Integrated antifouling multi-channel electrochemical immunosensor for combined synchronous detection of sepsis biomarkers

Abstract

The invention discloses an integrated antifouling multi-channel electrochemical immunosensor for combined synchronous detection of sepsis biomarkers. The multi-channel electrochemical sensor is rapidly prepared in a large scale by adopting a silk-screen printing mode; a high-sensitivity three-dimensional antifouling coating is constructed through a bovine serum albumin / carbon nanotube / glutaraldehyde rapid cross-linking method; and by integrating with portable electrochemical detection equipment, combined synchronous detection and remote data transmission of six markers of sepsis are realized, and the kit has good sensitivity and specificity and is successfully used for clinical actual sample detection. The integrated antifouling multi-channel electrochemical immunosensor for combined synchronous detection of sepsis biomarkers, established by the invention, can be used for accurate diagnosis, disease progress monitoring and effective prognosis evaluation of sepsis patients, and is helpful for customizing individualized diagnosis and treatment plans for the sepsis patients.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and more specifically, relates to an integrated anti-fouling multi-channel electrochemical immunosensor for the combined synchronous detection of sepsis biomarkers. The constructed integrated sensor is expected to be used for on-site rapid combined synchronous detection of sepsis-related markers, promote the development of electrochemical on-site detection technology, and provide new ideas for comprehensive and precise diagnosis and treatment of diseases based on "biomarker groups". Background Art

[0002] Sepsis is a multi-organ dysfunction syndrome caused by systemic immune dysfunction induced by pathogenic microorganisms such as bacteria and viruses. Due to its extremely high mortality rate, it is one of the most critical issues in the global medical field. After the host is infected, pathogenic microorganisms multiply rapidly and release a large amount of toxins, triggering the body's "inflammatory factor storm", and the patient's condition can take a sharp turn for the worse within 24 hours. However, sepsis is a clinical syndrome defined by a series of signs, symptoms, abnormal blood indicators and characteristic pathophysiological disorders. Its initial symptoms are not typical and specific, which often leads to untimely diagnosis and ultimately a high mortality rate of sepsis. Early rapid diagnosis and early initiation of appropriate intervention, accurate assessment of the severity of the disease to correctly guide the selection of individualized treatment plans are important foundations for infection treatment, which directly affect the treatment effect and quality of life of patients with sepsis.

[0003] The onset of sepsis involves pathophysiological changes in multiple organs and systems, and different biomarkers show different behaviors in the pathophysiological process of sepsis. C-reactive protein (CRP), serum amyloid A (SAA), procalcitonin (PCT), interleukin 6 (IL-6) and D-dimer (DD) are closely related to the severity of sepsis, and can reflect the occurrence and development of systemic inflammatory response in patients with sepsis. They are important sepsis-related biomarkers. CRP is an important inflammatory marker of bacterial infection. When the body is under stress, the CRP level increases rapidly, and the degree of increase is closely related to the severity of sepsis. SAA is an important inflammatory marker. When the body responds to inflammation, the SAA level increases rapidly, which can reflect the occurrence and development of inflammatory response in patients with sepsis. PCT is an important biomarker of systemic infection, which can reflect the degree of systemic infection and has been widely used as an indicator for evaluating the efficacy of clinical infectious diseases and antibiotic management. IL-6 is an important cytokine with multiple biological activities. When local and systemic inflammatory responses occur in the body, the IL-6 level in the body begins to increase first, and a continuous increase can reflect the worsening of the patient's condition, with high sensitivity. DD is the simplest specific product of fibrin degradation. Its increased level reflects abnormal coagulation function of the body. It is an important biomarker for evaluating the severity of sepsis and the risk of prognosis and death.

[0004] At present, the commonly used clinical detection methods for CRP, SAA, PCT, IL-6 and DD markers are mainly immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold immunochromatography, etc. These methods are used for early diagnosis and prognosis evaluation of clinical sepsis. The detection index is single and lacks high specificity or sensitivity. If you want to avoid these problems, you need to perform a joint analysis of multiple markers for each sample. At present, multiplexed biomarker analysis is usually performed in clinical research laboratories. These experiments rely on precise experimental operations and large detection equipment. Commercial bedside detection equipment is still bulky, and only a limited number of protein multiplexed immunoassays have been verified in bedside detection equipment. Therefore, for the sepsis "biomarker group" composed of CRP, SAA, PCT, IL-6 and DD, it is of great clinical value and practical significance to study and develop a new rapid combined synchronous detection method for early diagnosis, disease monitoring and prognosis evaluation of sepsis that is rapid, simple, economical, highly sensitive, specific and portable.

[0005] Multi-channel electrochemical immunoassay technology is a high-throughput, high-sensitivity, high-specificity rapid analysis technology. It combines highly sensitive electrochemical sensing technology with highly specific immune reactions. Different antigen or antibody probes modified on the surface of multi-channel electrodes react specifically with the target to be detected in the sample, capture the target molecules in the sample, and then use multi-channel electrochemical sensors to perform highly sensitive simultaneous quantitative detection of multiple biomarkers. This technology only requires a small amount of sample and can achieve on-site simultaneous detection and analysis of multiple biomarkers within 1 hour. It has the advantages of small sample requirements, rapidity, simplicity, high sensitivity, high throughput, low cost, miniaturization, and portability. It is highly in line with the development needs of multi-biomarker joint detection technology in clinical biological specimens.

[0006] However, the application of the combined simultaneous detection of the above-mentioned markers in clinical bedside still faces a series of technical challenges: (1) Currently, there are few commercial portable multi-channel electrochemical workstations and they are expensive. It is obviously unreasonable to use a desktop electrochemical workstation to carry out bedside detection in a nursing ward with limited space. It is necessary to develop a new type of portable multi-channel electrochemical detection equipment; (2) The commonly used clinical reference concentrations of CRP are 1-500 μg / mL, SAA is 1-200 μg / mL, PCT is 0.1-100 ng / mL, IL-6 is 0.01-5 ng / mL, and DD is 0.1-10 μg / mL. The clinical reference concentrations of these markers vary greatly. To reach 7 orders of magnitude, it is necessary to balance multiple linear ranges to achieve joint and synchronous analysis; (3) Diluting samples according to the clinical concentrations of multiple targets not only relies on complex experimental operations and professional devices, but also is cumbersome, which is not friendly to non-experimental personnel. It is necessary to develop a new electrochemical immunosensor with simple equipment and simple operation for on-site joint and synchronous detection of multiple markers; (4) In on-site testing, when clinical samples cannot be precisely diluted, overcoming the non-specific adsorption of a large amount of protein in serum or plasma to the electrode and reducing the contamination of the electrode interface by biological fluids are important prerequisites for achieving stable analysis of electrochemical immunosensors in biological fluids, and anti-fouling methods need to be studied. Therefore, it is a major challenge to develop sensitive, specific, small and portable, simple to operate, economical and environmentally friendly CRP, SAA, PCT, IL-6, and DD multi-channel electrochemical immunosensors that meet the needs of bedside testing.

[0007] Electrochemical sensor integration technology is an integrated technology that combines electrochemical principles with microelectronic device manufacturing. This technology integrates the circuits of electrochemical sensor modules, test instruction modules, signal acquisition modules, and information communication modules to manufacture miniaturized, portable, and intelligent detection equipment for use in a variety of non-laboratory environments such as on-site detection. Screen printing technology is an important step in completing the preparation of sensors in integrated devices. Sensors are prepared by printing complex and detailed circuits and structures on metal and polymer materials. In addition, screen printing technology also has many advantages, mainly in the following aspects: (1) Wide applicability. Screen printing technology is used for paper printing, and can also be used on the surfaces of electronic components, glass, ceramics, textiles, plastic products, and other materials. (2) Diverse ink selection. Screen printing can use various types of inks, such as water-based, oil-based, UV-curable inks, fluorescent inks, conductive inks, etc., to meet specific printing requirements and achieve different product functions through the combination of inks; (3) Repeatability. Screen printing is an economical and efficient solution for large-scale product production. Once the plate is made, it can be printed quickly and repeatedly, which is very suitable for commercial production of products and is easy to realize automated assembly line operations. These advantages make screen printing technology occupy an important position in many fields such as electronics, solar energy, and medical treatment. Its high efficiency, precision, and repeatability make it an important technology in the field of analysis. At present, screen printing can integrate a variety of electrochemical immunoassay technologies and methods into economical and practical materials and commercial portable devices, and realize large-scale production and practical application research. Therefore, the development of screen printing technology has solved the problem of consumables cost for integrated electrochemical sensing technology, and has provided a strong theoretical and technical basis for the establishment of a new method for the on-site combined simultaneous detection of sepsis markers using multi-channel electrochemical immunosensors.

