3D printing assembly type multi-channel electrochemical immunosensor for combined synchronous detection of sepsis biomarkers

Abstract

The invention discloses a 3D printing assembly type multi-channel electrochemical immunosensor for combined synchronous detection of sepsis biomarkers. The assembled conductive and non-conductive electrochemical biosensor substrate is prepared through a 3D printing technology, the operation steps are shortened by adopting a one-step identification strategy, the step complexity in operation is simplified, and the electrochemical biosensor is prepared by utilizing the low-crosstalk advantage of interface catalysis. The 3D printing assembled multi-channel electrochemical biosensor for combined synchronous detection of multiple sepsis biomarkers such as C-reactive protein, serum amyloid protein A, D-dimer and fibronectin is constructed, has the advantages of simplicity and convenience in operation, short time consumption, high sensitivity, high specificity and the like, can be used for clinical accurate, efficient and rapid sepsis diagnosis, and has a wide application prospect. A new thought is provided for joint detection of other biomarkers, and research and development of disease-related biomarker group detection are promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and more specifically, relates to a 3D printed assembled multi-channel electrochemical immunosensor for the combined synchronous detection of sepsis biomarkers. The constructed 3D printed multi-channel electrochemical biosensor is expected to be used for the rapid combined detection of sepsis-related biomarkers, promote the development of biomarker group research, and provide new ideas for the combined detection research of other disease-related biomarkers. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by the dysregulation of the body's response to infection. Its condition changes rapidly, with a high degree of severity and fatality rate, and it is one of the major diseases seriously threatening human health. Early diagnosis and accurate assessment of the treatment effect and quality of life of sepsis patients are crucial. The combined synchronous detection of sepsis biomarkers can timely, scientifically, and accurately monitor the evolution process of the physiological and pathological changes of individual patients, not only enabling the early rapid diagnosis of sepsis, the monitoring of the severity of the condition, and the effective assessment of the prognosis, but also guiding the formulation of individualized precise treatment plans for sepsis patients.

[0003] The pathogenesis of sepsis involves pathophysiological changes in multiple organs and systems, and different biomarkers show different manifestations in the pathophysiological process of sepsis. C-reactive protein (CRP), serum amyloid A (SAA), D-dimer (D-D), and fibronectin (FN) are closely related to the severity of sepsis, can reflect the occurrence and development process of the systemic inflammatory response in sepsis patients, and are important sepsis-related biomarkers. CRP is an important inflammatory biomarker for bacterial infections. When the body is in a stress state, the CRP level rapidly increases, and the increase amplitude is closely related to the severity of sepsis. SAA is an important inflammatory biomarker. The SAA level rapidly increases during the body's inflammatory response and can reflect the occurrence and development process of the inflammatory response in sepsis patients. D-D is the simplest specific fibrin degradation product, and its increased level reflects abnormal coagulation function in the body, which is an important biomarker for evaluating the severity of sepsis and the risk of prognostic death. FN is a high-molecular glycoprotein that participates in multiple physiological processes and is closely related to the severity of sepsis. It can be used as a supplement to the aforementioned indicators to effectively monitor the changes in the severity of sepsis and evaluate the prognosis. In addition, the combined detection of multiple biomarkers can improve the diagnostic and prognostic evaluation value of sepsis and help reduce the mortality of sepsis. For example, CRP and SAA have different sensitivities to non-bacterial infections and can be used to diagnose potential non-bacterial sepsis. However, during the treatment of patients, the plasma CRP or SAA of patients may always be at a relatively high level, and they are not sensitive in terms of the disease progression and prognostic evaluation of sepsis. In contrast, D-D and FN have good indications for the disease progression and poor prognosis of sepsis and can make up for the deficiencies of the first two biomarkers in prognostic evaluation. Therefore, the rapid, synchronous, and quantitative detection of the biomarkers CRP, SAA, D-D, and FN helps improve the treatment effect and quality of life of sepsis patients.

[0004] Currently, the commonly used detection methods for the biomarkers CRP, SAA, D-D, and FN in clinical practice mainly include immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence method, colloidal gold immunochromatography, etc. These methods are used for the early diagnosis and prognostic evaluation of clinical sepsis, but the detection indicators are single, lacking high specificity or sensitivity. If we want to avoid these problems and perform a combined analysis of multiple biomarkers for each sample, the cost is high and the cycle is long. At the same time, the above methods also rely on laboratory techniques or large-scale detection equipment, which are not user-friendly for non-laboratory personnel and are not conducive to the immediate bedside detection of sepsis patients. In summary, it is of great practical significance to develop a new method for the rapid, synchronous detection of the early diagnosis, disease monitoring, and prognostic evaluation of sepsis for CRP, SAA, D-D, and FN, which is rapid, simple, economical, highly sensitive, highly specific, and portable.

