A method and system for detecting blood drug concentration based on a multi-channel nanopore system

Abstract

The application relates to the technical field of measurement, and particularly discloses a blood drug concentration detection method and system based on a multi-channel nanopore system, which specifically comprises the following steps: S101, a weak current measurement circuit is constructed to generate an electric field force to drive a to-be-detected drug molecule in an electrolyte chamber to pass through a corresponding nanopore channel, and an electric current signal generated when the to-be-detected drug molecule passes through each nanopore channel is acquired, then the electric current signal is converted into a voltage signal and amplified and sampled to obtain corresponding to-be-analyzed data; S102, the to-be-analyzed data is acquired, and the to-be-analyzed data is preprocessed to obtain effective data and invalid data; S103, the number of times of passing through a pore of the to-be-detected drug molecule is judged based on the effective data; and S104, the concentration of the to-be-detected drug molecule is calculated according to the number of times of passing through a pore and a prestored frequency-concentration standard curve.

Description

Technical Field

[0001] This invention belongs to the field of measurement technology, and in particular relates to a method and system for detecting blood drug concentration based on a multi-channel nanopore system. Background Technology

[0002] In the fields of pharmaceuticals and medical diagnostics, rapid detection of drug concentrations and contents is frequently required, such as detecting the content of active ingredients in drug formulations or the drug concentration in patient serum. Nucleic acids, as important structural and functional substances in organisms, are currently target molecules for many anticancer and antiviral drugs.

[0003] The main drug detection methods currently include: chromatographic analysis techniques, such as gas chromatography and high performance liquid chromatography; spectroscopic analysis techniques, such as ultraviolet-visible spectrophotometry, infrared spectrophotometry, and fluorescence spectroscopy; electrochemical methods; surface plasmon resonance (SPR) methods; and immunoassay methods. Immunoassays, based on the specific binding of antigens and antibodies, enable rapid quantitative detection using enzymes, fluorescent substances, and radioactive isotope labels. However, they require antibody development for specific drugs, resulting in poor versatility, and are expensive to prepare, leading to high reagent costs. Chromatography separates drugs from other components using chromatographic columns and combines UV and mass spectrometry detectors for simultaneous detection of multiple drug components in complex samples. While highly versatile, it requires complex pretreatment, is time-consuming per test, has high equipment costs, and relies on professional operators. Spectroscopy utilizes the absorption characteristics of drug molecules at specific wavelengths of light, rapidly calculating concentrations by measuring absorbance changes. However, it is susceptible to interference, has slow analysis speed, and is limited by the spectral absorption properties of drug molecules, may require fluorescent labeling, has low sensitivity, and is only suitable for high-concentration drug detection, resulting in poor versatility. Electrochemical and SPR methods often require chemical modification of nucleic acid probes at electrode or chip interfaces, making the processing steps cumbersome. Mass spectrometry ionizes drug molecules and measures their mass-to-charge ratio for qualitative and quantitative analysis, offering ultra-high sensitivity. However, it requires expensive equipment, complex operation, highly purified samples to avoid matrix effects, and demanding pretreatment requirements. The aforementioned traditional methods are no longer sufficient to meet the needs of drug screening and disease diagnosis, and there is an urgent need to develop rapid, convenient, and highly sensitive drug detection methods.

[0004] Nanopores, as a single-molecule analytical technique, offer advantages such as high sensitivity, label-free operation, simplicity, low cost, and speed. Signals can be continuously and directly read during detection without the need for amplification, cloning, or complex sample processing. Based on this, existing technologies have proposed applying nanopores to drug detection.

[0005] For example, patent application CN105259229B discloses a single-molecule analysis method for drug detection. This method constructs a drug nanopore sensor using natural α-hemolysin. Target nucleic acid samples and drug molecules are sequentially added to an electrolytic cell. Changes in the nanopore current blocking signal before and after drug addition are recorded and analyzed. Before drug addition, the target nucleic acid sample passing through the nanopore causes a current blocking signal. When the nucleic acid binds to the drug and passes through the nanopore, the binding product triggers a characteristic current blocking signal. The blocking time, blocking current, and other parameters are significantly different from the signal of the original nucleic acid substrate. By monitoring the perforation frequency of this characteristic blocking signal, a relationship between the perforation frequency and the drug concentration can be established, allowing for quantitative analysis of the target drug. However, this method is an indirect method for drug concentration detection; it detects the current signal of the drug-nucleic acid complex passing through the nanopore. This not only increases the complexity of the method but also directly affects the final detection result due to the effectiveness of drug-nucleic acid binding, thus introducing interference factors.

[0006] Based on this existing technology, a method for direct detection of drug molecules has been proposed. For example, patent application CN115725685A discloses a method for detecting drug molecules based on biological micropores, comprising the following steps: S1, adding the sample to a micropore system, the micropore system comprising: micropores, an insulating membrane, a first medium, and a second medium, the micropores being MScS micropores, the micropores having a radially symmetrical, cylindrical heptamer structure, the heptamer structure containing 7 side openings and 1 bottom opening; the sample being added to the first medium; S2, applying a driving force to the first medium and the second medium, the drug molecules in the sample interacting with the micropores and generating an electrical signal; S3, analyzing the electrical signal to identify the drug molecules in the sample. This method provides a direct detection method for drug concentration.

[0007] However, regardless of whether the detection method is indirect or direct, the signal generated during drug-nucleic acid permeation or the current signal generated during drug-nucleic acid permeation is extremely weak. Although various weak current detection circuits have been proposed in the technology, such as CN112924745A, CN118033228A, and CN115725685A, none of them disclose how to further analyze and obtain drug concentration based on the microcurrent. Therefore, how to obtain drug concentration based on the weak current signal generated during drug permeation has become an urgent technical problem to be solved. Summary of the Invention

[0008] The purpose of this invention is to provide a method and system for detecting blood drug concentration based on a multi-channel nanopore system, which partially solves or alleviates the above-mentioned deficiencies in the prior art and enables rapid identification and quantitative analysis of drug molecule concentration.

[0009] To solve the aforementioned technical problems, the present invention specifically adopts the following technical solution:

[0010] A first aspect of the present invention is to provide a method for detecting blood drug concentration based on a multi-channel nanopore system, wherein the multi-channel nanopore system includes multiple nanopore channels, each nanopore channel having an electrolyte chamber disposed on both sides, and the nanopores being embedded in an insulating membrane between two electrolyte chambers; correspondingly, the method for detecting blood drug concentration specifically includes the following steps:

[0011] S101, a weak current measurement circuit is constructed to generate an electric field force to drive the drug molecule to be tested in the electrolyte chamber through the corresponding nanopore channel, and to acquire the current signal generated when the drug molecule to be tested passes through each nanopore channel. Then, the current signal is converted into a voltage signal and amplified and sampled to obtain the corresponding data to be analyzed.

[0012] S102, acquire the data to be analyzed, and preprocess the data to be analyzed to obtain valid data and invalid data;

[0013] S103, determine the number of times the drug molecule to be tested passes through the pores based on the effective data;

[0014] S104, the concentration of the drug molecule to be tested is calculated based on the number of pores and the pre-stored frequency-concentration standard curve;

[0015] Specifically, step S103 includes:

[0016] S1031, Calculate the mean and standard deviation based on all valid data within the first specified time period;

[0017] S1032, determine whether there are at least two consecutive valid data points within the first specified time period whose differences from the mean are greater than twice the standard deviation; if so, determine that the drug molecule to be tested has been poreped once.

