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CN-121994566-A - Fingertip blood myocardial marker detection method and system based on dynamic baseline

CN121994566ACN 121994566 ACN121994566 ACN 121994566ACN-121994566-A

Abstract

The invention relates to the technical field of biomedical detection and discloses a fingertip blood myocardial marker detection method based on a dynamic baseline, which comprises the following steps of S1, sample pretreatment, namely after a fingertip blood sample is collected by a fingertip blood taking needle, the sample is injected into a sample injection end of a microfluidic chip with a built-in gradient filter channel; the gradient filter channel of the micro-fluidic chip is used for separating erythrocytes from impurities in the sample, an anticoagulation coating is preset on the inner wall of the chip, and the sample is driven to flow through the gradient filter channel by the micro-pump arranged in the chip, so that the erythrocytes are separated from the impurities. By introducing a dynamic baseline calibration module, the influence of environmental parameter changes such as temperature, humidity, air pressure and the like on the system electrical baseline is sensed and compensated in real time, and baseline drift is controlled at an extremely low level, so that the detection system can keep a stable working state under different altitudes and climates, and the problems of unrepeated and unreliable detection results caused by environmental fluctuation of the conventional POCT equipment are fundamentally solved.

Inventors

  • ZHANG YAN
  • SONG YONGWEI
  • QIAN JUNJIANG

Assignees

  • 临海市第一人民医院医疗卫生服务共同体(临海市第一人民医院)

Dates

Publication Date
20260508
Application Date
20260225

Claims (10)