[0005] Electrochemical immunoassay technology is a rapid analysis technology with high sensitivity and high specificity. It combines highly sensitive electrochemical sensing technology with highly specific immune reactions. Through specific recognition reactions between antigen or antibody probes modified on the electrode surface and the target analytes in the sample, the target molecules in the sample are captured, and then the electrochemical sensor is used to highly sensitively quantify the biomarkers. However, the inclusion of multiple biomarkers will increase the operational complexity of the detection process, resulting in increased detection costs and workloads, making electrochemical immunosensors face similar challenges to traditional independent detection methods during the detection process. Therefore, there is a need to develop simpler and more efficient new methods for simultaneous detection of multiple biomarkers.

[0006] 3D printing, also known as rapid prototyping technology and additive manufacturing, is a cutting-edge technology that integrates digital modeling, mechatronics control, information technology, materials science, and chemistry. Its basic principle is as follows: First, a digital model is constructed and digitally layered - physically laminated, so that the three-dimensional model is divided into multiple single-layer structures, and the processing path and track information are set according to the model characteristics. Then, through methods such as fused deposition, stereolithography, or electron beam melting, the printed object is finally obtained. Compared with traditional processing methods, 3D printing technology has many advantages, mainly reflected in: (1) High design freedom. Traditional manufacturing methods have high restrictions on products. For example, there are strict requirements for processes such as product demolding and cutting, and many additional factors need to be considered during design, which is extremely inconvenient. 3D printing avoids these problems through layer-by-layer manufacturing, providing extremely high design freedom for device manufacturing and allowing very complex geometries to be easily created. (2) Strong mechanical repeatability. 3D printing can rapidly manufacture devices on a large scale, and the overall dimensional tolerances of multiple batches of products are within 0.05 - 2.5 mm, eliminating the differences caused by manual manufacturing. (3) Short cycle. Through 3D printing, "design is production" can be achieved. For a model with a complex structure, it only takes a few hours from design export to printing and production completion, greatly accelerating the design verification and iteration process, while traditional manufacturing processes may take several days or weeks to complete the production of a prototype. (4) Energy-saving and environmentally friendly: Based on the "net shaping" manufacturing of 3D printing, it is more environmentally friendly than traditional manufacturing industries. Almost all raw materials are used in the manufactured devices, and waste can be reduced to 90%. These characteristics of 3D printing technology provide important technical support for the heuristic design of on-site analysis technology and equipment manufacturing. In the initial stage of the development of new methods and new devices for simultaneous detection of multiple biomarkers, the advantages of 3D printing technology such as customized design, rapid prototyping production, and timely parameter optimization can be utilized to quickly obtain multi-channel electrochemical immunosensors that meet the experimental research requirements, thus contributing to the innovation and development of the technology for simultaneous electrochemical immunoanalysis of multiple biomarkers.

[0007] To overcome the problems of cumbersome operation steps and long time consumption in the synchronous detection of multiple sepsis biomarkers, the present invention designs and develops a 3D printed assembled multi-channel electrochemical immunosensor for the synchronous detection of sepsis biomarkers through 3D printing, and establishes a new method for the synchronous detection of sepsis markers CRP, SAA, D-D and FN based on the 3D printed assembled multi-channel electrochemical immunosensor. Through multiple immunoassays, one-step assembly and parallel operation, the operation process of synchronous detection of multiple markers is simplified. The signal is amplified by the catalytic oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) by bridged streptavidin-labeled polyhorseradish peroxidase (SA-pHRP) and detected on a multi-channel electrochemical sensor. The constructed sensor is successfully used for the synchronous detection of CRP, SAA, D-D and FN in simulated and real samples, and is highly correlated with the results of ELISA method, meeting the clinical detection requirements for these biomarkers. Summary of the Invention

[0008] 1. The object of the present invention is to provide a 3D printed assembled multi-channel electrochemical immunosensor for the synchronous detection of sepsis biomarkers.

[0009] 2. The 3D printed assembled multi-channel electrochemical immunosensor for the synchronous detection of sepsis biomarkers according to the present invention is prepared by designing and preparing a multi-channel electrode from scratch through a full 3D printing mode, further modifying a variety of specific probes on the surfaces of different channel electrodes as target recognition components, and using SA-pHRP catalyzing TMB as a bridged electrochemical signal amplification medium, which can realize the capture and synchronous detection of CRP, SAA, D-D and FN in the sample. It is characterized in that it is prepared by the following method, including the following steps: (1) Preparation of 3D printed assembled multi-channel electrode: First, design the three-dimensional diagrams of the multi-channel electrode module, reference module and detection cell module, then make them by fused deposition 3D printing, and further assemble them into a multi-channel electrode; (2) Preparation of 3D printed assembled multi-channel electrochemical immunosensor: Through the steps of electrochemical deposition of gold and specific probe coupling, immunosensing interfaces for C-reactive protein (CRP), serum amyloid A (SAA), D-dimer (D-D) and fibronectin (FN) are respectively prepared on different channels; (3) Synchronous detection of CRP, SAA, D-D and FN: The target substances in the sample are rapidly assembled with CRP, SAA, D-D and FN antibodies respectively by the double antibody sandwich one-step assembly method, and then the horseradish peroxidase of bridged streptavidin is further used, and the synchronous detection is realized based on the detection mode of the multi-channel electrochemical immunosensor, and the reaction of catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide is carried out on the electrode surface to generate a reduction current signal.