[0018] S1033, Count the number of times the drug molecule to be tested passes through the pore within the second specified time period; the second specified time period is longer than the first specified time period;

[0019] Before step S102, the following steps are also included:

[0020] Determine whether the current value corresponding to each nanopore channel is a fixed value within a preset first time period, and the fixed value is greater than 0 but less than the opening current I1;

[0021] If so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as blocked;

[0022] Alternatively, determine whether the current value corresponding to each nanopore suddenly increases to the first preset threshold and falls back to the second preset threshold within a preset second time period. If the current state of the corresponding nanopore channel is determined to be abnormal, mark it as a membrane rupture state.

[0023] Alternatively, determine whether the current value corresponding to each nanopore suddenly increases to the first preset threshold and remains at the current value. If so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as a membrane rupture state.

[0024] In some embodiments, step S102 specifically includes the following steps:

[0025] S1021, Obtain the status information of each data to be analyzed; the status information includes: the source of the nanopore channel, and the corresponding nanopore channel status; the nanopore channel status includes normal or abnormal.

[0026] S1022, Determine whether the current state of the nanopore channel corresponding to each piece of data to be analyzed is normal.

[0027] If normal, proceed to step S1023; if abnormal and in a ruptured membrane state, proceed to step S1025; if abnormal and in a blocked state, proceed to step S1024.

[0028] S1023, mark the corresponding data to be analyzed as valid data;

[0029] S1024, apply a reverse driving voltage of the first threshold to the corresponding nanopore channel to perform at least one hole kick to restore it to normal, and when it is restored to normal, update the current state of the nanopore channel to normal, execute step S101, and mark the data to be analyzed when the nanopore channel is in the normal state as valid data; while marking the data when it is in the blocked state as invalid data.

[0030] S1025 marks the corresponding data to be analyzed as invalid data and controls the closure of the corresponding nanopore channels.

[0031] In some embodiments, if within a third specified time period, it is determined that the number of times the corresponding nanopore channel is continuously marked as blocked is greater than or equal to a preset number of blockages, the corresponding nanopore channel is closed, and all data of the nanopore channel is marked as invalid data; the third specified time period is shorter than the second specified time period.

[0032] In some embodiments, in step S1024, a reverse driving voltage of the first threshold is applied to the corresponding nanopore channel multiple times (e.g., multiple times at preset time intervals) to perform continuous hole kicking n times. Then, based on the current data detected after hole kicking, it is determined whether the nanopore channel has returned to normal. If it has not returned to normal, it is kicked n times again and determined whether it has returned to normal. This process is repeated until it returns to normal. If it has not returned to normal after M times, the channel is closed. Wherein, n=N / M, and N is the preset hole kicking number threshold N.

[0033] In some embodiments, the weak current measurement circuit specifically includes: an overcurrent protection module, a switch matrix module, an R-TIA module, an overvoltage protection module, a signal processing module, a signal acquisition module, and a DAC module; wherein...

[0034] The input port of the overcurrent protection module is connected to the nanopore, the output port of the overcurrent protection module is connected in series with the input port of the switch matrix module, the output port of the switch matrix module is connected in series with the negative input port of the R-TIA module, the output terminal of the R-TIA module is connected to the input port of the overvoltage protection module, the output port of the overvoltage protection module is connected to the signal processing module, and the signal processing module is connected to the signal acquisition module.

[0035] The output of the DAC module is connected to the positive input of the operational amplifiers in the R-TIA module and the signal processing module, respectively.

[0036] In some embodiments, the weak current measurement circuit further includes an LPF module disposed between the overvoltage protection module and the signal processing module.

[0037] In some embodiments, the R-TIA module specifically comprises: an operational amplifier OPA1, a feedback resistor RF, and a compensation capacitor CF, wherein...

[0038] The negative input terminal of the operational amplifier OPA1 is connected to the output port of the switch matrix module, and also to terminal A of the feedback resistor RF and the compensation capacitor CF.

[0039] The positive input port of the operational amplifier OPA1 is connected to the output port of the DAC module;

[0040] The output port of the operational amplifier OPA1 is connected to the B terminal of the feedback resistor RF and the compensation capacitor CF, and is also connected to the input terminal of the overvoltage protection module.

[0041] A second aspect of the present invention is to provide a blood drug concentration detection system, comprising:

[0042] A multi-channel nanopore system includes multiple nanopore channels, each nanopore channel having an electrolyte chamber on each side, and the nanopores being embedded in an insulating membrane between the two electrolyte chambers;

[0043] A weak current measurement circuit, connected to the nanopores in the multi-channel nanopore system, is configured to generate an electric field to drive the drug molecule to be tested in the electrolyte chamber through the corresponding nanopore channel, and to acquire the current signal generated when the drug molecule to be tested passes through each nanopore channel. Then, the current signal is converted into a voltage signal and amplified and sampled to obtain the corresponding data to be analyzed.

[0044] The host computer, connected to the weak current measurement circuit, is configured to acquire the data to be analyzed from the weak current measurement circuit, preprocess the data to be analyzed to obtain valid data and invalid data; and determine the number of times the drug molecule to be tested passes through the pores based on the valid data, and then calculate the concentration of the drug molecule to be tested based on the number of times it passes through the pores and the pre-stored frequency-concentration standard curve.

[0045] The host computer specifically includes:

[0046] The preprocessing unit is used to acquire the data to be analyzed from the weak current measurement circuit and perform preprocessing to obtain valid data and invalid data;

[0047] The data analysis unit is used to determine the number of times the drug molecule to be tested is sieved based on the valid data obtained by the preprocessing unit. Specifically, the data analysis unit is used to calculate the mean and standard deviation based on all valid data within a first specified time period; and to determine whether there are at least two consecutive valid data within the first specified time period whose differences from the mean are greater than twice the standard deviation. If so, it is determined that the drug molecule to be tested has been sieved once; then, the number of times the drug molecule to be tested is sieved within a second specified time period is counted; the second specified time period is longer than the first specified time period.

[0048] The concentration calculation unit is used to calculate the concentration of the drug molecule to be tested based on the number of pores obtained by the data analysis unit and the pre-stored frequency-concentration standard curve.

[0049] In some embodiments, the data analysis unit is specifically configured to acquire the status information of each piece of data to be analyzed; the status information includes: the source of the nanopore channel and the corresponding nanopore channel status; the nanopore channel status includes normal or abnormal; and determine whether the current status of the nanopore channel corresponding to each piece of data to be analyzed is normal. If it is normal, the corresponding piece of data to be analyzed is marked as valid data; if it is abnormal and in a ruptured state, the corresponding piece of data to be analyzed is marked as invalid data, and the corresponding nanopore channel is controlled to be closed; if it is abnormal and in a blocked state, a reverse driving voltage of a first threshold is applied to the corresponding nanopore channel to perform at least one hole kick to restore it to normal, and when it is restored to normal, the current status of the nanopore channel is updated to normal, and the piece of data to be analyzed when the nanopore channel is in a normal state is marked as valid data; while the data in a blocked state is marked as invalid data.

[0050] In some embodiments, the weak current measurement circuit specifically includes: an overcurrent protection module, a switch matrix module, an R-TIA module, an overvoltage protection module, a signal processing module, a signal acquisition module, and a DAC module; wherein...