[0008] In addition, protein adsorption contamination in biological fluids is the biggest challenge in the on-site electrochemical detection of actual samples. The adsorption and clogging of protein interfering substances in biological fluids such as serum or plasma on the interface can affect the reproducibility and reliability of the data, and even mask the detection signal, resulting in sensor failure. Therefore, overcoming the problem of unstable electrochemical signals caused by actual samples is a major difficulty in the transformation of sensors from laboratory technology to the real world; at the same time, constructing an antifouling coating on the target recognition interface of electrochemical immunosensors is an important research direction in the field of electrochemical immunoassay. Unlike ELISA or immunofluorescence analysis, electrochemical immunosensors need to achieve the functions of antifouling and signal detection at the same interface. Insulating antifouling coatings constructed by physical adsorption or chemical deposition may reduce the sensitivity of electrochemical immunosensors. A study has developed a high-conductivity antifouling coating for electrochemical immunosensors based on a three-dimensional coating combining a conductive material skeleton with antifouling molecules, which can maintain the antifouling function of the electrode interface without compromising the electron transfer performance. However, the construction process of these composite antifouling coatings is often time-consuming and may take hours or even days, which hinders the mass production of electrochemical immunosensors. Therefore, overcoming the problem of electrochemical interface contamination by biological fluid samples will help develop a variety of electrochemical immunosensors suitable for real clinical samples.

[0009] In order to overcome the problems existing in the on-site electrochemical detection of biomarker groups, such as lack of on-site equipment, mismatch between detection performance and actual needs, and contamination of the sensor interface by clinical samples. The present invention proposes an integrated anti-fouling multi-channel electrochemical immunosensor for the joint and synchronous detection of sepsis biomarkers, which is used for the on-site joint and synchronous detection of CRP, SAA, PCT, IL-6, and DD levels in sepsis patients. First, a large-scale method is used to prepare a multi-channel electrochemical sensor; secondly, a highly sensitive three-dimensional anti-fouling coating is constructed by a bovine serum albumin / carbon nanotube / glutaraldehyde (BSA / CNT / GA) rapid thermal cross-linking method; then, the anti-fouling multi-channel electrochemical immunosensor is integrated into a portable electrochemical detection device to achieve on-site joint and synchronous detection of multiple markers. Based on the above-mentioned advantageous technologies, the developed integrated anti-fouling multi-channel electrochemical immunosensor has good sensitivity and specificity for five markers, CRP, SAA, PCT, IL-6 and DD, in multiple detection ranges of six orders of magnitude from 100 pg / mL to 100 μg / mL, and maintains a stable detection signal under 50% plasma or blood interference. At the same time, the method can accurately distinguish the CRP, SAA, PCT, IL-6 and DD levels of various simulated samples of normal people, non-bacterial infection patients, mild inflammation patients, non-infected patients, bacterial infection patients, sepsis patients with systemic inflammatory response, patients with poor prognosis of bacterial infection, and sepsis patients with poor prognosis of systemic inflammatory response. In addition, the integrated anti-fouling multi-channel electrochemical immunosensor can be used for on-site joint synchronous detection and remote data transmission, and the results of the desktop CHI1030C electrochemical workstation in clinical sample detection are basically consistent, and highly correlated with the ELISA results. The present invention can provide more complete information for the systemic inflammatory response state of patients with sepsis, and provide a more scientific and comprehensive theoretical basis for clinical diagnosis and treatment strategies. Summary of the invention

[0010] 1. The purpose of the present invention is to provide an integrated anti-fouling multi-channel electrochemical immunosensor for the combined simultaneous detection of sepsis biomarkers.

[0011] 2. The integrated anti-fouling multi-channel electrochemical immunosensor for the combined and synchronous detection of sepsis biomarkers described in the present invention adopts a large-scale method to prepare multi-channel electrodes, and is further modified to become a multi-channel electrochemical immunosensor with anti-biological fluid contamination function.

[0012] 3. The integrated anti-fouling multi-channel electrochemical immunosensor for the combined synchronous detection of sepsis biomarkers described in the present invention integrates the multi-channel electrochemical immunosensor with a portable electrochemical detection device to achieve on-site combined synchronous detection of multiple sepsis-related biomarkers such as CRP, SAA, PCT, IL-6, and DD.

[0013] 4. The integrated anti-fouling multi-channel electrochemical immunosensor for the combined and synchronous detection of sepsis biomarkers described in the present invention does not require additional operating steps during the combined and synchronous detection of multiple markers, and the time and manpower required to detect five markers are the same as those required to detect a single marker.

[0014] 5. The integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers described in the present invention comprises the following steps in sequence:

[0015] (1) Design and prepare multi-channel screen-printed electrodes;

[0016] (2) modifying an antifouling coating on the surface of a multi-channel electrode;

[0017] (3) modifying specific antibodies on the basis of a multi-channel electrode with an antifouling coating to prepare a multi-channel electrochemical immunosensor;

[0018] (4) assembling CRP, SAA, PCT, IL-6, and DD and their corresponding signal probes simultaneously;

[0019] (5) streptavidin-poly horseradish peroxidase (SA-pHRP);

[0020] (6) The multi-channel electrochemical immunosensor is connected to a detection device to perform electrochemical detection.

[0021] Advantages of the present invention:

[0022] Currently, there are few commercial integrated electrochemical detection devices, and most devices and multi-marker combined synchronous detection methods are still in the development stage. Based on new technologies such as electrochemical sensor integration technology, screen printing technology, and anti-fouling coating preparation technology, the present invention designs and manufactures a multi-channel electrochemical combined synchronous detection device entity for clinical field detection applications, providing important technical support for the development of subsequent field bedside diagnostic methods; at the same time, by integrating sepsis-related biomarker detection methods such as CRP, SAA, PCT, IL-6 and DD into the same solution system, the time and operation complexity required for the combined synchronous detection of multiple markers are simplified to the greatest extent, which is conducive to the subsequent development of equipment automation and large-scale production; in addition, unlike past retrospective clinical studies, the sepsis-related "biomarker group" information obtained through field detection technology is conducive to comprehensive and comprehensive prospective research on the progression of sepsis in patients, and helps promote the precise diagnosis and treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram of the preparation and on-site joint synchronous detection of an integrated antifouling multi-channel electrochemical immunosensor.

[0024] Figure 2 Design diagram of multi-channel screen-printed electrodes.

[0025] Figure 3 Schematic diagram for the fabrication of multi-channel screen-printed electrodes.

[0026] Figure 4 It is a three-dimensional model of the circuit board and circuit design schematic diagram of the portable electrochemical detection device, as well as the main interface and detection parameter setting interface of the device.

[0027] Figure 5 This is a real photo of the multi-channel screen-printed electrode.

[0028] Figure 6 Scanning electron microscope (SEM) images of various interfaces.

[0029] Figure 7 Figure 2 shows the results of cell adsorption tests on various interfaces.

[0030] Figure 8 These are the cyclic voltammetry test results for various interfaces.

[0031] Fig. 9 Electrochemical impedance spectroscopy (EIS) of channels 1 to 5 of the multichannel electrochemical immunosensor when assembled.

[0032] Fig.10 This is a schematic diagram of the current-time curve (it) of CRP detection in channel 1 and its result analysis.

[0033] Fig.11 It results for SAA, PCT, IL-6 and DD detection.

[0034] Fig.12 This study is a methodological investigation of CRP detection.

[0035] Fig.13 This study is a methodological investigation for SAA detection.

[0036] Fig.14 This is a methodological study on PCT detection.