[0010] Preferably, according to the physicochemical properties of CRP, SAA, D-D and FN, antibody probe pairs capable of simultaneously binding to the target are set. Based on the low interference characteristics of the sandwich immunoassay method and the advantages of the one-step assembly method, by simultaneously operating multiple-channel immunosensors, the multiple and repetitive operation steps in the detection are greatly reduced, and the combined synchronous detection of CRP, SAA, D-D and FN in plasma samples is realized.

[0011] Preferably, during the detection process, the linear range of CRP is 0.125 - 5.00 μg / mL, and the detection limit is 0.115 μg / mL; the linear range of SAA is 0.250 - 10.0 μg / mL, and the detection limit is 0.106 μg / mL; the linear range of D-D is 0.125 - 5.00 μg / mL, and the detection limit is 0.100 μg / mL; the linear range of FN is 5 - 500 ng / mL, and the detection limit is 3.14 ng / mL, which conforms to the reference value range of the four biomarkers in clinical detection.

[0012] 3. The 3D printing assembled multi-channel electrochemical immunosensor for the combined synchronous detection of sepsis biomarkers of the present invention simplifies the operation process of the combined synchronous detection of multiple biomarkers through the one-step assembly and parallel operation of multiple immunoassays, reduces the repetitive operation steps during the detection of multiple biomarkers, and realizes the detection of multiple biomarkers in one operation.

[0013] 4. The preparation method of the 3D printing assembled multi-channel electrochemical immunosensor for the combined synchronous detection of sepsis biomarkers of the present invention includes the following steps:

[0014] (1) Design a model through drawing software and fabricate physical modules through a 3D printer, including a multi-channel electrode module, a reference module, and a detection cell module;

[0015] (2) Modify nano-gold on the surface of the multi-channel electrode module by electrochemical reduction method, and respectively bind CRP, SAA, D-D and FN probes on different electrodes to prepare a multi-channel electrochemical immunosensor;

[0016] (3) First, perform BSA blocking pretreatment on the detection cell module, and then place the test sample and the biotinylated signal antibody (Ab 2 ) in the Ab 2 pool, and assemble the multi-channel electrochemical immunosensor into the Ab 2 pool for incubation;

[0017] (4) During the incubation in step (3), SA-pHRP is added to the SA-pHRP pool of the detection cell module in (3). After the incubation in step (3) is completed, the multi-channel electrochemical immunosensor is washed and assembled into the SA-pHRP pool for incubation;

[0018] (5) During the incubation in step (4), TMB is added to the TMB pool of the detection cell module in (3), and the reference module prepared in step (1) is assembled. After step (4) is completed, the multi-channel electrochemical immunosensor is washed and assembled into the TMB pool for electrochemical detection;

[0019] (6) Electrochemical detection uses the current-time curve (i-t) method.

[0020] Preferably, its manufacturing method sequentially includes the following specific steps:

[0021] (1) Design and fabrication of a 3D printed assembled multi-channel electrochemical immunosensor: First, use 123D software to design the three-dimensional models of each component and save them in stl format; then slice the three-dimensional models layer by layer, use JGcreat 2.5.0 to establish the printing sequence, and save the generated program in gcode format; then upload the gcode file to a 3D printer for automatic printing; finally, assemble the fabricated components according to the design scheme; The 3D printed multi-channel electrochemical immunosensor includes 3D printed electrode components, convex components, concave components, reference module support components and detection cell components, which are assembled into a multi-channel electrode module, a reference module and a detection cell module after printing;