[0051] The input port of the overcurrent protection module is connected to the nanopore, the output port of the overcurrent protection module is connected in series with the input port of the switch matrix module, the output port of the switch matrix module is connected in series with the negative input port of the R-TIA module, the output terminal of the R-TIA module is connected to the input port of the overvoltage protection module, the output port of the overvoltage protection module is connected to the signal processing module, and the signal processing module is connected to the signal acquisition module.

[0052] The output of the DAC module is connected to the positive input of the operational amplifier in the R-TIA module and the signal processing module 106, respectively.

[0053] In some embodiments, the weak current measurement circuit further includes an LPF module disposed between the overvoltage protection module and the signal processing module.

[0054] In some embodiments, the R-TIA module specifically comprises: an operational amplifier OPA1, a feedback resistor RF, and a compensation capacitor CF, wherein...

[0055] The negative input terminal of the operational amplifier OPA1 is connected to the output port of the switching matrix, and also to the feedback resistor RF and the A terminal of the compensation capacitor CF.

[0056] The positive input port of the operational amplifier OPA1 is connected to the output port of the DAC module;

[0057] The output port of the operational amplifier OPA1 is connected to the B terminal of the feedback resistor RF and the compensation capacitor CF, and is also connected to the input terminal of the overvoltage protection module.

[0058] Beneficial effects: Since the current generated by the drug molecules passing through the pores is too weak, this invention constructs a detection circuit with a high signal-to-noise ratio to detect the current signal and analyzes it to obtain the drug concentration.

[0059] The detection circuit of this invention employs a switch matrix module: supporting rapid switching between multiple probes or electrodes, suitable for high-throughput detection scenarios (such as 96-well plate parallel sequencing). DAC dynamic voltage regulation: real-time optimization of bias voltage to adapt to different molecules (such as DNA / protein), improving the signal-to-noise ratio.

[0060] The detection circuit of this invention uses R-TIA+LPF synergy, which has a high noise suppression capability: R-TIA directly converts pA level current and the gain is adjustable (e.g., 1GΩ feedback resistor); LPF (cutoff frequency 1kHz) filters out high-frequency interference, which is better than CN112924745A which only relies on feedback capacitor for band limiting.

[0061] The detection circuit of this invention employs a dual protection mechanism: the overcurrent protection module directly intercepts abnormal current (such as electrode short circuit) to prevent breakdown of the nanopore or damage to the amplifier; the overvoltage protection module prevents voltage over-limit caused by power fluctuations / static electricity (such as clamping to 3.3V). Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0063] Figure 1 This is a functional block diagram of a weak current detection circuit according to the present invention;

[0064] Figure 2 This is a functional block diagram of another embodiment of a weak current detection circuit according to the present invention;

[0065] Figure 3 This is a schematic diagram of an embodiment of a weak current measuring circuit according to the present invention;

[0066] Figure 4 This is a flowchart of an embodiment of a blood drug concentration detection method based on multi-channel nanopores according to the present invention;

[0067] Figure 5 This is a functional block diagram of an embodiment of a blood drug concentration detection system based on multi-channel nanopores according to the present invention.

[0068] Figure reference numerals: 101-Overcurrent protection module; 102-Switch matrix module; 103-R-TIA module; 104-Overvoltage protection module; 105-LPF module; 106-Signal processing module; 107-Signal acquisition module; 108-DAC module. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0070] In this document, suffixes such as "module," "part," or "unit" used to denote elements are used only for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "part," or "unit" may be used interchangeably.

[0071] In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0072] In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0073] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0074] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0075] As mentioned above, since the current signal generated when drug molecules pass through pores is very weak, this invention proposes a detection circuit for detecting the weak current generated when drug molecules pass through pores, and performs data processing based on the weak current data detected by the detection circuit to quickly analyze and obtain the drug concentration.

[0076] Example 1: As Figure 1 As shown, the weak current detection circuit of the present invention specifically includes:

[0077] The system includes a multi-channel nanopore switching matrix module 102, and connected to the switching matrix module 102 are an R-TIA module 103, an overvoltage protection module 104, a signal processing module 106, a signal acquisition module 107, and a DAC module 108; wherein,

[0078] The input terminal of the switch matrix module 102 is connected to the front-end sampling channel (or multi-channel nanopore channel). The output port of the switch matrix module 102 is connected in series with the negative input port of the R-TIA module 103. The output terminal of the R-TIA module 103 is connected to the input port of the overvoltage protection module 104. The output port of the overvoltage protection module 104 is connected to the signal processing module 106. The signal processing module 106 is connected to the signal acquisition module 107. The output terminal of the DAC module 108 is connected to the positive input terminal of the operational amplifier in the R-TIA module 103 and the signal processing module 106, respectively.

[0079] Among them, the switch matrix module 102 is used to select the front-end sampling channel; the R-TIA module 103 is used to amplify the sampled current signal and convert it into a voltage signal; the sampling module (i.e., ADC) is used to perform analog signal to digital signal conversion; and the DAC module 108 is used for the bias signal of the dynamically modulated signal.

[0080] In some embodiments, the switch matrix module 102 is a single-pole multiple-throw electronic switch. The front end of the switch matrix module 102 allows multiple nanopore input signals to be connected. The controller time-division multiplexes the R-TIA module 103 and subsequent circuits. When an abnormality occurs in a nanopore, it can quickly switch to other nanopores, avoiding redundancy in subsequent circuits, enhancing equipment reliability and reducing equipment costs.

[0081] In some embodiments, the R-TIA module 103 specifically comprises: an operational amplifier OPA1, a feedback resistor RF, and a compensation capacitor CF, wherein,

[0082] The negative input terminal of operational amplifier OPA1 is connected to the output port of switch matrix module 102, and also to the feedback resistor RF and the A terminal of compensation capacitor CF.

[0083] The positive input port of operational amplifier OPA1 is connected to the output port of DAC module 108;

[0084] The output port of operational amplifier OPA1 is connected to the B terminal of feedback resistor RF and compensation capacitor CF, and is also connected to the input terminal of overvoltage protection module 104.

[0085] Since drug molecules of different concentrations generate current signals with different degrees and frequencies of blockage when passing through nanopores, the detection circuit in this embodiment uses a DAC to generate an electric field (excitation voltage signal Vbias) to drive drug molecules through nanoscale protein pores. The current signal is converted into a voltage signal and amplified by an R-TIA. The amplified voltage signal is then sampled by an ADC to obtain the corresponding measurement results. The host computer or the signal processing module 106 integrated in the circuit identifies the drug concentration information in the sample based on the amplitude and frequency characteristics of the voltage signal in the measurement results, thereby realizing rapid identification and quantitative analysis of drug molecule concentration (the specific analysis method can be referred to in Embodiment 3, which will not be repeated here). This solution has the advantages of simple and reliable measurement circuit, low cost, easy operation, high-precision picoampere-level current resolution, and suitability for micro-samples.

[0086] See Figure 2 In some embodiments, an overcurrent protection module 101 is connected in series between the switch matrix module 102 and the multi-channel nanopore. The input port of the overcurrent protection module 101 is connected to the nanopore, and the output port of the overcurrent protection module 101 is connected to the input of the switch matrix module 102. When an abnormally large current signal appears in the front-end nanopore or a large current interference signal is coupled into the line, the overcurrent protection module 101 will automatically disconnect to protect the downstream circuit; if the output line of the downstream circuit is short-circuited, the overcurrent protection module 101 can quickly melt and protect the nanopore from the interference of the short-circuit signal.