[0037] Fig.15 This is a methodological investigation of IL-6 detection.

[0038] Fig.16 This study is a methodological investigation for DD detection.

[0039] Fig.17 The signal stability of channels 1-6 detected in different BSA concentration environments was investigated.

[0040] Fig.18 This is an investigation of the detection signals of channels 1-6 in plasma environments with different ratios.

[0041] Fig.19 The detection range of CRP, SAA, PCT, IL-6 and DD in 50% blood or 50% plasma was investigated.

[0042] Fig. 20 These are the combined simultaneous test results of nine plasma simulation samples.

[0043] Fig.21 The device required for on-site detection of the integrated anti-fouling multi-channel electrochemical immunosensor and the photo of the detection.

[0044] Fig. 22 Schematic diagram of the device structure of the portable electrochemical detection equipment.

[0045] Fig.23 This is the circuit diagram for the main control module, TTL-485 module and Bluetooth module of the portable electrochemical detection equipment.

[0046] Fig.24 This is the implementation circuit diagram of the current acquisition module, constant potential module, DAC module and ADC module of the portable electrochemical detection equipment.

[0047] Fig.25 The present invention is a circuit diagram for realizing a power management module of a portable electrochemical detection device, including a digital power supply module and an analog power supply module.

[0048] Fig.26 The detection range of an integrated antifouling multi-channel electrochemical immunosensor was investigated for on-site combined simultaneous detection of CRP, SAA, PCT, IL-6 and DD in 50% blood or 50% plasma.

[0049] Fig. 27 Comparison of CRP, SAA, PCT, IL-6 and DD results of plasma samples from patients with sepsis obtained using CHI660E electrochemical workstation and portable device.

[0050] Fig.28 The results of the integrated antifouling multi-channel electrochemical immunosensor detection of the present invention are compared with the ELISA results. DETAILED DESCRIPTION

[0051] In order to make the technical problem, technical solution and effect to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with embodiments and drawings.

[0052] The integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers described in the present invention is used for combined simultaneous detection of multiple sepsis-related biomarkers, and the specific operation steps are as follows:

[0053] (1) Preparation of instruments, reagents and solutions for testing:

[0054] The instruments used in this invention are from the following companies:

[0055]

[0056] The reagents used in this invention are from the following companies:

[0057]

[0058]

[0059] The solution used in the present invention is prepared as follows:

[0060] ①1 mol / L NaOH solution: weigh 0.4 mg of sodium hydroxide powder and dissolve it in 10 mL of ultrapure water.

[0061] ② Phosphate buffered saline (PBS, pH 7.4): Accurately weigh 2.9642 g NaH 2 PO 4 ·2H 2 O and 11.4988 g Na 2 HPO 4 Dissolve in 500 mL ultrapure water, adjust pH to 7.4 with 1 mol / L NaOH solution to obtain phosphate buffer stock solution. Take 20 mL of phosphate buffer stock solution and dilute it in 380 mL ultrapure water, add 2.3376 g NaCl and dissolve, and store at 4°C.

[0062] ③100 mg / mL BSA solution: Accurately weigh 1.000 g of BSA solid powder and dissolve it in PBS, store at 4°C and use within 7 days.

[0063] ④10 mg / mL BSA solution: dilute 1 mL of 100 mg / mL BSA solution in 9 mL PBS, store at 4°C and use within 7 days.

[0064] ⑤ Phosphate buffered saline Tween solution (PBST): dilute 200 μL of Tween 20 in 500 mL of PBS solution.

[0065] ⑥2-Morpholineethanesulfonic acid (MES) solution: Accurately weigh 0.2130 g of MES powder and dissolve it in 100 mL of ultrapure water. Adjust the pH to 6.0 with 1 mol / L NaOH solution as indicated by a pH meter and store at 4°C for later use.

[0066] ⑦1-Ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) solution: accurately weigh 0.1534 g EDC·HCl powder, dissolve it in 1 mL MES solution to prepare 0.8 mol / L EDC·HCl solution, dispense it into 25 μL / tube and store it at -20℃ for later use; accurately weigh 0.0230 mg NHS powder, dissolve it in 1 mL MES solution to prepare 0.2 mol / L NHS solution, dispense it into 25 μL / tube and store it at -20℃ for later use; when using, thaw 0.8 mol / L EDC·HCl solution and 0.2 mol / L NHS solution, mix them in a volume ratio of 1:1, and use them immediately.

[0067] ⑧5wt% glutaraldehyde GA solution: Take 100 μL of 50wt% GA solution, dilute it in 900 μL ultrapure water, and store it at 4°C away from light.

[0068] (2) Design and preparation of multi-channel screen-printed electrodes:

[0069] First, draw the conductive layer and the insulating layer separately on the drawing software. The specific plan is as follows: Figure 2 As shown in the figure, 6×6 identical conductive lines of multi-channel screen-printed electrodes are drawn in an A4-sized layer. Each multi-channel screen-printed electrode has eight conductive lines, corresponding to six working electrodes, one pseudo-reference electrode and one counter electrode. The starting points of the eight conductive lines are all at the bottom of the multi-channel screen-printed electrode and arranged with a center spacing of 2.54mm, so as to facilitate the integration of the electrodes into the portable device interface. The ends of all conductive lines are concentrated in a circular range with a diameter of 1cm, among which the conductive line of the pseudo-reference electrode is the first from the right, and the conductive line of the counter electrode is the fourth from the left. The end points of the two electrodes are both in the middle of the circular range. The end of the pseudo-reference electrode line is in a ring shape, and the end of the counter electrode line is in a circular shape with a diameter of 2mm. The ends of the conductive lines of the six working electrodes are all in a circular shape with a diameter of 2mm, distributed in six directions with the same distance from the pseudo-reference electrode. There is a 2cm×2cm square area at the upper left and lower right of the entire conductive layer A4 image, which is used to align the positions when the conductive layer and the insulating layer are printed successively.

[0070] The design of the insulating layer is to prevent the infiltration of liquid outside the sensor test area during the detection process. Draw 6×6 identical insulating layers of multi-channel screen-printed electrodes in an A4-sized layer. Each insulating layer of a multi-channel screen-printed electrode is rectangular, and a circular area with a diameter of 1 cm is hollowed out in the middle. The hollowed-out area just overlaps with the circular range where the ends of the eight conductive lines of the conductive layer are concentrated. The insulating layer does not cover the beginning and end of the conductive layer line. There is a 2cm×2cm square area at the upper left and lower right of the insulating layer, which is used to align the positions of the conductive layer and the insulating layer when printing the conductive layer and the insulating layer one after another.

[0071] After the design drawing is completed, the production of the screen printing plate is handed over to Shenzhen Deliou Screen Printing Equipment Co., Ltd. for customization. The screen printing plate is produced in accordance with Figure 3 Printing is performed according to the steps shown: first, a conductive layer screen printing stencil is fixed on a printing frame, and an A4-sized polyethylene terephthalate (PET) sheet is placed at the bottom of the conductive layer screen printing stencil, and the position of the PET sheet is adjusted so that the printing area is located in the middle of the sheet; then, conductive ink is added to the conductive layer screen printing stencil and evenly applied on the printing area of ​​the conductive layer screen printing stencil; then, the conductive layer screen printing stencil is pressed above the PET sheet, and a pointed bottom glue brush is pressed against the conductive layer screen printing stencil and moved in the printing area, while maintaining a certain downward pressure, so that the ink on the conductive layer screen printing stencil is squeezed onto the bottom PET sheet through the hollow part of the printing area, forming a conductive layer pattern of the multi-channel electrochemical immunosensor; finally, the PET sheet printed with the conductive layer is placed in a blast drying oven, and the conductive ink on the sheet is dried at 50°C to obtain a PET sheet printed with a conductive layer. The PET sheet is placed at the bottom of the insulating layer screen printing plate, and the position of the PET sheet is adjusted so that the two squares in the insulating layer printing area are aligned with the squares in the conductive layer of the PET sheet. The insulating green light-curing ink is added to the insulating layer screen printing plate and printed in the same way. The printed PET sheet is placed under a light-emitting diode (LED) light curing lamp at a distance of 20 cm for 1 minute. After the insulating layer is completely cured, the multi-channel screen-printed electrodes of the entire plate are cut into single pieces with a paper cutter for later use.