[0022] (2) Interface assembly of a 3D printed assembled electrochemical immunosensor for the combined synchronous detection of CRP, SAA, D-D and FN: The assembly methods of the SAA, D-D and FN electrochemical immunosensor interfaces are the same as those of the CRP electrochemical immunosensor interface. The assembly process of the CRP electrochemical immunosensor interface is as follows: The 3D printed electrode is polished on coarse and fine sandpapers with an alumina (Al 2 O 3 ) suspension and then ultrasonically treated in ethanol and ultrapure water for 30 s each, and washed with ultrapure water; The 3D printed electrode is immersed in a 2.8 mmol / L chloroauric acid (HAuCl 4 ) solution, and nano-gold is deposited using the current-time curve (i-t) method, with a scanning voltage of -0.2 V and a scanning time of 500 s; The 3D printed electrode is washed with ultrapure water and placed in 0.5 mol / L sulfuric acid (H 2 SO 4)CV scans were performed in the solution with a scanning potential of 0 - 1.5 V, a scanning rate of 1 V / s, and 60 scanning segments; the electrode was washed with ultrapure water and dried with nitrogen. Then, 3 μL of a 10 mmol / L phosphate buffered saline (PBS) solution containing 100 μg / mL of the capture antibody for CRP (CRP Ab 1 ) was dropped onto the electrode surface, and incubated in the dark at room temperature for 16 h with the container sealed; the 3D printed electrode was washed with 10 mmol / L PBS and immersed in 100 μL of a 10 mg / mL BSA solution, and incubated at room temperature for 30 min. After washing with 10 mmol / L PBS, the CRP electrode was formed and set aside. The electrochemical immunosensor interfaces for SAA, D-D, and FN were assembled in the same way as that for the CRP electrochemical immunosensor interface. After being assembled using the capture antibody for SAA (SAA Ab1), the capture antibody for D-D (D-D Ab1), and the capture antibody for FN (FN Ab1) respectively, the SAA electrode, D-D electrode, and FN electrode were formed and set aside;

[0023] (3) Simultaneous detection of CRP, SAA, D-D, and FN: The simultaneous detection methods for SAA, D-D, and FN are the same as that for CRP. The CRP detection method is as follows: all areas in the detection cell module were soaked in a 10 mg / mL BSA solution for 30 min and dried with nitrogen; 5 μL of the sample was mixed with 5 μL of a 10 mg / mL BSA solution containing 20 μg / mL of the biotinylated signal antibody for CRP (CRP Ab 2 ) and added to the CRP detection Ab 2 pool in the detection cell module; the multi-channel electrochemical immunosensor was aligned with the position of the Ab 2 pool and assembled into the Ab 2Incubate in the pool at room temperature for 50 min. Remove the multi-channel electrochemical immunosensor, wash it with 10 mmol / L PBS solution, and dry it with nitrogen. Take 10 μL of a 10 mg / mL BSA solution containing 0.5 μg / mL SA-pHRP and add it to the SA-pHRP pool. Align the washed multi-channel electrochemical immunosensor with the position of the SA-pHRP pool and assemble it into the SA-pHRP pool in the detection cell module. Incubate at room temperature for 30 min. Remove the multi-channel electrochemical immunosensor, wash it with 10 mmol / L PBS solution, and dry it with nitrogen. Take 400 μL of enhanced TMB solution and add it to the TMB pool. Align the washed multi-channel electrochemical immunosensor with the position of the TMB pool and assemble it into the TMB pool in the detection cell module. Immerse the CRP electrode obtained in step (2) into the solution of the TMB pool. At the same time, assemble the reference module to the detection cell module and detect using a CHI1030C multi-channel potentiostat. The simultaneous detection method for SAA, D-D, and FN is the same as the simultaneous detection method for CRP, and the biotinylated signal antibodies for SAA (SAA Ab 2 ), D-D (D-D Ab2), and FN (FN Ab2) are used respectively;

[0024] (4) Electrochemical detection parameters: Simultaneously detect CRP, SAA, D-D, and FN by multi-channel i-t method. The detection voltage is -0.2 V, the detection time is 100 s, collect the i-t results, and record the current value at 100 s.

[0025] Advantages of the present invention:

[0026] Currently, there are few reports on similar simultaneous electrochemical detection methods and physical devices for CRP, SAA, D-D, and FN. The present invention takes advantage of the design and manufacturing advantages of 3D printing technology to design from scratch a physical entity of a multi-channel electrochemical simultaneous detection device, providing a basic version for the subsequent expansion and optimization of detection methods. In addition, the present invention simplifies the operation process of simultaneous detection of multiple biomarkers through multiple immunoassay one-step assembly parallel operation, and realizes the detection of multiple biomarkers in one operation. The simultaneous detection of CRP, SAA, D-D, and FN based on the same device evaluates the patient's condition from multiple perspectives of sepsis diagnosis and prognosis, helps to form a combined evaluation thinking of disease biomarkers, and promotes the formation of a simultaneous detection method for a "biomarker group" related to sepsis. Description of the Drawings

[0027] Figure 1 It is a schematic diagram of the preparation of the 3D printed multi-channel electrochemical immunosensor of the present invention and the simultaneous detection of CRP, SAA, D-D, and FN.

[0028] Figure 2 This is the design diagram of the 3D printed electrode of the present invention.

[0029] Figure 3 This is the design diagram of the support component of the 3D printed reference module of the present invention.