[0087] Preferably, the overcurrent protection module 101 can be implemented using a fuse or a controllable current limiting circuit. The protection current threshold of the overcurrent protection module 101 can be adjusted as needed, but this embodiment does not impose any specific limitations.

[0088] In other embodiments, the weak current measurement circuit further includes an LPF module 105 disposed between the overvoltage protection module 104 and the signal processing module 106.

[0089] See Figure 3In specific implementation, ① is a signal detection branch, where OpAmp1, Rf, and Cf constitute a transimpedance amplifier circuit as a first-stage signal amplification circuit to convert the current signal into a voltage signal; OpAmp2 is a subtractor circuit as a second-stage signal amplification circuit to amplify the signal a second time and eliminate the noise effect generated by Vbias; LPF is an RC low-pass filter circuit as an anti-aliasing filter at the front end of the ADC to filter out high-frequency noise and improve the signal-to-noise ratio; ADC is an analog-to-digital converter to convert the analog voltage signal into a digital signal; DAC is a digital-to-analog converter that, in conjunction with the OpAmp buffer circuit, generates the excitation voltage signal Vbias; ② fluid chip (flow The nanopore structure, supported by a nanopore cell, provides the electrochemical environment required for electrophoresis. The diagram illustrates three nanopore channels, each containing a membrane capacitor, nanopore proteins, rate-controlling proteins, and electrodes. These electrodes are connected to CH1, CH2, ..., CHn (n being the channel number) of the measurement circuit. ④ The digital output measures the measurement results, identifying drug concentration information in the sample based on the amplitude and frequency characteristics of the voltage signal in the measurement results. ⑤ The FPGA digital signal processing unit performs digital filtering on the acquired data uploaded by the ADC, then uploads the processed data to the PC host computer, while simultaneously sending the excitation voltage signal command required by the DAC. ⑥ The PC host computer receives and processes the data uploaded by the FPGA, realizing the functions of detecting valid data, storing data, analyzing data, calculating concentration, and determining results.

[0090] In some embodiments, after the front-end nanopore signal is converted from a microcurrent signal to a microvoltage signal by the R-TIA module 103, it may generate spike interference. All voltage interference signals, including but not limited to spikes, can be absorbed and limited by the overvoltage protection module 104. This avoids abnormal signal interference with the sampling data and, furthermore, avoids misjudgment during signal analysis.

[0091] In some embodiments, the voltage output by the DAC module 108 is applied to the positive input terminals of the operational amplifiers in the R-TIA module 103 and the signal processing module 106, respectively. The voltage output by the DAC module 108 can dynamically adjust the positive bias voltage of the operational amplifiers in the R-TIA module 103 and the signal processing module 106. Even when the nanopores are blocked, they can be quickly removed (for example, applying positive pressure causes the measured substance to move in the positive direction, and applying negative pressure causes the measured substance to move in the negative direction, thereby achieving rapid removal of the nanopores), thus enhancing the reliability of the device.

[0092] In some embodiments, the signal processing module 106 (e.g., FPGA) analyzes the detected current data to obtain the drug concentration. Specifically, the signal processing module 106 includes:

[0093] A preprocessing unit is used to acquire the data to be analyzed from the weak current measurement circuit and perform preprocessing to obtain valid data and invalid data. Specifically, the preprocessing unit is used to acquire the status information of each data to be analyzed. The status information includes: the source of the nanopore channel and the corresponding nanopore channel status. The nanopore channel status includes normal or abnormal. The unit determines whether the current status of the nanopore channel corresponding to each data to be analyzed is normal. If it is normal, the corresponding data to be analyzed is marked as valid data. If it is abnormal and in a ruptured state, the corresponding data to be analyzed is marked as invalid data, and the corresponding nanopore channel is controlled to be closed. If it is abnormal and in a blocked state, the DAC module is controlled to apply a reverse driving voltage of a first threshold to the corresponding nanopore channel to perform at least one hole kick to restore it to normal. When it is restored to normal, the current status of the nanopore channel is updated to normal, and the data to be analyzed when the nanopore channel is in a normal state is marked as valid data. The data in a blocked state is marked as invalid data.

[0094] The data analysis unit is used to determine the number of times the drug molecule to be tested is sieved based on the valid data obtained by the preprocessing unit. Specifically, the data analysis unit is used to calculate the mean and standard deviation based on all valid data within a first specified time period; and to determine whether there are at least two consecutive valid data within the first specified time period whose differences from the mean are greater than twice the standard deviation. If so, it is determined that the drug molecule to be tested has been sieved once; then, the number of times the drug molecule to be tested is sieved within a second specified time period is counted; the second specified time period is longer than the first specified time period.

[0095] The concentration calculation unit is used to calculate the concentration of the drug molecule to be tested based on the number of pores obtained by the data analysis unit and the pre-stored frequency-concentration standard curve.

[0096] Furthermore, the data analysis unit is also used to determine whether the number of times the corresponding nanopore channel is continuously marked as blocked within the third specified time period is greater than or equal to the preset number of blockages. If so, the control switch matrix module will close the corresponding nanopore channel and mark all data of the nanopore channel as invalid data. The third specified time period is shorter than the second specified time period.

[0097] Furthermore, the data analysis unit is also used to control the DAC module to apply corresponding bias voltages to the R-TIA module and operational amplifier OPA2 multiple times when it is determined that the current state of the corresponding nanopore channel is abnormal and blocked, so as to perform continuous hole kicking n times. Then, based on the current data detected after hole kicking, it is determined whether the nanopore channel has returned to normal. If it has not returned to normal, it is kicked n times again and determined whether it has returned to normal. This process is repeated until it returns to normal. If it has not returned to normal after M times, the channel is closed. Wherein, n=N / M, and N is the preset threshold number of hole kicking times N.

[0098] In other embodiments, the signal processing module further includes:

[0099] The state analysis unit is used to determine whether the current value corresponding to each nanopore channel acquired by the signal acquisition module is a fixed value within a preset first time period, and the fixed value is greater than 0 but less than the opening current I1; if so, the current state of the corresponding nanopore channel is determined to be abnormal and marked as blocked, and the DAC module is controlled to apply a reverse driving voltage of the first threshold to the nanopore channel to perform a hole kick; or, it determines whether the current value corresponding to each nanopore suddenly increases to the first preset threshold and falls back to the second preset threshold within a preset second time period, and if so, the current state of the corresponding nanopore channel is determined to be abnormal and marked as ruptured; or, it determines whether the current value corresponding to each nanopore suddenly increases to the first preset threshold and remains at the current value, and if so, the current state of the corresponding nanopore channel is determined to be abnormal and marked as ruptured.

[0100] In other embodiments, the data analysis unit is also used to obtain the number of blocked nanopores in each nanopore group. When the number of blocked nanopores in any nanopore group reaches a preset threshold, the current state of all nanopores in that group is marked as blocked, and the DAC module is controlled to apply a reverse bias voltage to all nanopores in that group to kick the nanopores (as mentioned above, the reverse bias voltage is applied n times consecutively at preset time intervals of 0.5s or 1s). The kicking success rate of the group is calculated (i.e., the percentage of successfully kicked nanopores to the total number of nanopores in the group). If the kicking success rate is greater than the preset success rate threshold, kicking is stopped, and all blocked or ruptured nanopore channels in the group are closed, with only normally functioning nanopore channels used as the source of data to be analyzed. If the kicking success rate is less than the preset power rate, all nanopores in the group are controlled to be closed. The nanopore group is obtained by pre-grouping according to a preset grouping rule. Specifically, the grouping rule is to obtain a nanopore group with any nanopore as the origin and K adjacent nanopores as the radius.

