a program code executable by a computing system. Thus, they can be stored in a storage system and executed by the computing system, or they can be made into individual integrated circuit modules, or multiple modules or steps therein can be made into a single integrated circuit module for implementation. Thus, the present invention is not limited to any specific combination of hardware and software. Example 3

[0186] As attached Fig.11 As shown, on the other hand, the present application also proposes a rapid nucleic acid detector, including a picoampere-level multi-channel fluorescence signal detection micro-integrated system as described in the above-mentioned Example 1.

[0187] Preferably, it also includes:

[0188] A charging and discharging module, used to provide charging and discharging services;

[0189] At least 2 heating and temperature control modules to provide heating services for nucleic acid testing;

[0190] Multi-channel LED control circuit, used to provide different types of LED response services;

[0191] The communication interface Type-C is used to communicate with the detection terminal, including: transmitting the sampling value of the fluorescent signal generated by the nucleic acid detection molecule detection to the detection terminal;

[0192] The charging and discharging module, the heating and temperature control module, the multi-channel LED control circuit and the communication interface Type-C are electrically connected to the MCU control board respectively.

[0193] In order to use low-cost optical detection solutions on AirPOC (a multi-channel fluorescence signal detector, also a type of rapid nucleic acid detector), achieve rapid mass production, reduce the difficulty of mass production, and obtain reliable multi-channel data, the present invention uses low-cost devices (cost <500 yuan) through optoelectronic integration and innovative control logic to achieve high-sensitivity (pA level, or single molecule), multiple (multiple light signal sources or multiple pathogens), and rapid (<30min) nucleic acid molecule detection on a handheld instrument, making molecular detection POCT a reality, thereby solving the problem of excessive background signals or even exceeding the limit, and collecting reliable multi-channel optical signal data.

[0194] The nucleic acid rapid detector, in addition to adopting the "picoampere-level multi-channel fluorescence signal detection micro-integrated system" of the present invention, also has the following functional modules.

[0195] During specific implementation, as an optional implementation scheme of the present application, optionally, it also includes:

[0196] A charging and discharging module, used to provide charging and discharging services;

[0197] 9-way heating and temperature control module, used to provide heating services for nucleic acid testing;

[0198] Multi-channel LED control circuit, used to provide different types of LED response services;

[0199] The communication interface Type-C is used to communicate with the detection terminal, including: transmitting the sampling value of the fluorescent signal generated by the nucleic acid detection molecule detection to the detection terminal;

[0200] The charging and discharging module, the 9-way heating and temperature control module (representing only a multi-way assembly solution of this embodiment), the multi-way LED control circuit (arranged accordingly) and the communication interface Type-C are electrically connected to the MCU control board respectively.

[0201] The charging and discharging module is a conventional component module of electronic devices, and therefore, the configuration functions of existing electronic devices may be referred to.

[0202] The heating and temperature control module can achieve corresponding temperature heating under the control of the MCU program, and can provide a corresponding high-temperature environment for molecular movement during nucleic acid testing.

[0203] The multi-channel LED control circuit can provide corresponding LED response services for the nucleic acid detector. For example, if the MCU determines that the AD value reaches the set value, an LED response indicator signal is issued; at this time, if the LED indicator connected to the multi-channel LED control circuit outputs the result through the MCU internal yin and yang judgment algorithm, a signal response can be made to display the corresponding judgment result; correspondingly, if the preset value is not reached, the MCU can control the corresponding LED indicator to display the corresponding judgment result; in the process of continuous adjustment of the AD value, the MCU can control the LED signal light to flash continuously until the AD value is locked, and the corresponding LED indicator can be controlled to display green continuously, etc. For the LED display function, an LED display screen can also be added, and the MCU can process the corresponding calculation results, the detection value of the judgment result (fluorescence signal), etc., and send and display them on the LED screen. Therefore, different LED response services can be configured accordingly, and connected to the control board where the MCU is located through the multi-channel LED control circuit, and controlled by the MCU.

[0204] The communication interface Type-C can communicate with the detection terminal, and the results of the sampling, analysis and calculation of the nucleic acid detector can be transmitted to the detection terminal through the communication interface. The detection terminal uses the software program to input the detection sampling value, draw the corresponding amplification curve, and display the corresponding detection curve results in time on the detection terminal. The detection terminal (PC or other host computer) can also configure the MCU through the communication interface, such as entering the corresponding set values, parameter adjustment and configuration, etc.