[0030] Figure 4 This is the design diagram of the 3D printed convex component of the present invention.

[0031] Figure 5 This is the design diagram of the 3D printed concave component of the present invention.

[0032] Figure 6 This is the design diagram of the 3D printed detection cell of the present invention.

[0033] Figure 7 These are the physical diagrams of all 3D printed components and the physical diagram after assembly.

[0034] Figure 8 These are the electrochemical impedance spectroscopy (EIS) characterizations of the assembly of all channel electrodes.

[0035] Figure 9 These are the i-t characterizations of the detection of corresponding targets for all channels.

[0036] Figure 10 These are the methodological investigations for the detection of CRP in channel 1.

[0037] Figure 11 These are the methodological investigations for the detection of SAA in channel 2.

[0038] Figure 12 These are the methodological investigations for the detection of D-D in channel 3.

[0039] Figure 13 These are the methodological investigations for the detection of FN in channel 4.

[0040] Figure 14 These are the results of the combined synchronous detection of a biomarker combination simulation sample using a 3D printed assembled multi-channel electrochemical immunosensor.

[0041] Figure 15 These are the results of the combined synchronous detection of biomarkers in real clinical plasma samples using a 3D printed assembled multi-channel electrochemical immunosensor.

[0042] Figure 16 These are the correlation analyses of the biomarker detection results of a 3D printed assembled multi-channel electrochemical immunosensor and the biomarker results measured by the ELISA method. Detailed implementation manners

[0043] To make the technical problems, technical solutions and effects to be solved by the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments and drawings.

[0044] A 3D printed assembled multi-channel electrochemical immunosensor for the simultaneous detection of sepsis biomarkers, which is used for the simultaneous detection of multiple sepsis-related biomarkers. The specific operation steps are as follows:

[0045] (1) Preparation of instruments, reagents and solutions for detection:

[0046] All the instruments used in the present invention are from the following companies:

[0047]

[0048] All the reagents used in the present invention are from the following companies:

[0049]

[0050]

[0051] The solutions used in the present invention are prepared as follows:

[0052] ① 10 mmol / L phosphate buffered saline (PBS, pH 7.4): Weigh 2.9642 g of NaH 2 PO 4 ·2H 2 O and 11.4988 g of Na 2 HPO 4 and dissolve them in 500 mL of ultrapure water. Adjust the pH to 7.4 with NaOH solution to obtain the mother liquor of phosphate buffer. Take 20 mL of the mother liquor of phosphate buffer and dilute it in 380 mL of ultrapure water. Add 2.3376 g of NaCl and dissolve it, and store it at 4°C.

[0053] ② 10 mg / mL BSA solution: Weigh 1.000 g of BSA solid powder and dissolve it in 10 mL of PBS to obtain a 100 mg / mL BSA solution. Take 1 mL of the 100 mg / mL BSA solution and dilute it in 9 mL of 10 mmol / L PBS. The BSA solution is stored at 4°C and used within 7 days.

[0054] ③ 0.5 mol / L H 2 SO 4 solution: Take 10.86 mL of H 2 SO 4 and dilute it in 400 mL of ultrapure water.

[0055] ④ 2.8 mmol / L HAuCl 4Solution: Dissolve 1 g of HAuCl 4 powder in 55.9 mL of 0.5 mol / L H 2 SO 4 solution to obtain 50 mmol / L HAuCl 4 solution. Take 1 mL of 50 mmol / L HAuCl 4 solution and dilute it in 16.8 mL of 0.5 mol / L H 2 SO 4 solution, and store it in the dark at 4 °C for later use.

[0056] (2) Design and fabrication of 3D printed assembled multi-channel electrochemical immunosensor: First, use 123D software to design the 3D models of each component and save them in stl format; then slice the 3D models layer by layer, use JGcreat 2.5.0 to establish the printing sequence, and save the generated program in gcode format; then upload the gcode file to the 3D printer for automatic printing; finally, assemble the fabricated components according to the design scheme.

[0057] The 3D printer works basically as follows:

[0058]

[0059] The 3D printed multi-channel electrochemical immunosensor includes 3D printed electrode components, convex components, concave components, reference module support components and detection cell components, and their 3D engineering drawings are respectively as Figure 2-6 shown. After printing, they are assembled into a multi-channel electrode module, a reference module and a detection cell module.