[0101] Example 2: Figure 4As shown, based on the aforementioned weak current detection circuit, this invention provides a method for detecting blood drug concentration based on a multi-channel nanopore system, specifically including the following steps:

[0102] S101, a weak current measurement circuit is constructed to generate an electric field force to drive the drug molecules to be tested in the electrolyte chamber through the corresponding nanopore channels, and to acquire the current signal generated when the drug molecules to be tested pass through each nanopore channel. Then, the current signal is converted into a voltage signal and amplified and sampled to obtain the corresponding data to be analyzed.

[0103] In some embodiments, the weak current measurement circuit adopts the weak current measurement circuit of Embodiment 1 above. Its working principle can be referred to Embodiment 1 above, and will not be repeated here.

[0104] In some embodiments, the multi-channel nanopore system includes multiple nanopore channels, each nanopore channel having an electrolyte chamber on each side, and the nanopores being embedded in an insulating membrane between the two electrolyte chambers.

[0105] Preferably, the nanopore system is an MScCG nanopore system, in which the nanopores through which molecules pass are pre-modified, as described in CN115725685A, so that only one drug molecule can pass through the nanopores at a time. Therefore, there is no need to pre-treat or modify the drug molecules in the sample to be tested.

[0106] In some embodiments, before converting the current signal into a voltage signal in step S101 or before executing step S102, the method further includes the step of: identifying the state of each nanopore channel based on the detected current signal; specifically, determining whether the current collected within a preset first time period (e.g., within 30 seconds) is a preset fixed value or suddenly increases to a first preset threshold.

[0107] If the current collected over a long period of time (such as within the preset first time period of 30 seconds) is a preset fixed value (between 0 current and the opening current I1), the current state of the corresponding nanopore channel is determined to be abnormal, and its abnormality type is marked as pore blockage state.

[0108] If the current suddenly increases to the first preset threshold, for example, 1250pA, and the current quickly drops back to the second preset threshold, for example, around 0pA, within a preset second time period (for example, within 1-3 seconds), or if the current suddenly increases to the first preset threshold, for example, 1250pA, and remains at that value, then the current state of the corresponding nanopore channel is determined to be abnormal, and its abnormality type is marked as membrane rupture state.

[0109] S102, acquire the data to be analyzed, and preprocess the data to be analyzed to obtain valid data and invalid data.

[0110] In some embodiments, step S102 specifically includes the following steps:

[0111] S1021, Obtain the status information of each piece of data to be analyzed.

[0112] In some embodiments, the status information includes: the source of the nanopore channel, and the corresponding nanopore channel status (e.g., the current status corresponding to each data point to be analyzed); the nanopore channel status includes normal or abnormal.

[0113] S1022 Determine whether the current state of the nanopore channel corresponding to each of the data to be analyzed is normal. If it is normal, proceed to step S1023; if it is abnormal and in a blocked state, proceed to step S1024; if it is abnormal and in a ruptured state, proceed to step S1025.

[0114] S1023 marks the corresponding data to be analyzed as valid data.

[0115] S1024 applies a first threshold voltage, such as a reverse driving voltage of 180mV-200mV (preferably 180mV), to the corresponding nanopore channel to kick the hole and restore it to normal, and executes step S101.

[0116] In practice, after completing one hole kick, the current data of the channel needs to be acquired again, and the channel is judged to be normal based on the current data (for example, whether the hole current of the channel is normal, if so, it means that it has recovered to normal). If it has recovered to normal, its status is updated to normal; otherwise, the hole kick is performed again, and the current data collected after the second hole kick is used to judge whether it has recovered to normal.

[0117] Because there are various reasons why nanopores can become blocked, sometimes a single attempt to kick the pore may not be enough to restore it to normal operation, thus requiring multiple consecutive attempts. Of course, if multiple attempts to kick the same nanopore fail (for example, if a nanopore is identified as blocked and the number of consecutive attempts exceeds a preset threshold N), the driving voltage for that nanopore is turned off, thereby shutting down the nanopore channel. In this case, the nanopore is no longer used, and the data collected by it is no longer considered a valid data source.

[0118] Specifically, after each hole-kicking process, it can be checked whether the system has returned to normal. If it has not returned to normal, the hole-kicking process is repeated until the number of hole-kickings reaches N. If the system still has not returned to normal, the nanopore channel is closed.

[0119] In other embodiments, the channel can be continuously kicked n times (e.g., applying the first threshold voltage multiple times consecutively, preferably with an interval of 0.5s or 1s each time), and then it can be determined whether it has returned to normal. If it has not returned to normal, the channel is kicked n times again, and the result is determined whether it has returned to normal. This process is repeated until it returns to normal. If it has not returned to normal after M times, the channel is closed. Here, n = N / M. By using a continuous grouping method for kicking the channel, the number of judgments is reduced, which reduces the amount of computation. Preferably, n is 2 times, N is 6 times, and M = 3.

[0120] Furthermore, a nanopore may be blocked multiple times. That is, even if the nanochannel is restored to normal after a period of time, the nanopore may be blocked again. Therefore, this embodiment further includes the following steps:

[0121] If, within a third specified time period, the number of times a corresponding nanopore channel is continuously marked as blocked is greater than or equal to a preset number of blocking times, the corresponding nanopore channel will be closed, and all data from that nanopore channel will be marked as invalid data. The third specified time period is shorter than the second specified time period.

[0122] For example, if the nanopore is blocked 3 times in 15 seconds, then the corresponding nanopore channel will be closed and all data of that nanopore channel will be marked as invalid data.

[0123] In some embodiments, when the nanopore channel is in a normal state, a drug molecule passing through the pore once will generate a current waveform, such as a current pulse signal with a certain amplitude and period. If a current pulse signal (i.e., valid data) is detected, it is also necessary to determine whether its amplitude is within a preset valid signal amplitude threshold and whether its period is within a preset valid signal period threshold. If so, it is determined to be valid data; otherwise, it is determined to be invalid data.

[0124] S1025 marks the corresponding data to be analyzed as invalid data and controls the closure of the corresponding nanopore channels.

[0125] S103, determine the number of times the drug molecule to be tested passes through the pores based on valid data.

[0126] In some embodiments, step S103 specifically includes:

[0127] S1031, calculate the mean and standard deviation based on all valid data from all channels within a first specified time period (e.g., 1 second).

[0128] S1032, determine whether there are at least two consecutive valid data points within the first specified time period whose differences from the mean are greater than twice the standard deviation. If so, determine that the drug molecule to be tested has passed through the pore once, and execute step S1033; otherwise, execute step S1031 again.

[0129] S1033, the number of times the drug molecule to be tested passes through the pores within the second specified time period (e.g., 1 minute per unit time) is statistically obtained.

[0130] In some embodiments, the second specified time period is longer than the first specified time period.

[0131] S104. The concentration of the drug molecule to be tested is calculated based on the number of pore passes and the pre-stored frequency-concentration standard curve.