[0072] (3) Preparation of multi-channel electrochemical immunosensor:

[0073] ① Preparation of bovine serum albumin / carbon nanotube (BSA / CNT) complex: Take 2 mL of 1 mg / mL carbon nanotube (CNT) aqueous solution, mix it with an equal volume of 10 mg / mL BSA solution, ultrasonicate for 1 h, and react in a boiling water bath for 5 min to obtain a black liquid (bovine serum albumin / carbon nanotube (BSA / CNT) complex), which was stored at 4°C for later use.

[0074] ② Rapid assembly of bovine serum albumin / carbon nanotube / glutaraldehyde (BSA / CNT / GA) conductive antifouling coating: 10 μL of 5 wt% GA solution was mixed with 90 μL of BSA / CNT complex, and 1.5 μL was immediately dropped on each working electrode area in the multi-channel screen-printed electrode, reacted at 85 °C for 5 min, and then the BSA / CNT / GA coating-modified electrode was washed with ultrapure water and blown dry with nitrogen.

[0075] ③ Interface assembly of multi-channel electrochemical immunosensor: The interface assembly method of multi-channel electrochemical immunosensor for simultaneous detection of CRP, SAA, PCT, IL-6, and DD is consistent. The interface assembly process of CRP electrochemical immunosensor is used as an example. First, 2 μL of EDC / NHS solution was added to the CRP detection channel of the BSA / CNT / GA coated modified electrode, incubated at room temperature in the dark for 40 minutes, washed with PBS, and dried with nitrogen. Subsequently, 2 μL of CRP Ab was added. 1 PBS, incubate at room temperature in the dark for 45 minutes, wash with PBS, and blow dry the electrode with nitrogen. Finally, add 100 μL of 100 mg / mL BSA solution to all areas of the electrode, incubate at room temperature in the dark for 30 minutes, wash with PBS, blow dry with nitrogen to prepare the CRP electrochemical immunosensor, and store it in PBS at 4°C for later use. The SAA, PCT, IL-6 and DD electrochemical immunosensors were prepared in the same way, thereby preparing a multi-channel electrochemical immunosensor; CRP Ab in different channels in the multi-channel electrochemical immunosensor 1 、SAA Ab 1 、PCT Ab 1 IL-6Ab 1 and DD Ab 1 The concentrations were 250 μg / mL, 500 μg / mL, 250 μg / mL, 500 μg / mL and 500 μg / mL, respectively.

[0076] (4) Simultaneous detection of CRP, SAA, PCT, IL-6 and DD:

[0077] CRP Ab was added into the mixture using 10 mg / mL BSA solution. 2 、SAA Ab 2 、PCT Ab 2 IL-6Ab 2 and DD Ab 2The concentrations were prepared into 20μg / mL, 20μg / mL, 20μg / mL, 20μg / mL and 40μg / mL, and 5μL of each was mixed to make 25μL of premixed solution, which was then mixed with 25μL of the sample to be tested and added dropwise to the multi-channel electrochemical immunosensor to cover all channels. The samples were incubated at room temperature in the dark for 40 minutes, and the electrodes on each channel were washed with PBST and dried with nitrogen. Subsequently, 50μL of SA-pHRP solution was added dropwise to the electrode to cover all channels, incubated at room temperature in the dark for 10 minutes, and washed with PBST solution. Finally, 150μL of enhanced TMB solution was added dropwise to the electrodes on each channel to cover all channels, and used immediately for electrochemical detection.

[0078] (5) Electrochemical detection procedure:

[0079] When testing on the Chenhua CHI1030C electrochemical workstation, the multi-channel current-time curve (IT) method was used to simultaneously detect five markers, namely CRP, SAA, PCT, IL-6 and DD. The detection voltage was -0.3 V, the detection time was 100 s, the IT curves of six channels were collected, and the current value at 100 s was recorded.

[0080] When testing on a portable electrochemical device, the multi-channel IT method is also used for testing. The detection voltage is -0.3V, the detection channels are 1-6 channels, and the detection time is 100s. The final result is read out by the detection device or mobile terminal, and the IT curve data is stored by the detection device.

[0081] Figure 4 A in Figure 4 B in the figure represents the three-dimensional simulation diagram of the circuit board and the circuit design diagram. All electrodes have a corresponding single-chip microcomputer to execute input potential instructions and collect output voltage signals. The collected analog signals are amplified by operational amplifiers and converted into current signals through precision resistors. In addition, the detection equipment is designed with a connection module with a control panel and Bluetooth, which is used to apply potential instructions to the equipment and transmit the collected current signals during portable on-site detection. The main interface and parameter setting interface of the portable electrochemical detection equipment are shown in the figure. Figure 4 C in Figure 4As shown in D in the figure. The main interface shows that the detection method of the device is the time current method, and records the set detection time, detection potential, and real-time current readings of channels 1 to 6. Use the green button in the center below the main interface to start or pause the detection process. The device will automatically stop detecting after reaching the set detection time. In the parameter setting interface, you can set a variety of parameters including test time, test potential, sampling period, voltage range, detection channel, and data file name. The detection time can be set in the range of 0 to 1000s, the detection potential range is -2.0 to +2.0V, and the minimum sampling period is set to 0.3s / time. Up to 6 channels can be detected simultaneously, and the current range that the device can accurately record is 20nA to 20μA.

[0082] Figure 5 The actual picture of the multi-channel screen-printed electrode is shown. Through the screen printing technology, the conductive layer and the insulating layer are printed on the PET sheet in turn. The prepared single-piece electrode has a size of 4cm×2cm and a thickness of 0.1mm. At the same time, since the PET substrate is relatively soft, the prepared multi-channel electrochemical sensor has good flexibility and can be used to develop flexible multi-channel electrochemical sensors. In addition, each batch of customized printing plates can be used for more than 30 A4-sized PET sheets, that is, more than 1,000 multi-channel sensors can be prepared. The average cost of each electrode is less than 0.1 yuan, and it can still be produced using automated equipment, further reducing costs and having obvious price advantages.

[0083] Figure 6 The morphology of different coatings was shown by SEM. Figure 6 A in the figure is the prepared screen-printed electrode, which shows a large number of granular structures and is doped with flake structures. Figure 6 B in the figure is the BSA coating, and the interface is relatively smooth; Figure 6 C in the figure is the BSA / GA coating, which has many more pore structures than the BSA interface; Figure 6 D in the figure is a CNT coating with a large number of strip-like structures; Figure 6 E in the figure is the BSA / CNT coating, which is a relatively flat coating with strip-like structures and a small amount of pores in the middle; Figure 6 F in the figure is a BSA / CNT / GA coating, which is a three-dimensional network structure formed by stacking strip structures. The above results show that the addition of GA helps to form a pore structure in the BSA interface, CNT can serve as the skeleton of the three-dimensional coating, and only the simultaneous addition of CNT, BSA and GA can form a three-dimensional network with high porosity.

[0084] Figure 7The results of the antifouling performance of each coating were investigated. MCF-7 breast cancer cells were selected as the model cells for the anti-cell adhesion experiment. The unmodified electrode interface showed obvious fluorescence under the microscope after cell staining, indicating that there was significant cell adhesion on the electrode surface. The electrode modified with only CNT coating had a small amount of fluorescence, while all electrodes modified with BSA had only a small amount of fluorescence, indicating that the BSA coating played an important role in the formation of the antifouling coating and significantly reduced cell adhesion.