[0060] (3) Interface assembly of 3D printed assembled electrochemical immunosensor for the combined synchronous detection of CRP, SAA, D-D and FN: The interface assembly methods of CRP, SAA, D-D and FN electrochemical immunosensors are the same. The interface assembly process is illustrated by taking the interface assembly process of the CRP electrochemical immunosensor as an example. Polish the 3D printed electrode on coarse and fine sandpapers with Al 2 O 3 suspension and then polish it, and ultrasonically clean it in ethanol and ultrapure water for 30 s in sequence, and wash it with ultrapure water. Immerse the 3D printed electrode in 2.8 mmol / L HAuCl 4 solution, and deposit nano-gold using the i-t method, with a scanning voltage of -0.2 V and a scanning time of 500 s. Wash the 3D printed electrode with ultrapure water and place it in 0.5 mol / L H 2 SO 4Cyclic voltammetry (CV) scanning was performed in the solution with a scanning potential of 0 - 1.5 V, a scanning rate of 1 V / s, and 60 scanning segments. The electrode was washed with ultrapure water and dried with nitrogen. Then, 3 μL of a 10 mmol / L PBS solution containing 100 μg / mL CRP Ab 1 was dropped onto the electrode surface and incubated in the dark at room temperature for 16 h with the container sealed. The 3D printed electrode was washed with 10 mmol / L PBS and immersed in 100 μL of a 10 mg / mL BSA solution and incubated at room temperature for 30 min, then washed with 10 mmol / L PBS to obtain a CRP electrode for standby. Similarly, SAA Ab 1 , D-D Ab 1 and FN Ab 1 were modified on the electrode surface using the aforementioned method to obtain SAA electrodes, D-D electrodes and FN electrodes for standby.

[0061] (4) Simultaneous detection of CRP, SAA, D-D and FN: The simultaneous detection methods for CRP, SAA, D-D and FN are the same. Taking the CRP detection method as an example. All areas in the detection cell were soaked with 10 mg / mL BSA for 30 min and dried with nitrogen. 5 μL of the sample was mixed with 5 μL of a 10 mg / mL BSA solution containing 20 μg / mL CRP Ab 2 and added to the Ab 2 pool for CRP detection. The multi-channel electrochemical immunosensor (CRP electrode) was aligned with the position of the Ab 2 pool and assembled into the Ab 2 pool in the detection cell module, incubated at room temperature for 50 min, then the multi-channel electrochemical immunosensor (CRP electrode) was removed, washed with 10 mmol / L PBS solution and dried with nitrogen. 10 μL of a 10 mg / mL BSA solution containing 0.5 μg / mL SA-pHRP was added to the SA-pHRP pool. From the Ab 2Align the cleaned multi-channel electrochemical immunosensor (CRP electrode) from the pool with the position of the SA-pHRP pool in the detection cell module, assemble it into the SA-pHRP pool in the detection cell module, incubate at room temperature for 30 min, remove the multi-channel electrochemical immunosensor (CRP electrode), and wash it with 10 mmol / L PBS solution, then dry it with nitrogen. Take 400 μL of enhanced TMB solution and add it to the TMB pool. Align the cleaned multi-channel electrochemical immunosensor (CRP electrode) from the SA-pHRP pool with the position of the TMB pool, and assemble the cleaned multi-channel electrochemical immunosensor (CRP electrode) from the SA-pHRP pool into the TMB pool in the detection cell module, immerse the CRP electrode in the solution of the TMB pool. At the same time, assemble the reference module (containing silver / silver chloride electrode) into the detection cell module and detect it using a CHI1030C multi-channel potentiostat. Similarly, obtain the situation where the SAA electrode, D-D electrode, and FN electrode are immersed in the solution of the TMB pool, and at the same time, assemble the reference module (containing silver / silver chloride electrode) into the detection cell module and detect it using a CHI1030C multi-channel potentiostat;

[0062] (5) Electrochemical detection parameters: Simultaneously detect CRP, SAA, D-D, and FN by multi-channel i-t method, the detection voltage is -0.2 V, the detection time is 100 s, collect the i-t results, and record the current value at 100 s.

[0063] Figure 2 For the unified 3D-printed electrode style, all electrodes are in an "L" shape structure, the electrode length is 13.75 mm, the width is 5 mm, and the area for the electrochemical immunosensor interface is a 2 mm × 3 mm rectangular bottom surface.

[0064] Figure 3 The size of the reference module support part is 14 mm × 12 mm × 7 mm, and two rectangular channels of 2 cm × 3 cm are designed in the middle of the support part.

[0065] Figure 4 The size of the convex module is 30 mm × 16.75 mm × 8 mm, and there is a cube protrusion of 4 mm × 4 mm × 3 mm at each of its left and right ends, and four concave areas are set in the middle for 3D-printed electrode assembly.

[0066] Figure 5 The size of the concave module is 30 mm × 16.75 mm × 4 mm, and there is a cube defect of 4 mm × 4 mm × 3 mm at each of its left and right ends, and four concave areas are also set in the middle. The concave module and Figure 4 The convex module are fitted together through the complementary parts at both ends to form four rectangular channels of 2 mm × 3 mm for electrode assembly and fixation for 3D-printed electrode assembly.