[0132] In one specific embodiment, there are three channels: CHA, CHB, and CHC, all of which are effective channels (i.e., even if there is blockage, the number of blockages is less than the preset number of blockages). Within a unit of time (e.g., 1 minute), the effective pulse count for CHA is 30 (e.g., if two consecutive effective data points within 1 second each have a difference greater than twice the standard deviation from the mean, forming one effective pulse, and there are 30 pulses within 1 minute); the effective pulse count for CHB is 40 (e.g., if two consecutive effective data points within 1 second each have a difference greater than twice the standard deviation from the mean, forming one effective pulse, and there are 40 pulses within 1 minute); and the effective pulse count for CHC is 50 (e.g., if two consecutive effective data points within 1 second each have a difference greater than twice the standard deviation from the mean, forming one effective pulse, and there are 50 pulses within 1 minute). Therefore, the average number of pulses is (30 + 40 + 50) / 3 = 40. This average is then substituted into the standard curve formula for concentration versus frequency to calculate the drug concentration. The effective pulse count actually refers to the effective voltage pulse signal after the current signal is converted into a voltage signal; of course, in some other embodiments, it may also refer to the effective current pulse signal, that is, the effective data can refer to both current signal and voltage signal, and the judgment principle is the same.

[0133] As mentioned earlier, each nanopore channel may be blocked a different number of times, meaning the blocking frequency of each nanopore channel during the detection process is different. This results in inconsistent pulse counts for each nanopore channel within the same time period. Therefore, the blocking time is removed when calculating the pulse count, and then the number of pulses per unit time is calculated.

[0134] For example: if channel A samples for 10 minutes and blocks the hole for 9 minutes, a total of 100 pulses are collected; then the effective pulse count per unit time (1 minute) of channel A is 100 / (10-9) = 100.

[0135] In other embodiments, if a high-throughput nanopore system is used, i.e., instead of individual sample addition to each nanopore, all nanopores are added through the same sample inlet and connected to the same inlet channel, then adjacent nanopores may experience similar blockages. Therefore, to avoid the computational burden and complexity of individually judging each nanopore and applying a reverse bias voltage to kick out each pore, the following steps are also included:

[0136] The nanopores are grouped according to a preset grouping rule; specifically, the grouping rule is to obtain a nanopore group with any nanopore as the origin and K adjacent nanopores as the radius.

[0137] If the number of blocked nanopores in any nanopore group reaches a preset threshold, the current state of all nanopores in that group is marked as blocked, and a reverse bias is applied to all nanopores in that group to kick the nanopores (as mentioned above, this is done n times consecutively at preset time intervals of 0.5s or 1s). The kicking success rate of that group (i.e., the percentage of successfully kicked nanopores to the total number of nanopores in that group) is calculated. If the kicking success rate is greater than the preset success rate threshold, kicking is abandoned for channels that have not yet been successfully kicked, and all blocked or ruptured nanopore channels in that group are closed, with only normally functioning nanopore channels used as the source of data to be analyzed. If the kicking success rate is less than the preset power rate, all nanopores in that group are closed. Furthermore, before closing a group or abandoning kicking, it is determined whether the number of all effective nanopore channels in the nanopore system has reached a preset high-throughput condition (such as an effective channel number threshold). If it has, the group is closed; if not, kicking is performed again until the number of effective channels reaches the high-throughput preset condition.

[0138] For example, if N1 (greater than or equal to a preset threshold number) nanopores in a group are found to be blocked, but N2 (e.g., N2 = N1 / 10) nanopores are still in a normal state, then a reverse bias voltage is applied to the entire group of nanopores to kick them out. After kicking out the nanopores, the state of N3 nanopores is determined to return to normal based on the current signal. Then the kicking rate P0 = N3 / N1.

[0139] If N3 = N1 + N2, then P0 > 1, indicating that the hole was successfully kicked.

[0140] If N3=N1, then P0=1, indicating that a small number of nanopores failed to kick the hole. Control the continuous kicking process again. If P0=1 is determined again, the continuous kicking process is repeated until the preset number of kicks is reached or P0>1 is reached.

[0141] If N3 < N1, then P0 < 1, indicating that most nanopores failed to kick in. Therefore, all nanopores in this group should be shut down.

[0142] In some embodiments, the weak current measurement circuit specifically includes: an overcurrent protection module, a switch matrix module, an R-TIA module, an overvoltage protection module, a signal processing module, a signal acquisition module, and a DAC module; wherein, the input port of the overcurrent protection module is connected to a nanopore, the output port of the overcurrent protection module is connected in series with the input port of the switch matrix module, the output port of the switch matrix module is connected in series with the negative input port of the R-TIA module, the output terminal of the R-TIA module is connected to the input port of the overvoltage protection module, the output port of the overvoltage protection module is connected to the signal processing module, and the signal processing module is connected to the signal acquisition module; the output terminal of the DAC module is connected to the positive input terminals of the operational amplifiers in the R-TIA module and the signal processing module 106, respectively.

[0143] In some embodiments, the R-TIA module specifically comprises: an operational amplifier OPA1, a feedback resistor RF, and a compensation capacitor CF, wherein...

[0144] The negative input terminal of the operational amplifier OPA1 is connected to the output port of the switch matrix module, and also to terminal A of the feedback resistor RF and the compensation capacitor CF.

[0145] The positive input port of the operational amplifier OPA1 is connected to the output port of the DAC module;

[0146] The output port of the operational amplifier OPA1 is connected to the B terminal of the feedback resistor RF and the compensation capacitor CF, and is also connected to the input terminal of the overvoltage protection module.

[0147] In other embodiments, the weak current measurement circuit further includes an LPF module disposed between the overvoltage protection module and the signal processing module.

[0148] Example 3: Figure 5 As shown, the present invention also provides a blood drug concentration detection system based on multi-channel nanopores, specifically comprising:

[0149] A multi-channel nanopore system includes multiple nanopore channels, each nanopore channel having an electrolyte chamber on each side, and the nanopores being embedded in an insulating membrane between the two electrolyte chambers;

[0150] A weak current measurement circuit, connected to the nanopores in the multi-channel nanopore system, is configured to generate an electric field to drive the drug molecule to be tested in the electrolyte chamber through the corresponding nanopore channel, and to acquire the current signal generated when the drug molecule to be tested passes through each nanopore channel. Then, the current signal is converted into a voltage signal and amplified and sampled to obtain the corresponding data to be analyzed.

[0151] The host computer, connected to the weak current measurement circuit, is configured to acquire the data to be analyzed from the weak current measurement circuit, preprocess the data to be analyzed to obtain valid data and invalid data; and determine the number of times the drug molecule to be tested passes through the pores based on the valid data, and then calculate the concentration of the drug molecule to be tested based on the number of times it passes through the pores and the pre-stored frequency-concentration standard curve.

[0152] In some embodiments, the host computer specifically includes:

[0153] The preprocessing unit is used to acquire the data to be analyzed from the weak current measurement circuit and perform preprocessing to obtain valid data and invalid data;

[0154] The data analysis unit is used to determine the number of times the drug molecule to be tested passes through the pores based on the effective data obtained from the preprocessing unit.

[0155] The concentration calculation unit is used to calculate the concentration of the drug molecule to be tested based on the number of pores obtained by the data analysis unit and the pre-stored frequency-concentration standard curve.