[0205] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A picoampere-level multi-channel photoelectric signal detection micro-integrated system for nucleic acid molecule amplification fluorescence signal POCT detection, characterized in that: include: A light-transmitting reaction container, used for nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is an enzymatic reaction; At least one light source device, used to excite the fluorescein in the nucleic acid amplification reaction to generate at least one fluorescent signal, the light source device is no larger than 10 mm 3 ; At least one photosensitive (PD) device, used to sense the fluorescent signal and output a corresponding PD signal, the volume of the photosensitive (PD) device is no larger than 100 mm 3 ; An MCU control board, used for outputting digital-to-analog (DAC) signals and collecting analog-to-digital (ADC) signals, the MCU control board includes an MCU microprocessing unit, used for implementing a peeling algorithm and a signal collection function, wherein the peeling algorithm is based on an MCU peeling control logic; the peeling control logic includes: a. Set a pico value in MCU; b. A DAC value is provided by the MCU; c. outputting a DA buffer value by a buffer circuit based on the DAC value; d. Output ADC value after passing through the amplification circuit according to the PD signal value and DA buffer value; e. Determine whether the ADC value reaches the set value: If yes, the DAC signal is locked and stably output, the peeling operation is completed, and the output ADC signal value is the detected fluorescence signal value; If not, adjust the MCU to output the corrected DAC value, perform the peeling operation from step d to step e, until the ADC value reaches the set peeling value, then lock and stably output the corrected DAC value, complete the peeling operation, and the output ADC signal value is the detected fluorescence signal value; and / or f. If necessary, repeat steps a to e of the second stage peeling operation; At least one set of DA buffer circuits, the buffer circuit comprising a first follower circuit and a second follower circuit, the first follower circuit outputs a reference voltage, the second follower circuit buffers the DAC signal and generates a digital-to-analog buffer signal after dividing the reference voltage: DA buffer signal; At least one stage of amplification circuit, used for superimposing the PD signal and the DA buffer signal to obtain a peeled ADC signal; The photosensitive (PD) device and the DA buffer circuit are respectively connected to the amplifier circuit on the PCB; The average PCB area occupied by a single channel of the buffer circuit and amplifier circuit is no more than 500mm 2 ; The DA buffer circuit and the amplifier circuit are electrically connected to the MCU control board respectively.

2. The picoampere-level multi-channel photoelectric signal detection micro-integrated system according to claim 1, characterized in that: The peeling algorithm comprises: ， in: u(t) is the control output at time t, which is the DAC value output by the MCU control board; K P is a proportional gain, used to adjust the response strength of the MCU control board to the current error; K i is the integral gain, used to eliminate steady-state errors; K d is the differential gain, which is used to predict the error trend and reduce the overshoot and oscillation of the system; is the error at time t, calculated as the difference between the set target pico value and the actual optical signal currently collected by the MCU control board: , where SP is the set value of the skin value, PV(t) is the process variable, that is, the current integral term, which is the accumulation of error over time and is used to eliminate steady-state errors: The integral term is the accumulation of error over time and is used to eliminate steady-state error: ； The derivative term is a prediction of the error rate of change and is used to predict the error trend: ； PV(T) is a process variable calculated by the MCU control board using the trimmed mean algorithm through multiple data collected within a certain period of time. The calculation is as follows: Let X = {x1, x2, x3, ..., xn} be a data set of a certain original optical signal collected, which contains n values; Find the m maximum values ​​max(X) and minimum values ​​min(X) in the original data set and remove them to get the data set Y; Calculate the sum S of the remaining values: ,in: x Represents each element in the set Y; Calculate the number of remaining values, k is the number of elements in the data set Y: K = nm; Calculate the Y mean of the data set: PV(t)=S / k.

3. The picoampere-level multi-channel photoelectric signal detection micro-integrated system according to claim 1, characterized in that: The DA buffer circuit includes an operational amplifier chip U6B, a resistor R40, a resistor R42, a resistor R44, a capacitor C27 and a resistor R45, wherein: The positive input terminal MCU_DA_PD_Zero (DAC value output by MCU) of the operational amplifier chip U6B is connected to the MCU DA output pin of the MCU control board, the negative input terminal is connected to one end of the resistor R40, and the output terminal is connected to the other end of the resistor R40; the input terminal of the resistor R42 is connected to the output terminal of the operational amplifier chip U6B, and the output terminal is connected to the input terminal of the resistor R45; the output terminal of the resistor R45 is grounded; the input terminal of the resistor R44 is connected in parallel to the output terminal of the resistor R42, and the output terminal is connected to the reference power supply; the input terminal of the capacitor C27 is connected in parallel to the output terminal of the resistor R42, and the output terminal is grounded.

4. The picoampere-level multi-channel fluorescence signal detection micro-integrated system according to claim 3, characterized in that: The operational amplifier chip U6B is an operational amplifier chip powered by a single power supply of at least 3V and with a gain bandwidth of at least 1Mhz.