[0085] Figure 8 Figure A in the figure shows the cyclic voltammetry test results of various coatings, which are used to compare their electron transfer performance. The multi-channel SPCE of the unmodified coating shows obvious oxidation peaks and reduction peaks, but the potential difference between the two peaks is as high as 0.906V; while the oxidation-reduction peak potential difference of the electrode modified with CNT coating is reduced to 0.665V; in addition, no obvious redox signal appears at the interface modified with BSA coating, BSA / GA coating, and BSA / CNT coating; while the oxidation peak-reduction peak potential difference of the BSA / CNT / GA coating electrode interface is 0.803V. The above results show that CNT is the main factor in increasing the conductivity of the interface, and BSA is the main factor in inhibiting the conductivity of the interface, and the inhibitory effect of BSA on the interface signal is more obvious than the conductivity enhancement effect of CNT, which may be due to the accumulation of BSA on the interface to form a dense structure, thereby inhibiting the electron transfer efficiency of the interface, which is consistent with the SEM observation results of the BSA / CNT interface. When GA is added to the BSA / CNT / GA coating, BSA forms a network cross-linking to prevent excessive accumulation, making the BSA / CNT / GA coating have good electrical conductivity and excellent antifouling properties. Figure 8 Figure B shows the results of CV detection of the electrode modified with BSA / CNT / GA coating after incubation in 10 mg / mL BSA solution or plasma for 1 hour. The CV curves before and after incubation are basically the same, indicating that the two solutions have no significant effect on the electrochemical behavior of the BSA / CNT / GA coating electrode. Therefore, the BSA / CNT / GA coating has good anti-interference ability and is suitable for the construction of subsequent multi-channel electrochemical immunosensors.

[0086] Fig. 9 This is the electrochemical impedance spectrum (EIS) of the antifouling multi-channel electrochemical immunosensor assembly process. A to E in the figure are the assembly results of CRP, SAA, PCT, IL-6, and DD channels, respectively. Since the EIS change trend caused by the assembly of multiple immunosensor interfaces is consistent, only the CRP assembly process is taken as an example for analysis. The results show that the impedance of the printed electrode is about 11kΩ, and the electrode impedance remains almost unchanged after the BSA / CNT / GA coating is assembled; CRP Ab 1After assembly, the impedance value increased by about 13kΩ; after BSA blocking, the impedance value increased to about 15kΩ; CRP Ab 2 The impedance value increased to about 19 kΩ after one-step assembly with CRP, and the impedance value increased to about 24 kΩ after SA-pHRP assembly. The above results show that the CRP electrochemical immunosensor was successfully assembled. Since the assembly process of SAA, PCT, IL-6 and DD is the same as that of CRP, it is considered that the antifouling multi-channel electrochemical immunosensor was successfully assembled.

[0087] Fig.10 The "hook effect" in CRP detection was investigated. When there is no target, the sensor shows a current of about 0.4μA, which is the background signal for CRP detection; when the CRP concentration is 0.5μg / mL, the sensor shows a response current of about 3μA; and when the CRP concentration is as high as 10μg / mL, the sensor shows a response current of only 2μA, which is higher than the background current but lower than the response current of 0.5μg / mL CRP. The results show that the "hook effect" exists in the clinical concentration range of CRP detection, and the signal intensity is negatively correlated with the CRP concentration. Therefore, it is proposed to detect CRP using the negative correlation between signal and concentration in the "hook effect" area.

[0088] Fig.11 The results of the feasibility analysis of electrochemical detection of SAA, PCT, IL-6 and DD. A to D in the figure are the results of SAA, PCT, IL-6 and DD, respectively. Taking SAA as an example for discussion, when there is no target, the electrochemical sensor shows a current of about 0.15μA, which is the background signal for SAA detection, while the sensor shows a response current of about 1.8μA to 10μg / mL SAA, indicating that the electrochemical sensor is feasible for SAA detection. Similarly, the blank group and target group of PCT detection showed response currents of about 0.15μA and about 4.5μA, respectively, the blank group and target group of IL-6 detection showed response currents of about 0.4μA and about 2.2μA, respectively, and the blank group and target group of DD detection showed response currents of about 0.5μA and about 2.0μA, respectively. The above results show that the anti-fouling multi-channel electrochemical immunosensor is feasible for the combined and simultaneous detection of CRP, SAA, PCT, IL-6 and DD.

[0089] Fig.12 The sensitivity, specificity and reproducibility of CRP detection were investigated. Fig.12A in the figure is the IT result of CRP in the concentration gradient, where (a) to (h) are the IT results of 0, 100, 50.0, 25.0, 10.0, 5.00, 1.00 and 0.500 μg / mL CRP. In the range of 0.500 to 100 μg / mL, the reduction current signal decreases with the increase of CRP concentration. The four-parameter logistic function equation is fitted through the current-concentration relationship diagram to obtain Fig.12 The B curve in the figure shows that the sensor current and CRP concentration show a good curve relationship. 2 =0.998, the detection limit is 1.40 μg / mL, which falls within the clinical range of CRP (1-500 μg / mL). Fig.12 As shown in C in the figure, the CRP concentration is 0.5 μg / mL, and the interferents include 10 μg / mL SAA, 50 ng / mL PCT, 10 ng / mL IL-6, and 0.5 μg / mL DD. The biomarker concentrations are based on the levels of sepsis patients. The electrochemical immunosensor only produces a significant signal when CRP is present in the sample, and only produces a weak background signal for other interferents. In addition, the reproducibility of CRP detection is shown in Fig.12 As shown in D, the response currents of multiple test groups to 0.5 μg / mL CRP were basically consistent, with a relative standard deviation (RSD) of 1.93%. The above results show that the CRP electrochemical immunosensor has good sensitivity, specificity and reproducibility, and meets the clinical detection needs.

[0090] Fig.13 The sensitivity, specificity and reproducibility of SAA detection were investigated. Fig.13 A in the figure is the IT result of SAA with concentration gradient, and (a) to (j) are the IT results of 0, 0.250, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0, 50.0 and 100 μg / mL SAA. In the range of 0.500 to 100 μg / mL, the reduction current increases with the increase of SAA concentration. The four-parameter logistic function equation is fitted through the current-concentration relationship diagram to obtain Fig.13 As shown in the curve B in the figure, the sensor current and SAA concentration have a good curve relationship, R2 = 0.999, and the detection limit is 0.345 μg / mL, which falls within the clinical range of SAA (1-200 μg / mL). At the same time, the SAA detection specificity analysis results are shown in Fig.13As shown in C, the SAA concentration is 10 μg / mL, and the concentration of interferents is based on the level of sepsis patients, including 10 μg / mL CRP, 50 ng / mL PCT, 10 ng / mL IL-6, and 0.5 μg / mL DD. The electrochemical immunosensor only produces a significant signal when SAA is present in the sample, and only a weak background signal is generated for other interferents. In addition, the reproducibility of SAA detection is shown in Fig.13 As shown in D, the response currents of multiple test groups to 10 μg / mL SAA are basically consistent, with an RSD of 2.82%. The above results show that the SAA electrochemical immunosensor has good sensitivity, specificity and reproducibility, and meets the needs of clinical detection.

[0091] Fig.14 The sensitivity, specificity and reproducibility of PCT detection were investigated. Fig.14 A in the figure is the IT result of concentration gradient PCT, where (a) to (j) are the IT results of 0, 0.250, 0.500, 1.00, 2.50, 5.00, 10.0, 20.0, 40.0 and 100 ng / mL PCT. In the concentration range of 0.250-100 ng / mL, the reduction current increases with the increase of PCT concentration. The four-parameter logistic function equation is fitted through the current-concentration response relationship diagram to obtain Fig.14 As shown in the curve B in the figure, the sensor current has a good curve relationship with the SAA concentration, R 2 =0.980, the detection limit is 0.882 ng / mL, which falls within the PCT clinical range (0.1-100 ng / mL). Fig.14 As shown in C, the PCT concentration is 10 ng / mL, and the concentration of interferents is based on the level of sepsis patients, including 10 μg / mL CRP, 10 μg / mL SAA, 10 ng / mL IL-6, and 0.5 μg / mL DD. The electrochemical immunosensor only produces a significant signal when the sample contains PCT, and only produces a weak background signal for other interferents. In addition, the reproducibility of PCT detection is shown in Fig.14 As shown in D, the response currents of multiple test groups to 10 ng / mL PCT were basically consistent, with an RSD of 2.39%. The above results show that the PCT electrochemical immunosensor has good sensitivity, specificity and reproducibility, and meets the clinical detection needs.