[0156] In some embodiments, the data analysis unit is specifically configured to acquire status information for each piece of data to be analyzed; the status information includes: the source of the nanopore channel and the corresponding nanopore channel status; the nanopore channel status includes normal or abnormal; and determine whether the nanopore channel status corresponding to each piece of data to be analyzed is normal. If it is normal, the corresponding piece of data to be analyzed is marked as valid data; if it is abnormal, the corresponding piece of data to be analyzed is marked as invalid data, and the corresponding nanopore channel is controlled to be closed.

[0157] Furthermore, the data analysis unit is also used to determine whether the number of times the corresponding nanopore channel is continuously marked as blocked within the third specified time period is greater than or equal to the preset number of blockages. If so, the control switch matrix module will close the corresponding nanopore channel and mark all data of the nanopore channel as invalid data. The third specified time period is shorter than the second specified time period.

[0158] Furthermore, the data analysis unit is specifically used to control the DAC module to apply a corresponding bias voltage to the R-TIA module and operational amplifier OPA2 when it is determined that the current state of the corresponding nanopore channel is abnormal and blocked, in order to perform a hole kick. Then, based on the current data detected after the hole kick, it is determined whether the nanopore channel has returned to normal. If it has not returned to normal, a reverse drive voltage is applied again to perform a hole kick, and it is determined whether it has returned to normal. This process is repeated until it returns to normal. If it still has not returned to normal after a preset threshold of N hole kicks, the system is turned off. The channel is closed; or, when the current state of the corresponding nanopore channel is determined to be abnormal and blocked, the DAC module is controlled to apply the corresponding bias voltage to the corresponding nanopore channel multiple times at a preset time interval to perform n consecutive knocks. Then, based on the current data detected after the knock, it is determined whether the nanopore channel has returned to normal. If it has not returned to normal, the knock is performed again n times and it is determined whether it has returned to normal. This process is repeated until it returns to normal. If it has not returned to normal after M times, the channel is closed; where n = N / M, and N is the preset threshold number of knocks N.

[0159] In other embodiments, the host computer further includes: a state analysis unit, used to determine whether the current value corresponding to each nanopore channel acquired by the signal acquisition module is a fixed value within a preset first time period, and the fixed value is greater than 0 but less than the opening current I1; if so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as a blocked state, and simultaneously apply a reverse driving voltage of a first threshold to the nanopore channel to perform a hole kick; or, determine whether the current value corresponding to each nanopore suddenly increases to a first preset threshold and falls back to a second preset threshold within a preset second time period, and if so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as a membrane rupture state; or, determine whether the current value corresponding to each nanopore suddenly increases to a first preset threshold and consistently maintains the current value, and if so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as a membrane rupture state.

[0160] In other embodiments, the data analysis unit is also used to obtain the number of blocked nanopores in each nanopore group. When the number of blocked nanopores in any nanopore group reaches a preset threshold, the current state of all nanopores in that group is marked as blocked, and the DAC module is controlled to apply a reverse bias voltage to all nanopores in that group to kick the nanopores (as mentioned above, the reverse bias voltage is applied n times consecutively at preset time intervals of 0.5s or 1s). The kicking success rate of the group is calculated (i.e., the percentage of successfully kicked nanopores to the total number of nanopores in the group). If the kicking success rate is greater than the preset success rate threshold, kicking is stopped, and all blocked or ruptured nanopore channels in the group are closed, with only normally functioning nanopore channels used as the source of data to be analyzed. If the kicking success rate is less than the preset power rate, all nanopores in the group are controlled to be closed. The nanopore group is obtained by pre-grouping according to a preset grouping rule. Specifically, the grouping rule is to obtain a nanopore group with any nanopore as the origin and K adjacent nanopores as the radius.

[0161] In some embodiments, the weak current measurement circuit includes the modules described in Embodiment 1 above. However, in this embodiment, the weak current detection circuit does not possess the aforementioned data processing function. Specifically, it includes: an overcurrent protection module, a switch matrix module, an R-TIA module, an overvoltage protection module, a signal processing module, a signal acquisition module, and a DAC module. The input port of the overcurrent protection module is connected to a nanopore; the output port of the overcurrent protection module is connected in series with the input port of the switch matrix module; the output port of the switch matrix module is connected in series with the negative input port of the R-TIA module; the output terminal of the R-TIA module is connected to the input port of the overvoltage protection module; the output port of the overvoltage protection module is connected to the signal processing module; and the signal processing module is connected to the signal acquisition module. The output terminal of the DAC module is connected to the positive input terminals of the operational amplifiers in the R-TIA module and the signal processing module, respectively.

[0162] In some embodiments, the R-TIA module specifically comprises: an operational amplifier OPA1, a feedback resistor RF, and a compensation capacitor CF, wherein...

[0163] The negative input terminal of the operational amplifier OPA1 is connected to the output port of the switch matrix module, and also to terminal A of the feedback resistor RF and the compensation capacitor CF.

[0164] The positive input port of the operational amplifier OPA1 is connected to the output port of the DAC module;

[0165] The output port of the operational amplifier OPA1 is connected to the B terminal of the feedback resistor RF and the compensation capacitor CF, and is also connected to the input terminal of the overvoltage protection module.

[0166] In other embodiments, the weak current measurement circuit further includes an LPF module disposed between the overvoltage protection module and the signal processing module.

[0167] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0168] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) and includes several instructions to cause a computer terminal (which may be a mobile phone, computer, server, or network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0169] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for detecting blood drug concentration based on a multi-channel nanoporous system, characterized in that, The multi-channel nanopore system includes multiple nanopore channels, each nanopore channel having an electrolyte chamber on each side, and the nanopores are embedded in an insulating membrane between the two electrolyte chambers; correspondingly, the blood drug concentration detection method specifically includes the following steps: S101, a weak current measurement circuit is constructed to generate an electric field force to drive the drug molecule to be tested in the electrolyte chamber through the corresponding nanopore channel, and to acquire the current signal generated when the drug molecule to be tested passes through each nanopore channel. Then, the current signal is converted into a voltage signal and amplified and sampled to obtain the corresponding data to be analyzed. S102, acquire the data to be analyzed, and preprocess the data to be analyzed to obtain valid data and invalid data; S103, determine the number of times the drug molecule to be tested passes through the pores based on the effective data; S104, the concentration of the drug molecule to be tested is calculated based on the number of pore passes and the pre-stored frequency-concentration standard curve; Specifically, step S103 includes: S1031, Calculate the mean and standard deviation based on all valid data within the first specified time period; S1032, determine whether there are at least two consecutive valid data points within the first specified time period whose differences from the mean are greater than twice the standard deviation; if so, determine that the drug molecule to be tested has been poreped once. S1033, Count the number of times the drug molecule to be tested passes through the pore within the second specified time period; the second specified time period is longer than the first specified time period; Before step S102, the following steps are also included: Determine whether the current value corresponding to each nanopore channel is a fixed value within a preset first time period, and the fixed value is greater than 0 but less than the opening current I1; if so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as blocked. Alternatively, determine whether the current value corresponding to each nanopore suddenly increases to the first preset threshold and falls back to the second preset threshold within a preset second time period. If so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as a membrane rupture state. Alternatively, determine whether the current value corresponding to each nanopore suddenly increases to the first preset threshold and remains at the current value. If so, determine that the current state of the corresponding nanopore channel is abnormal and mark it as a membrane rupture state.