  1. 1. The fingertip blood myocardial marker detection method based on the dynamic baseline is characterized by comprising the following steps of: S1, sample pretreatment, namely, after a fingertip blood sample is collected by a fingertip blood taking needle, injecting the sample into a sample injection end of a microfluidic chip with a built-in gradient filter channel, wherein the gradient filter channel of the microfluidic chip is used for separating erythrocytes and impurities in the sample, and an anticoagulation coating is preset on the inner wall of the chip; S2, dynamic baseline calibration, namely setting a baseline acquisition unit, an environmental parameter sensing unit and a baseline dynamic correction unit, acquiring dark current signals of an optical detection unit under the conditions of no sample and no excitation light source irradiation through the baseline acquisition unit, and converting the dark current signals into initial electric signal baselines of a detection system in an empty load state through a signal acquisition circuit, wherein a sampling point of the baseline acquisition unit is positioned on a signal path between an output end of a photomultiplier and a front-stage low-noise pre-amplification module; S3, performing specific detection, namely quantitatively injecting the pretreated serum sample to be detected into a reaction tank, and simultaneously adding a preset amount of myocardial marker specific antibodies of a labeled fluorescent probe, wherein an oscillation mixing structure is arranged in the reaction tank, and after the oscillation mixing structure is uniformly mixed, incubating for a preset time under the constant temperature condition of 37 ℃ to form a reaction compound; S4, signal processing and result output are carried out, a signal processing unit and a result output unit are arranged, background interference of fluorescent signals is subtracted based on the dynamic calibration baseline obtained in the step S2, wherein the correction precision of the dynamic calibration baseline enables the detection variation Coefficient (CV) of the system to a 0.1ng/mL cTnI standard substance to be less than or equal to 5% under the condition that environmental parameters change by +/-10%, noise reduction processing and signal enhancement are carried out through the signal processing unit, quantitative analysis of myocardial markers is completed through comparison with a preset myocardial marker standard curve, and the detection result is output through the result output unit.
  2. 2. The method of claim 1, wherein the gradient filtering channel of the microfluidic chip in the step S1 is of a three-layer modified nanofiber membrane superposition structure, hydrophilic modification groups are grafted on the surfaces of all the nanofiber membranes, the gradient decreasing pore diameters from a sample inlet end to a sample outlet end are 8-10 μm, 2-5 μm and 0.5-1 μm, red blood cells, large-size impurities and small-molecule impurities are intercepted in a targeted manner respectively, the anticoagulation coating is a heparin-chitosan crosslinked composite coating, a micro-nano concave-convex structure is formed on the surface of the coating to improve anticoagulation stability, the sample inlet end, the sample outlet end and the inner wall of a connecting pipeline of the microfluidic chip are covered with the heparin-chitosan crosslinked composite anticoagulation coating, the gradient filtering channel is formed by three layers of independent modified nanofiber membranes, each membrane is bonded in a reserved groove on a PDMS substrate after being activated by oxygen plasma, the membrane surfaces are not covered with the coating to avoid blocking the nanopore, the sample treatment flow is regulated to be 5-10 μm/min by a micropump, and the temperature of the chip is maintained at 25-30 ℃ for 3-5 DEG in a closed loop treatment process.
  3. 3. The method of claim 1, wherein the environmental parameter sensing unit and the baseline dynamic correction unit in the step S2 adopt a real-time linkage feedback mechanism, the environmental parameter sensing unit synchronously collects temperature, humidity and air pressure parameters according to a sampling frequency of 10Hz and carries out multi-parameter fusion pretreatment, when the variation of any environmental parameter exceeds a preset threshold value, the baseline dynamic correction unit starts a grading correction strategy, specifically, the parameter variation amplitude is divided into three stages, the correction response speed is set to 3-7 ms/time when the first stage is changed, the correction amplitude is set to 0.001-0.003V, the correction response speed is set to 2-5 ms/time when the second stage is changed, the correction amplitude is set to 0.004-0.006V, the correction response speed is set to 0.5-2 ms/time when the third stage is changed, the correction amplitude is 0.008-0.012V, wherein the preset threshold values of the temperature, the humidity and the air pressure variation are respectively + -0.5 ℃ RH + -5 hPa, and the corrected dynamic calibration baseline is required to meet the baseline drift amount of less than or equal to 0.01V.
  4. 4. The method of claim 1, wherein the central muscle marker in the step S3 comprises cTnI, CK-MB and NT-proBNP, corresponding specific antibodies are mixed according to a proportion of 1:1:1, each antibody is subjected to affinity purification treatment, the fluorescent probe is a time-resolved fluorescent microsphere with a surface modified targeting group, the targeting group is precisely matched with a specific binding site of the myocardial marker, the excitation wavelength of the fluorescent microsphere is 340nm, the emission wavelength is 615nm, the fluorescence lifetime is more than or equal to 100 mu S, a sectional constant temperature strategy is adopted in the incubation process, wherein the sectional constant temperature strategy comprises the steps of firstly incubating for 10min at a constant temperature of 37 ℃ and then incubating for 5-10min at a temperature of 40 ℃, the temperature fluctuation range of each stage of sectional constant temperature incubation is less than or equal to +/-0.5 ℃, and the continuous oscillation of 50-80r/min is maintained through an oscillation mixed structure built in a reaction tank in the incubation process.