[0092] Fig.15 The sensitivity, specificity and reproducibility of IL-6 detection were investigated. Fig.15A in the figure is the IT result of IL-6 with concentration gradient, where (a) to (k) are the IT results of 0, 0.0125, 0.0250, 0.0625, 0.125, 0.250, 0.625, 1.25, 2.50, 5.00 and 10.0 ng / mL IL-6. In the concentration range of 0.0125 to 10.0 ng / mL, the reduction current increases with the increase of IL-6 concentration. The four-parameter logistic function equation is fitted through the current-concentration response relationship diagram to obtain Fig.15 As shown in the curve B in the figure, the sensor current has a good curve relationship with the IL-6 concentration, R 2 =0.972, the detection limit is 0.0225 ng / mL, which falls within the clinical range of IL-6 (0.01-5 ng / mL). Fig.15 As shown in C, the IL-6 concentration is 5 ng / mL, and the concentration of interfering substances is based on the level of sepsis patients, including 10 μg / mL CRP, 10 μg / mL SAA, 50 ng / mL PCT, and 0.5 μg / mL DD. The electrochemical immunosensor only produces a significant signal when the sample contains IL-6, and only produces a weak background signal for other interfering substances. In addition, the reproducibility results of IL-6 detection are shown in Fig.15 As shown in D, the response currents of multiple test groups to 5 ng / mL IL-6 were basically consistent, with an RSD of 3.74%. The above results show that the IL-6 electrochemical immunosensor has good sensitivity, specificity and reproducibility, and meets the clinical detection needs.

[0093] Fig.16 The sensitivity, specificity and reproducibility of DD detection were investigated. Fig.16 A is the IT result of concentration gradient DD, where (a) to (j) are the IT results of 0, 0.0050, 0.010, 0.025, 0.10, 0.25, 0.50, 1.0, 2.0 and 5.0 μg / mL DD. In the concentration range of 0.0250-5.00 μg / mL, the reduction current increases with the increase of DD concentration. The four-parameter logistic function equation is fitted through the current-concentration response relationship diagram to obtain Fig.16 As shown in the curve B in the figure, the sensor current has a good curve relationship with the DD concentration, R 2 =0.972, the detection limit is 0.0618 μg / mL, which falls within the DD clinical range (0.1-10 μg / mL). Fig.16As shown in C, the DD concentration is 0.5 μg / mL, and the concentration of interferents is based on the level of sepsis patients, including 10 μg / mL CRP, 10 μg / mL SAA, 50 ng / mL PCT, and 10 ng / mL IL-6. The results show that the electrochemical immunosensor produces a significant signal only when the sample contains DD, and only a weak background signal is generated for other interferents. In addition, the reproducibility results of DD detection are shown in Fig.16 As shown in D, the response currents of multiple test groups to 0.5 μg / mL DD were basically consistent, with an RSD of 2.34%. The above results show that the DD electrochemical immunosensor has good sensitivity, specificity and reproducibility, and meets the clinical detection needs.

[0094] Fig.17 Using bovine serum albumin as an interfering protein, the detection performance of the multi-channel electrochemical immunosensor under high protein concentration was investigated. A to E in the figure are CRP, SAA, PCT, IL-6, and DD channels, respectively. The specific detection concentrations are 100ng / mL CRP, 10μg / mL SAA, 10ng / mL PCT, 2.5ng / mL IL-6, and 2μg / mL DD. The detection results of the target group without BSA were set to 1, and the data were normalized. When the BSA concentration increased, the response signals of all markers did not decay significantly, and even increased slightly, while the background signal remained stable. In addition, in order to prevent the anti-fouling illusion that may be caused by the reduction of recognition efficiency and probe adsorption events, we added a BSA channel as a reference on the basis of the 5 marker channels to monitor the non-specific adsorption of marker proteins at the interface. The results are shown in the figure. Fig.17 As shown in F, with the increase of BSA concentration, the BSA interface signal did not change significantly, and the target group and blank group signals were very close, indicating that potential non-specific adsorption events of the target in the electrochemical sensor can be ignored. The total protein concentration of human plasma is about 75 mg / mL, which is lower than the 100 mg / mL BSA concentration. This result proves that the prepared anti-fouling multi-channel electrochemical immunosensor has excellent anti-protein adsorption performance.

[0095] Fig.18The traditional BSA coating was compared with the BSA / CNT / GA coating to investigate the effect of plasma content on marker detection. A to E in the figure are CRP, SAA, PCT, IL-6, and DD, respectively. Considering that the CRP signal detection mode is different from that of other markers, 0.5μg / mL CRP was used as the blank group, 50μg / mL CRP was used as the target group, and the background signal of the non-plasma group was used as the normalization benchmark. The concentrations of other markers were set to 10μg / mL CRP, 10μg / mL SAA, 10ng / mL PCT, 5ng / mL IL-6, and 2μg / mL DD, and the target signal of the non-plasma group was used as the normalization benchmark. In the BSA / CNT / GA interface, the detection signal showed a gradual decrease with the increase of plasma content. In the complete plasma sample, the signal of all markers was attenuated to about 70% of the plasma-free group; while in the BSA coating, the detection signal showed a trend of first increasing and then decreasing with the change of the target concentration. At the same time, the signal of the BSA / CNT / GA coating was higher than that of the BSA / GA coating. When the plasma concentration was 100%, the response signals of the BSA coating group for CRP, SAA, PCT, IL-6 and DD were only 37.8%, 11.6%, 14.6%, 12.0% and 19.3% of those of the BSA / CNT / GA coating group, respectively. The signal attenuation phenomenon may be caused by the interference of complex components such as proteins or ionic salts in plasma on the immune recognition process, as well as the contamination of the sensor interface by plasma. In addition, in the BSA channel, the signal of the BSA / CNT / GA coating group did not change significantly in plasma, while the signal of the BSA coating group attenuated with the increase of plasma concentration. This indicates that plasma has limited effect on the electron transfer of the BSA / CNT / GA coating, and is more likely to significantly interfere with the antigen-antibody recognition process. However, the BSA channel is still contaminated in high-concentration plasma, which excludes the possibility of signal interference caused by nonspecific adsorption of markers. The above results can fully demonstrate the great advantages of BSA / CNT / GA coating in balancing plasma antifouling and conductivity.

[0096] Fig.19The detection performance of the sensor in plasma or whole blood was investigated. Multiple targets were detected simultaneously in 50% plasma and 50% blood. Figures A to E are the current-concentration relationship diagrams for CRP, SAA, PCT, IL-6 and DD detection, respectively. The results showed that in 50% plasma samples, the detection range of all markers did not change significantly, and the detection limits of CRP, SAA, PCT, IL-6 and DD were 2.14μg / mL, 0.275μg / mL, 0.212ng / mL, 0.121ng / mL and 0.110μg / mL, respectively. In 50% blood samples, the change trend of the current-concentration relationship diagram was basically consistent with that in 50% plasma, and the detection limits of CRP, SAA, PCT, IL-6 and DD were 0.804μg / mL, 0.635μg / mL, 0.191ng / mL, 0.166ng / mL and 0.584μg / mL, respectively. The dotted lines in the figure indicate the clinical diagnostic reference thresholds of each biomarker after dilution, and all dotted lines are within the detection range of the immunosensor. The above results show that the anti-fouling multi-channel electrochemical immunosensor can effectively and simultaneously detect multiple biomarkers in 50% plasma or 50% blood containing high-concentration proteins. The sensitivity of all biomarkers meets the clinical reference thresholds, indicating that the sensor is suitable for direct or appropriately diluted detection of clinical samples.