2. The method for detecting blood drug concentration based on a multi-channel nanoporous system according to claim 1, characterized in that, Step S102 specifically includes the following steps: S1021, Obtain the status information of each data to be analyzed; the status information includes: the source of the nanopore channel, and the corresponding nanopore channel status; the nanopore channel status includes normal or abnormal. S1022, Determine whether the current state of the nanopore channel corresponding to each piece of data to be analyzed is normal. If normal, proceed to step S1023; if abnormal and in a ruptured membrane state, proceed to step S1025; if abnormal and in a blocked state, proceed to step S1024. S1023, mark the corresponding data to be analyzed as valid data; S1024, apply a reverse driving voltage of the first threshold to the corresponding nanopore channel to perform at least one hole kick to restore it to normal, and when the corresponding nanopore channel is restored to normal, update the current state of the nanopore channel to normal, execute step S101, and mark the data to be analyzed when the nanopore channel is in the normal state as valid data; while marking the data when it is in the blocked state as invalid data. S1025 marks the corresponding data to be analyzed as invalid data and controls the closure of the corresponding nanopore channels.

3. The method for detecting blood drug concentration based on a multi-channel nanoporous system according to claim 2, characterized in that, If, within a third specified time period, the number of times a corresponding nanopore channel is continuously marked as blocked is greater than or equal to a preset number of blockages, the corresponding nanopore channel will be closed, and all data of that nanopore channel will be marked as invalid data; the third specified time period is shorter than the second specified time period.

4. The method for detecting blood drug concentration based on a multi-channel nanoporous system according to claim 2, characterized in that, In step S1024, a reverse driving voltage of the first threshold is continuously applied to the corresponding nanopore channel multiple times to perform consecutive hole kicking n times. Then, based on the current data detected after hole kicking, it is determined whether the nanopore channel has returned to normal. If it has not returned to normal, it is kicked again n times and determined whether it has returned to normal. This process is repeated until it returns to normal. If it has not returned to normal after M times, the channel is closed. Here, n = N / M, and N is the preset hole kicking number threshold N.

5. The method for detecting blood drug concentration based on a multi-channel nanoporous system according to claim 1, characterized in that, The weak current measurement circuit specifically includes: an overcurrent protection module, a switch matrix module, an R-TIA module, an overvoltage protection module, a signal processing module, a signal acquisition module, and a DAC module; wherein... The input port of the overcurrent protection module is connected to the nanopore, the output port of the overcurrent protection module is connected in series with the input port of the switch matrix module, the output port of the switch matrix module is connected in series with the negative input port of the R-TIA module, the output terminal of the R-TIA module is connected to the input port of the overvoltage protection module, the output port of the overvoltage protection module is connected to the signal processing module, and the signal processing module is connected to the signal acquisition module. The output of the DAC module is connected to the positive input of the operational amplifiers in the R-TIA module and the signal processing module, respectively.

6. The method for detecting blood drug concentration based on a multi-channel nanoporous system according to claim 5, characterized in that, The weak current measurement circuit also includes an LPF module disposed between the overvoltage protection module and the signal processing module.

7. The method for detecting blood drug concentration based on a multi-channel nanoporous system according to claim 5, characterized in that, The R-TIA module specifically comprises: an operational amplifier OPA1, a feedback resistor RF, and a compensation capacitor CF, wherein... The negative input terminal of the operational amplifier OPA1 is connected to the output port of the switch matrix module, and also to terminal A of the feedback resistor RF and the compensation capacitor CF. The positive input port of the operational amplifier OPA1 is connected to the output port of the DAC module; The output port of the operational amplifier OPA1 is connected to the B terminal of the feedback resistor RF and the compensation capacitor CF, and is also connected to the input terminal of the overvoltage protection module.

8. A blood drug concentration detection system, characterized in that, include: A multi-channel nanopore system includes multiple nanopore channels, each nanopore channel having an electrolyte chamber on each side, and the nanopores being embedded in an insulating membrane between the two electrolyte chambers; A weak current measurement circuit, connected to the nanopores in the multi-channel nanopore system, is configured to generate an electric field to drive the drug molecule to be tested in the electrolyte chamber through the corresponding nanopore channel, and to acquire the current signal generated when the drug molecule to be tested passes through each nanopore channel. Then, the current signal is converted into a voltage signal and amplified and sampled to obtain the corresponding data to be analyzed. The host computer, connected to the weak current measurement circuit, is configured to acquire the data to be analyzed from the weak current measurement circuit, preprocess the data to be analyzed to obtain valid data and invalid data; and determine the number of times the drug molecule to be tested passes through the pores based on the valid data, and then calculate the concentration of the drug molecule to be tested based on the number of times it passes through the pores and the pre-stored frequency-concentration standard curve. The host computer specifically includes: The preprocessing unit is used to acquire the data to be analyzed from the weak current measurement circuit and perform preprocessing to obtain valid data and invalid data; The data analysis unit is used to determine the number of times the drug molecule to be tested is sieved based on the valid data obtained by the preprocessing unit. Specifically, the data analysis unit is used to calculate the mean and standard deviation based on all valid data within a first specified time period; and to determine whether there are at least two consecutive valid data within the first specified time period whose differences from the mean are greater than twice the standard deviation. If so, it is determined that the drug molecule to be tested has been sieved once; then, the number of times the drug molecule to be tested is sieved within a second specified time period is counted; the second specified time period is longer than the first specified time period. The concentration calculation unit is used to calculate the concentration of the drug molecule to be tested based on the number of pores obtained by the data analysis unit and the pre-stored frequency-concentration standard curve.

9. A blood drug concentration detection system according to claim 8, characterized in that, The data analysis unit is specifically configured to acquire the status information of each piece of data to be analyzed; the status information includes: the source of the nanopore channel and the corresponding nanopore channel status; the nanopore channel status includes normal or abnormal; and to determine whether the current status of the nanopore channel corresponding to each piece of data to be analyzed is normal. If it is normal, the corresponding piece of data to be analyzed is marked as valid data; if it is abnormal and in a ruptured state, the corresponding piece of data to be analyzed is marked as invalid data, and the corresponding nanopore channel is controlled to be closed; if it is abnormal and in a blocked state, a reverse driving voltage of a first threshold is applied to the corresponding nanopore channel to perform at least one hole kick to restore it to normal, and when it is restored to normal, the current status of the nanopore channel is updated to normal, and the piece of data to be analyzed when the nanopore channel is in a normal state is marked as valid data; while the data in a blocked state is marked as invalid data.

10. A blood drug concentration detection system according to claim 8, characterized in that, The weak current measurement circuit specifically includes: an overcurrent protection module, a switch matrix module, an R-TIA module, an overvoltage protection module, a signal processing module, a signal acquisition module, and a DAC module; wherein... The input port of the overcurrent protection module is connected to the nanopore, the output port of the overcurrent protection module is connected in series with the input port of the switch matrix module, the output port of the switch matrix module is connected in series with the negative input port of the R-TIA module, the output terminal of the R-TIA module is connected to the input port of the overvoltage protection module, the output port of the overvoltage protection module is connected to the signal processing module, and the signal processing module is connected to the signal acquisition module. The output of the DAC module is connected to the positive input of the operational amplifiers in the R-TIA module and the signal processing module, respectively. And / or, the weak current measurement circuit further includes an LPF module disposed between the overvoltage protection module and the signal processing module.

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