  5. 5. The method according to claim 1, wherein the signal processing unit in the step S4 adopts a two-stage lock-in amplifying structure, the front stage is a low-noise pre-amplifying module, the rear stage is a high-gain main amplifying module, the amplifying power can be adaptively and graded by 10 3 -10 5 times according to the fluorescence signal intensity, the standard curve is pre-constructed by a myocardial marker mixed standard substance with a series of concentration gradients, the concentration range is 0.01-100ng/mL, the gradient is set to 0.01, 0.1, 1, 10, 50, 100ng/mL, the correlation coefficient R 2 of the standard curve is more than or equal to 0.995, the quantitative analysis process adopts a signal multi-point acquisition screening structure, specifically, 5-8 acquisition points are set to different areas of the same reaction compound, each acquisition point is provided with an independent signal acquisition branch and continuously acquires 3 times of fluorescence signals, the signal screening module adopts a three times standard deviation (3 sigma) criterion to reject abnormal signal values, the average mu and standard sigma of three times of the same acquisition points are calculated, any measurement value exceeds [ mu-3 sigma, mu+3 sigma ] range, the average value is regarded as the average value of all the abnormal signal values, and the average value is taken as the effective average value of all the average signal values.
  6. 6. The fingertip blood myocardial marker detection system based on the dynamic baseline is characterized by comprising a sample preprocessing module, a dynamic baseline calibration module, a specificity detection module, a signal processing module and a result output module, wherein the modules are sequentially connected in a linkage manner along a detection flow; The sample pretreatment module comprises a fingertip blood collection assembly, a micro-fluidic chip and a micro-pump, wherein the collection assembly comprises a fingertip blood collection needle and a sample collection tube, an outlet of the sample collection tube is in sealing butt joint with a sample inlet end of the micro-fluidic chip through a sealing joint, and the micro-pump is connected between the sample collection tube and the micro-fluidic chip in series to drive a sample to flow through the chip; The dynamic baseline calibration module comprises a baseline acquisition unit, an environment parameter sensing unit and a baseline dynamic correction unit, wherein the baseline acquisition unit is used for acquiring dark current signals of the optical detection unit under the conditions of no sample and no excitation light source irradiation, and converting the dark current signals into initial electric signal baselines of the detection system in an empty load state through a signal acquisition circuit; The specificity detection module comprises a reaction tank, a constant temperature control unit and an optical detection unit, wherein the reaction tank is communicated with a sample outlet end of a chip through a pipeline, an oscillation mixing structure is built in, and the outside is wrapped by the constant temperature control unit to maintain 37 ℃; the signal processing module is respectively connected with the dynamic baseline calibration module and the specificity detection module and comprises a baseline deduction unit, a noise reduction unit and a signal amplification unit, and is used for sequentially performing baseline deduction, noise reduction and signal enhancement processing on the fluorescent signal, and then comparing the fluorescent signal with a preset standard curve to finish the quantitative analysis of the myocardial markers; The result output module is connected with the signal processing module, displays the analysis result in a digital and curve form, and has the functions of data storage and export.
  7. 7. The system of claim 6, wherein the microfluidic chip is integrally formed by soft lithography using PDMS material, the nanofiber membrane is prepared by electrostatic spinning, the thickness is 20-30 μm, the anticoagulation coating is formed by layer-by-layer self-assembly, the thickness is 50-100nm, the sample inlet end, the sample outlet end and the inner wall of the connecting pipeline outside the filtering channel are covered, and the surface of the nanofiber membrane in the gradient filtering channel is not covered.
  8. 8. The system according to claim 6, wherein the environmental parameter sensing unit comprises a temperature sensor, a humidity sensor and an air pressure sensor, wherein the sensors are uniformly distributed in a detection cavity of the detection system, the measurement precision of the temperature sensor is +/-0.1 ℃, the measurement precision of the humidity sensor is +/-2% RH, the measurement frequency of the air pressure sensor is +/-1 hPa, the sampling frequency of the sensors is 10Hz, and the baseline dynamic correction unit takes an FPGA chip as a core processing unit.
  9. 9. The system of claim 6, wherein the optical detection unit comprises a pulse LED excitation light source, a filter set, a photomultiplier and a signal acquisition circuit, wherein the central wavelength of the excitation filter is 340nm, the half bandwidth is 10nm, the central wavelength of the emission filter is 615nm, the half bandwidth is 15nm, and the response time of the photomultiplier is less than or equal to 1ns.
  10. 10. The system of claim 6, further comprising a power supply module, a wireless communication module and a signal early warning output module, wherein the power supply module is a 2000mAh rechargeable lithium battery, supports 5V/2A stable output, provides matched working power for each module through a power management circuit, the wireless communication module is electrically connected with the signal processing module by adopting a Bluetooth 5.0 protocol to realize real-time transmission of detection data, the signal early warning output module is connected with the wireless communication module, pre-stores normal reference thresholds of different muscle markers, and is internally provided with an acousto-optic early warning component and a signal storage unit, and when the detection value exceeds the corresponding preset threshold, early warning prompt is automatically sent out through sound and popup window forms, and meanwhile, history storage and export of the detection data are realized.