[0067] Figure 6 The detection cell module has dimensions of 52mm × 35mm × 13mm. The cell body includes four Ab 2 cells, four SA-pHRP cells, and one TMB cell. Each Ab 2 cell and SA-pHRP cell is a cubic space of 4mm × 3mm × 3.75mm, used to hold liquid during incubation and detection; on each of the left and right sides of each cell, a cubic space of 8mm × 3mm × 3.75mm is designed for assembly with a multi-channel electrochemical immunosensor.

[0068] Figure 7 In [it], A is a solid diagram of all components prepared by 3D printing, and B is the multi-channel electrode module, reference module, and detection cell module assembled after further cutting and polishing of these components. These results indicate that the detection device is reasonably designed and successfully fabricated.

[0069] Figure 8 In [it], A is the EIS of the PLA electrode. The semicircle width is the electrode resistance value, and the impedance of this electrode is about 15 kΩ; Figure 8 In [it], B - E are the EIS characterizations of the assembled CRP, SAA, D - D, and FN channels respectively. Since they have the same trend, B in Figure 8 is taken as an example for analysis. In Figure B, a - e are the EIS results of depositing nano-gold, assembling Ab 1 , blocking BSA, one-step assembling the target and Ab 2 , and assembling SA-pHRP in sequence. Nano-gold has conductivity, making the electrode impedance significantly smaller than that of PLA. With the assembly of non-conductive protein substances, the electrode impedance gradually increases. These results indicate the successful assembly of the CRP, SAA, D - D, and FN channels.

[0070] Figure 9 In [it], A - D are the detection feasibility analyses of the CRP, SAA, D - D, and FN electrochemical immunosensors in sequence. Figure 9 In A of [it], the CRP electrochemical immunosensor shows a background signal of 300 nA for the blank group and a reduction response signal of about 8000 nA for the 2.5 μg / mL CRP target group; Figure 9 In B of [it], the SAA electrochemical immunosensor shows a background signal of 1000 nA for the blank group and a reduction response signal of about 13500 nA for the 10 μg / mL SAA target group; Figure 9 In C of [it], the D - D electrochemical immunosensor shows a background signal of 500 nA for the blank group and a reduction response signal of about 4500 nA for the 5.0 μg / mL D - D target group; Figure 9In D, the FN electrochemical immunosensor showed a background signal of approximately 300 nA for the blank group and a reduction response signal of approximately 15000 nA for the 0.5 μg / mL FN target group. The above results indicate that the detection of CRP, SAA, D-D, and FN electrochemical immunosensors based on the one-step method is feasible.

[0071] Figure 10 The detection sensitivity, specificity, and reproducibility of the CRP electrochemical immunosensor were investigated. Figure 10 In A, the i-t results of the concentration gradient CRP are shown. In the range of 0.125 - 5.00 μg / mL, the reduction current signal increased with the increase in CRP concentration. Further fitting the response current-concentration relationship, a linear relationship as shown in Figure 10 B was obtained, and the linear equation was I (μA) = 2.22C CRP (μg / mL) + 3.36, and the detection limit was 0.115 μg / mL (≥3σ). Figure 10 In C, the specificity analysis results of the CRP electrochemical immunosensor are shown. The CRP concentration was 2.5 μg / mL, and the interferents included 50 μg / mL SAA, 50 ng / mL PCT, 1 ng / mL IL-6, and 0.5 μg / mL D-D. The interferent concentrations refer to the plasma biomarker levels of sepsis patients. The immunosensor only obtained a significant response signal in the test group containing CRP, while a weak background signal was obtained in other interferent groups. Figure 10 In D, the detection reproducibility of the CRP electrochemical immunosensor was investigated by performing multiple repeated detections of 2.5 μg / mL CRP in different batches. The response currents of multiple test groups to the target were basically the same, and the RSD was 4.51%. These results indicate that the CRP electrochemical immunosensor has good sensitivity, specificity, and reproducibility and can obtain stable detection results.

[0072] Figure 11 The detection sensitivity, specificity, and reproducibility of the SAA electrochemical immunosensor were investigated. Figure 11 In A, the i-t results of the concentration gradient SAA are shown. In the range of 0.250 - 10.0 μg / mL, the reduction current signal increased with the increase in SAA concentration. Further fitting the response current-concentration relationship, a linear relationship as shown in Figure 11 B was obtained, and the linear equation was I (μA) = 1.31C SAA (μg / mL) + 1.22, and the detection limit was 0.106 μg / mL (≥3σ). Figure 11C in it is the specific analysis result of the SAA electrochemical immunosensor. Among them, the SAA concentration is 5.0 μg / mL, and the interferents include 50 μg / mL CRP, 50 ng / mL PCT, 1 ng / mL IL-6, and 0.5 μg / mL D-D. The interferent concentrations refer to the plasma biomarker levels of sepsis patients. The immunosensor only obtained a sensitive response signal in the test group containing SAA, while a weak background signal was obtained in other interferent groups. Figure 10 D in it investigated the detection reproducibility of the SAA electrochemical immunosensor by performing multiple repeated detections of 5.0 μg / mL SAA in different batches. The response currents of multiple test groups to the target were basically the same, and the RSD was 4.24%. These results indicate that the SAA electrochemical immunosensor has good sensitivity, specificity, and reproducibility, and can obtain stable detection results.