5. The picoampere-level multi-channel photoelectric signal detection micro-integrated system according to claim 1, characterized in that: The amplifier circuit includes a resistor R7, a capacitor C3, a resistor R3, a resistor R4, a resistor R1, an operational amplifier chip U1A, a resistor R13, a resistor R17, an operational amplifier chip U2A, a resistor R8, a resistor R9, a capacitor C1, a resistor R14 and a capacitor C7, wherein: The current signal source DA_PD_Zero (DAC buffer value) acts on the negative input terminal of the operational amplifier chip U1A through the resistor R7, the positive input terminal of the operational amplifier chip U1A is grounded, and the output terminal of the operational amplifier chip U1A is connected to the input terminal of the resistor R13; the resistor R3 and the resistor R4 are connected in series, the input terminal of the resistor R3 is connected to the negative input terminal of the operational amplifier chip U1A, and the output terminal of the resistor R4 is connected to the output terminal of the resistor R13; the two ends of the capacitor C3 are respectively connected in parallel to the input terminal of the resistor R3 and the output terminal of the resistor R4; one end of the resistor R1 is connected to the output terminal of the resistor R3, and the other end is grounded; The current after being operationally amplified by the operational amplifier chip U1A acts on the positive input terminal of the operational amplifier chip U2A; the input terminal of the resistor R17 is connected to the output terminal of the resistor R13, and the output terminal is connected to D-0.01V; the output terminal of the operational amplifier chip U2A is connected to the input terminal of the resistor R14, and the negative input terminal of the operational amplifier chip U2A is connected to the output terminal of the resistor R8; the input terminal of the resistor R8 is grounded, and the output terminal is connected to the input terminal of the resistor R9, and the output terminal of the resistor R9 is connected to the input terminal of the resistor R14; the two ends of the capacitor C1 are respectively connected in parallel to the output terminal and the input terminal of the resistor R9; the input terminal of the capacitor C7 is connected to the input terminal of the resistor R14, and the output terminal is grounded; the output terminal of the resistor R14 outputs the final acquisition signal MCU_AD_PD0.

6. The picoampere-level multi-channel photoelectric signal detection micro-integrated system according to claim 5, characterized in that: The operational amplifier chip U1A is an operational amplifier chip with a single power supply of at least 1.8V and a gain bandwidth of at least 10Mhz.

7. The picoampere-level multi-channel photoelectric signal detection micro-integrated system according to claim 5, characterized in that: The operational amplifier chip U2A is an operational amplifier chip with a single power supply of at least 1.8V and a gain bandwidth of at least 1.2Mhz.

8. The picoampere-level multi-channel photoelectric signal detection micro-integrated system according to claim 5, characterized in that: The positive input terminal of the power supply of the operational amplifier chip U1A is connected to the positive pole of the power supply: D+3.3V, and the negative input terminal of the power supply is grounded.

9. A picoampere-level multi-channel photoelectric signal detection method, implemented based on a picoampere-level multi-channel photoelectric signal detection micro-integrated system according to any one of claims 1 to 8, characterized in that: The steps include: The MCU control board outputs DAC to the DA buffer circuit; After buffering the initial DAC signal output by the MCU through the DA buffer circuit, a voltage signal source DA_PD_Zero (DAC buffer value) ranging from -8mV to +550mV is provided for collecting the PD signal; The fluorescent signal generated by the nucleic acid amplification reaction is sensed and detected by a photosensitive (PD) device and the corresponding PD signal is output to the amplification circuit, wherein the fluorescent signal includes an initial light signal and a background fluorescent signal generated in the RT stage and the PCR stage in sequence; The PD signal and the initial DAC signal are amplified by an amplifying circuit to obtain and output a final acquisition signal MCU_AD_PD0 to the MCU control board; Through the MCU control board, the peeling function is performed in the RT stage and the PCR stage based on the preset MCU peeling control logic.

10. A rapid nucleic acid detector, characterized in that: include: A picoampere-level multi-channel fluorescence signal detection micro-integrated system according to any one of claims 1 to 8.

11. A rapid nucleic acid detector according to claim 10, characterized in that: Also includes: A charging and discharging module, used to provide charging and discharging services; At least 2 heating and temperature control modules to provide heating services for nucleic acid testing; Multi-channel LED control circuit, used to provide different types of LED response services; The communication interface Type-C is used to communicate with the detection terminal, including: transmitting the sampling value of the fluorescent signal generated by the nucleic acid detection molecule detection to the detection terminal; The charging and discharging module, the heating and temperature control module, the multi-channel LED control circuit and the communication interface Type-C are electrically connected to the MCU control board respectively.

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