Description

Fingertip blood myocardial marker detection method and system based on dynamic baseline Technical Field The invention relates to the technical field of biomedical detection, in particular to a fingertip blood myocardial marker detection method and system based on a dynamic baseline. Background Rapid and accurate detection of cardiac markers (such as cTnI, CK-MB, NT-proBNP) is critical for early diagnosis, risk stratification and efficacy monitoring of acute cardiovascular diseases, and traditional laboratory detection methods such as enzyme-linked immunosorbent assay (ELISA) typically require venous blood sampling, sample centrifugation, multi-step manual operation and large-scale instrumental analysis, resulting in long detection cycle, high requirements for the operating environment, and difficulty in meeting the instantaneous demands of point-of-care rapid detection (POCT). In recent years, POCT technology based on micro-fluidic and optical detection provides a new approach for rapid detection of myocardial markers, however, the prior art still has two obvious defects in practical application: Firstly, the environmental adaptability is poor, the detection baseline is unstable, the optical and electrical detection modules of the conventional POCT equipment are easily influenced by environmental temperature, humidity and air pressure fluctuation, so that the signal baseline of the detection system is shifted, the baseline shift can seriously interfere weak signals of particularly low-concentration targets, the detection result is inaccurate and the repeatability is poor, and the reliable application of the equipment in non-standard laboratory environments such as families, ambulances, basic clinics and the like is limited. Secondly, sample pretreatment is tedious, the integration level with a detection system is low, for trace whole blood samples such as fingertip blood, the effective plasma/serum separation is an accurate quantitative premise, the prior art relies on external separation steps or simple filter membranes, and the problems of low separation efficiency, easy blockage, easy hemolysis or insufficient anticoagulation and the like exist, so that an efficient, closed and automatic integrated flow cannot be formed with a subsequent biospecific recognition and signal detection module, and the overall speed of detection and the simplicity of operation are influenced. Disclosure of Invention The invention aims to provide a fingertip blood myocardial marker detection method and a fingertip blood myocardial marker detection system based on a dynamic baseline so as to solve the problems in the background art. In order to achieve the purpose, the invention provides the following technical scheme that the fingertip blood myocardial marker detection method based on the dynamic baseline comprises the following steps: S1, sample pretreatment, namely, after a fingertip blood sample is collected by a fingertip blood taking needle, injecting the sample into a sample injection end of a microfluidic chip with a built-in gradient filter channel, wherein the gradient filter channel of the microfluidic chip is used for separating erythrocytes and impurities in the sample, and an anticoagulation coating is preset on the inner wall of the chip; S2, dynamic baseline calibration, namely setting a baseline acquisition unit, an environmental parameter sensing unit and a baseline dynamic correction unit, acquiring dark current signals of an optical detection unit under the conditions of no sample and no excitation light source irradiation through the baseline acquisition unit, and converting the dark current signals into initial electric signal baselines of a detection system in an empty load state through a signal acquisition circuit, wherein a sampling point of the baseline acquisition unit is positioned on a signal path between an output end of a photomultiplier and a front-stage low-noise pre-amplification module; S3, performing specific detection, namely quantitatively injecting the pretreated serum sample to be detected into a reaction tank, and simultaneously adding a preset amount of myocardial marker specific antibodies of a labeled fluorescent probe, wherein an oscillation mixing structure is arranged in the reaction tank, and after the oscillation mixing structure is uniformly mixed, incubating for a preset time under the constant temperature condition of 37 ℃ to form a reaction compound; S4, signal processing and result output are carried out, a signal processing unit and a result output unit are arranged, background interference of fluorescent signals is subtracted based on the dynamic calibration baseline obtained in the step S2, wherein the correction precision of the dynamic calibration baseline enables the detection variation Coefficient (CV) of the system to a 0.1ng/mL cTnI standard substance to be less than or equal to 5% under the condition that environmental parameters change by +/-10%, noise reduction