[0097] Fig. 20 With 50% plasma as the detection background, the combined synchronous detection performance of the multi-channel electrochemical immunosensor was investigated by adding different combinations of standard products. A to I in the figure are blank group, SAA group, IL-6 group, DD group, CRP+SAA group, CRP+SAA+PCT group, CRP+SAA+DD group, CRP+SAA+PCT+IL-6 group, CRP+SAA+PCT+IL-6+DD group, a total of 9 simulation combinations. Among them, the SAA group is used to simulate non-bacterial infection, the IL-6 group is used to simulate early inflammation, the DD group is used to simulate thrombosis, the CRP+SAA group is used to simulate bacterial infection, the CRP+SAA+PCT group is used to simulate severe systemic inflammatory response or sepsis, and the CRP+SAA+PCT+IL-6 and CRP+SAA+PCT+IL-6+DD groups are used to simulate sepsis with poor prognosis. The results showed that in the blank group, the antifouling multi-channel electrochemical immunosensor only showed an obvious response signal in the CRP channel, which came from the low concentration of CRP in plasma and was consistent with the Fig.19After adding SAA, IL-6 and DD respectively, the corresponding channels produced response signals, while the signals of other channels were not affected, which indicates that the signal crosstalk between multiple channels is low. In the combined detection of CRP and SAA, changes in other channel signals were also observed. At this time, the SAA channel signal was similar to that of the SAA group alone, while the CRP signal was attenuated, which is consistent with the "hook effect" detection theory. Similarly, when three to five marker combinations are detected simultaneously, significant current responses are only generated in their respective corresponding channels. There are certain differences in the current values ​​of each channel, which is mainly due to the different affinities of each probe for the target. At the same time, we also found that the current response values ​​of each channel in the combination are consistent with Fig.19 The current responses of the various markers described in the above are similar. The above results fully demonstrate the excellent performance of the anti-fouling multi-channel electrochemical immunosensor in actual sample detection, and can provide strong support for the diagnosis and identification of diseases through the joint and synchronous detection of multiple biomarkers.

[0098] Fig.21 A portable electrochemical detection device designed and manufactured by the present invention is demonstrated, which includes a power supply, a circuit board, a human-computer interaction interface, and a mobile terminal for signal reception. The mobile terminal can be any brand of mobile phone for daily use. The size of the circuit board part is 10cm×10cm, and the display screen can be stacked with the circuit board, so that the device takes up very little space when not in use. In addition, the screen-printed electrode has been designed according to the requirements of the integrated circuit interface, so that it can be easily integrated into the circuit for multi-channel joint synchronous detection.

[0099] Figure 22-25The schematic diagram of the integrated circuit in the invention includes a main control module, a TTL-485 module, a Bluetooth module, a current acquisition module, a constant potential module, a DAC module, an ADC module, a digital power supply module and an analog power supply module, and a complete system framework for electrochemical detection is constructed. The core of the main control module is composed of an STM32F405 microcontroller, which is responsible for coordinating the communication and data processing of each module. The power management module (including a digital power supply module and an analog power supply module) provides a stable power input for the entire system to ensure the normal operation of each module. As a detection end, a multi-channel electrochemical immunosensor is connected to a constant potential module to complete the collection of electrochemical signals. The constant potential module receives a voltage signal from the main control module through a DAC module to keep the sensor working in a constant potential state. At the same time, the current acquisition module connects a multi-channel electrochemical immunosensor for collecting current signals during the detection process, and converts the analog signal into a digital signal through an ADC module and transmits it to the main control module for processing. The system also integrates a TTL-485 module and a Bluetooth module for realizing wired or wireless communication with external devices and mobile terminals. The mobile terminal can receive detection data through a Bluetooth module and realize remote control functions. In addition, the human-computer interaction interface is connected to the TTL-485 module through the main control module, providing key operation and display feedback, which is convenient for users to monitor and control the system operation in real time. The overall system completes the whole process from data acquisition, processing to transmission through the collaborative work between modules, which is suitable for efficient data monitoring and management in electrochemical detection scenarios.

[0100] Fig.26 In order to combine the portable electrochemical detection equipment with the antifouling multi-channel electrochemical immunosensor, an integrated antifouling multi-channel electrochemical immunosensor was constructed for the simultaneous detection of multiple markers in 50% plasma and 50% blood. A to E in the figure are the results of CRP, SAA, PCT, IL-6 and DD, respectively. The detection range of the markers based on the integrated sensor did not change significantly. In 50% plasma, the detection limits of CRP, SAA, PCT, IL-6 and DD were 1.77μg / mL, 61.6ng / mL, 0.188ng / mL, 15.9pg / mL and 0.174μg / mL, respectively. In 50% blood, the detection limits of these markers were 12.6μg / mL, 0.119μg / mL, 0.250ng / mL, 72.4pg / mL and 5.70ng / mL, respectively. The above results indicate that the integrated antifouling multi-channel electrochemical immunosensor can maintain highly sensitive simultaneous detection of multiple biomarkers under high protein interference, and the detection limit is consistent with the clinical reference threshold, which can be used for on-site detection of clinical samples.

[0101] Fig. 27The results of the combined simultaneous detection of biomarkers in plasma samples from 7 patients with sepsis using an integrated anti-fouling multi-channel electrochemical immunosensor are compared with the results detected by Chenhua CHI1030C. A to E in the figure are the results of CRP, SAA, PCT, IL-6 and DD, respectively. These results show that the levels of multiple markers obtained by the two devices are highly consistent.

[0102] Fig.28 To show Fig.24 The correlation analysis of the combined simultaneous detection results of biomarkers and ELISA results is shown in the figure. A to E in the figure are the results of CRP, SAA, PCT, IL-6 and DD respectively. The detection results of the five markers are highly consistent with the ELISA results, and the correlation coefficients are: R 2 =0.991, SAA's R 2 =0.975, PCT R 2 =0.888, R of IL-6 2 =0.899 and DD's R 2 =0.912, which indicates that the two methods have equal value in medical diagnosis.

Claims

1. An integrated antifouling multi-channel electrochemical immunosensor for the combined simultaneous detection of sepsis biomarkers, characterized in that: The method is prepared by the following steps: (1) designing and preparing a multi-channel screen-printed electrode; (2) modifying an antifouling coating on the surface of the multi-channel screen-printed electrode; (3) modifying specific antibodies on the basis of the multi-channel screen-printed electrode with the antifouling coating to prepare a multi-channel electrochemical immunosensor; (4) simultaneously assembling signal probes corresponding to C-reactive protein (CRP), serum amyloid A (SAA), procalcitonin (PCT), interleukin 6 (IL-6) and D-dimer (DD); (5) coupling the signal probes with streptavidin-poly horseradish peroxidase (SA-pHRP); and (6) connecting the multi-channel electrochemical immunosensor to a detection device for electrochemical detection.

2. An integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to claim 1, characterized in that: The multi-channel electrode was prepared by screen printing technology, and after anti-fouling coating cross-linking and antibody modification, a multi-channel electrochemical immunosensor with anti-biological liquid pollution function was prepared.

3. An integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to claim 1 or 2, characterized in that: The multi-channel electrochemical immunosensor was integrated with the portable electrochemical detection device to realize the on-site joint and synchronous detection of multiple sepsis-related biomarkers such as CRP, SAA, PCT, IL-6, and DD.

4. An integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to any one of claims 1 to 3, characterized in that: A complete portable system framework for electrochemical detection was constructed; the system framework included a main control module, a TTL-485 module, a Bluetooth module, a current acquisition module, a constant potential module, a DAC module, an ADC module, and a power management module. The power management module included a digital power supply module and an analog power supply module. A complete system framework for electrochemical detection was constructed; the core of the main control module was composed of an STM32F405 microcontroller, which was responsible for coordinating the communication and data processing of each module; the power management module provided a stable power input for the entire system to ensure the normal operation of each module. The multi-channel electrochemical immunosensor was used as the detection end and was connected to the constant potential module to complete the acquisition of electrochemical signals. The constant potential module received the voltage signal from the main control module through the DAC module to keep the multi-channel electrochemical immunosensor working at Constant potential state; at the same time, the current acquisition module is connected to the multi-channel electrochemical immunosensor to collect the current signal during the detection process, and converts the analog signal into a digital signal through the ADC module and transmits it to the main control module for processing; the complete system framework of electrochemical detection also integrates the TTL-485 module and the Bluetooth module to realize wired or wireless communication with external devices and mobile terminals. The mobile terminal can receive the detection data through the Bluetooth module and realize the remote control function. The human-computer interaction interface is connected to the TTL-485 module through the main control module, providing button operation and display feedback, which is convenient for users to monitor and control the system operation in real time. The modules in the complete system framework of electrochemical detection can work together to complete the whole process from data acquisition, processing to transmission, which is suitable for efficient data monitoring and management in electrochemical detection scenarios.

5. An integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to any one of claims 1 to 4, characterized in that: When multiple markers are detected simultaneously, there are no additional operation steps, and the time and manpower required to detect 5 markers are the same as those for detecting a single marker.

6. An integrated anti-fouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to any one of claims 1 to 5, characterized in that: The assay has good sensitivity, specificity and reproducibility for the five markers CRP, SAA, PCT, IL-6 and DD in multiple detection ranges of six orders of magnitude from 100 pg / mL to 100 μg / mL.

7. A method for preparing an integrated antifouling multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers, characterized in that: The specific steps are as follows: (1) Design and preparation of multi-channel screen-printed electrodes: First, the conductive layer and the insulating layer were drawn separately on the drawing software. In the A4-sized layer, 6×6 identical conductive lines of the multi-channel screen-printed electrodes were drawn. Each multi-channel screen-printed electrode had eight conductive lines, corresponding to six working electrodes, one pseudo-reference electrode and one counter electrode. The starting ends of the eight conductive lines were all installed at the bottom of the multi-channel screen-printed electrode and arranged at a center spacing of 2.54 mm, so as to facilitate the integration of the electrode into the portable device interface. The ends of all the conductive lines were concentrated in a circular range with a diameter of 1 cm, among which the conductive line of the pseudo-reference electrode was the first one from the right, and the conductive line of the counter electrode was the fourth one from the left. The end points of the two electrodes were both in the middle of the circular range. The end of the pseudo-reference electrode line was in a ring shape, and the end of the counter electrode line was in a circular shape with a diameter of 2 mm. The ends of the conductive lines of the six working electrodes were all in a circular shape with a diameter of 2 mm, distributed in six directions with the same distance from the pseudo-reference electrode. There was a 2 cm×2 cm square area at the upper left and lower right of the A4 image of the entire conductive layer, which was used to align the positions when the conductive layer and the insulating layer were printed successively. The design of the insulating layer is to prevent the liquid from infiltrating outside the sensor test area during the detection process. 6×6 identical insulating layers of multi-channel screen-printed electrodes are drawn in an A4-sized layer. The insulating layer of each sensor is rectangular, and a circular area with a diameter of 1 cm is hollowed out in the middle. The hollowed-out area just overlaps with the circular range where the ends of the eight conductive lines of the conductive layer are concentrated; the insulating layer does not cover the beginning and end of the conductive layer line; there is a 2 cm×2 cm square area at the upper left and lower right of the insulating layer, which is used to align the positions when the conductive layer and the insulating layer are printed successively; After the design drawing is completed, the screen printing plate is printed according to the following steps: first, the conductive layer screen printing plate is fixed on the printing frame, and an A4-sized polyethylene terephthalate (PET) sheet is placed at the bottom of the screen printing plate. The position of the PET sheet is adjusted so that the printing area is located in the middle of the sheet; then, the conductive ink is added to the screen printing plate and evenly applied on the printing area; then, the screen printing plate is pressed above the PET sheet, and the pointed bottom glue brush is pressed against the screen printing plate and moved on the printing area while maintaining a certain downward pressure, so that the ink on the screen printing plate is squeezed onto the bottom PET sheet through the hollow part of the printing area, forming a conductive layer pattern of the multi-channel electrochemical immunosensor; finally, the PET sheet printed with the conductive layer is placed in a blast drying oven for 50 ℃ drying the conductive ink on the sheet to obtain a PET sheet with a printed conductive layer; placing the PET sheet at the bottom of the insulating layer screen printing plate, adjusting the position of the PET sheet so that the two squares of the insulating layer printing area of ​​the insulating layer screen printing plate are aligned with the squares of the conductive layer of the PET sheet, adding insulating green light-curing ink to the insulating layer screen printing plate, and printing in the same way, placing the printed PET sheet under a light-emitting diode (LED) light-curing lamp at a distance of 20 cm for 1 min, and after the insulating layer is completely cured, using a paper cutter to cut the multi-channel screen-printed electrodes of the entire sheet into single pieces for standby use; (2) Preparation of multi-channel electrochemical immunosensor: ① Preparation of bovine serum albumin / carbon nanotube (BSA / CNT) complex: Take 2 mL of 1 mg / mL carbon nanotube (CNT) aqueous solution, mix it with an equal volume of 10 mg / mL bovine serum albumin (BSA) solution, ultrasonicate for 1 h, and react in a boiling water bath for 5 min to obtain a black liquid, which is stored at 4 °C for later use; ② Rapid assembly of bovine serum albumin / carbon nanotube / glutaraldehyde (BSA / CNT / GA) conductive antifouling coating: 10 μL of 5wt% glutaraldehyde (GA) solution was mixed with 90 μL of BSA / CNT complex, and 1.5 μL was immediately dripped on each working electrode area of ​​the multi-channel screen-printed electrode, reacted at 85 °C for 5 min, and then the electrode was washed with ultrapure water to obtain the BSA / CNT / GA coating modified electrode, and then dried with nitrogen; ③ Interface assembly of multi-channel electrochemical immunosensor: The interface assembly method of the multi-channel electrochemical immunosensor for simultaneous detection of SAA, PCT, IL-6, and DD is consistent with that of the multi-channel electrochemical immunosensor for CRP detection. The assembly process of the multi-channel electrochemical immunosensor for CRP detection is as follows: First, 2 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS) solution was added to the CRP detection channel of the BSA / CNT / GA coated modified electrode, incubated at room temperature in the dark for 40 min, washed with phosphate buffered saline (PBS), and dried with nitrogen gas; Subsequently, 2 μL of CRP capture antibody in PBS solution was added, incubated at room temperature in the dark for 45 min, and the electrode was washed with PBS and dried with nitrogen gas; Finally, 100 μL of 100 mg / mL BSA solution was added to all areas of the electrode, incubated at room temperature in the dark for 30 min, washed with PBS, and dried with nitrogen to prepare the CRP electrochemical immunosensor, which was stored in PBS at 4 °C for later use. The SAA, PCT, IL-6 and DD electrochemical immunosensors were prepared in the same way, thus preparing a multi-channel electrochemical immunosensor; the concentrations of the capture antibody of CRP, the capture antibody of SAA, the capture antibody of PCT, the capture antibody of IL-6 and the capture antibody of DD in different channels of the multi-channel electrochemical immunosensor were 250 μg / mL, 500 μg / mL, 250 μg / mL, 500 μg / mL and 500 μg / mL, respectively; (3) Simultaneous detection of CRP, SAA, PCT, IL-6 and DD: Use 10 mg / mL BSA solution to prepare the concentrations of biotin-labeled signal antibodies of CRP, SAA, PCT, IL-6, and DD to 20 μg / mL, 20 μg / mL, 20 μg / mL, 20 μg / mL, and 40 μg / mL, respectively, and take 5 μL of biotin-labeled signal antibodies to mix the five biotin-labeled signal antibodies to prepare 25 μL of premixed solution, which is then mixed with 25 μL of the sample to be tested and dropped onto the multi-channel electrochemical immunosensor to cover all channels, incubate at room temperature in the dark for 40 min, wash the electrodes with phosphate buffered saline Tween solution, and blow dry with nitrogen gas; Then, 50 μL of streptavidin-polyhorseradish peroxidase solution was dripped on the electrodes of all channels to cover all channels, incubated at room temperature in the dark for 10 min, and washed with phosphate buffered saline Tween solution; finally, 150 μL of enhanced 3,3',5,5'-tetramethylbenzidine (TMB) solution was dripped on the electrodes of all channels to cover all channels, and immediately used for electrochemical detection; (4) Electrochemical detection procedure: When testing on the Chenhua CHI1030C electrochemical workstation, the multi-channel current-time curve (it) method was used to simultaneously detect five markers, CRP, SAA, PCT, IL-6, and DD. The detection voltage was −0.3 V, the detection time was 100 s, the it curves of six channels were collected, and the current value at 100 s was recorded; When testing on a portable electrochemical device, the multi-channel IT method is also used for detection. The detection voltage is −0.3 V, the detection channels are 1-6 channels, and the detection time is 100 s. The final result is read out by the detection device or mobile terminal, and the IT curve data is stored by the detection device.

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