[0073] Figure 12 The detection sensitivity, specificity, and reproducibility of the D-D electrochemical immunosensor were investigated. Figure 12 A in it is the i-t result of D-D with a concentration gradient. In the range of 0.125 - 5.00 μg / mL, the reduction current signal increased with the increase in D-D concentration. Further fitting the relationship between the response current and concentration, a linear relationship as shown in Figure 12 B in it was obtained. The linear equation is I (μA) = 0.85C D-D (μg / mL) + 0.59, and the detection limit is 0.100 μg / mL (≥3σ). Figure 12 C in it is the specific analysis result of the D-D electrochemical immunosensor. Among them, the D-D concentration is 2.5 μg / mL, and the interferent concentrations are 50 μg / mL CRP, 50 μg / mL SAA, 50 ng / mL PCT, and 1 ng / mL IL-6 respectively. The interferent concentrations refer to the plasma biomarker levels of sepsis patients before adding them to the detection system. The immunosensor only obtained a sensitive response signal in the test group containing D-D, while a weak background signal was obtained in other interferent groups. Figure 12 D in it investigated the detection reproducibility of the D-D electrochemical immunosensor by performing multiple repeated detections of 2.5 μg / mL D-D in different batches. The response currents of multiple test groups to the target were basically the same, and the RSD was 5.03%. The results indicate that the D-D electrochemical immunosensor has good sensitivity, specificity, and reproducibility, and can obtain stable detection results.

[0074] Figure 13 The detection sensitivity, specificity, and reproducibility of the FN electrochemical immunosensor were investigated. Figure 13A in it is the i-t result of the concentration gradient of FN. In the range of 5 - 500 ng / mL, the reduction current signal increases with the increase of FN concentration. Further fitting the relationship between the response current and concentration, a linear relationship as shown in Figure 13 B in it is obtained, and the linear equation is I (μA) = 0.027C FN (ng / mL) + 0.66, and the detection limit is 3.14 ng / mL (≥3σ). Figure 13 C in it is the specific analysis result of the FN electrochemical immunosensor. Among them, the FN concentration is 0.25 μg / mL, and the concentrations of the interferents are 50 μg / mL CRP, 50 μg / mL SAA, 50 ng / mL PCT, 1 ng / mL IL-6, and 0.5 μg / mL D-D respectively. The concentrations of the interferents refer to the biomarker levels in the plasma of sepsis patients. The immunosensor only obtains a sensitive response signal in the test group containing FN, while a weak background signal is obtained in other interferent groups. Figure 13 D in it examines the detection reproducibility of the FN electrochemical immunosensor by performing multiple repeated detections of 0.25 μg / mL FN in different batches. The response currents of multiple test groups to the target are basically the same, and the RSD is 3.72% ( Figure 13 D in it). The results show that the FN electrochemical immunosensor has good sensitivity, specificity, and reproducibility, and can obtain stable detection results.

[0075] Figure 14 By adding different markers to the 10 mg / mL BSA solution to simulate the combined level changes of markers related to diseases. The experiment set up an SAA group (100 μg / mL SAA + 400 μg / mL FN), a CRP + SAA group (100 μg / mL CRP + 100 μg / mL SAA + 400 μg / mL FN), a D-D group (5 μg / mL D-D + 400 μg / mL FN), and a CRP + SAA + D-D + FN group (100 μg / mL CRP + 100 μg / mL SAA + 5 μg / mL D-D + 100 μg / mL FN). The detection results show that several combinations cited can be simultaneously detected by the immunosensor and the signal crosstalk between markers is low, indicating that this combined synchronous detection method can be applied to the simultaneous analysis of multiple markers in simulated samples.

[0076] Figure 15Plasma samples of 7 sepsis patients were subjected to simultaneous detection of a panel of biomarkers. The test results of CRP, SAA, D-D, and FN are shown in sequence as A - D. At present, there is no consensus on the diagnostic threshold of D-D for prognostic assessment of sepsis. According to a series of previous clinical studies on sepsis, the reference value for D-D detection was set at 2 μg / mL; the normal FN level is about 200 - 600 μg / mL, so the diagnostic threshold of FN for sepsis was set at 200 μg / mL. Figure 15 In A of Figure 15 Among the CRP and SAA test results shown in B of Figure 15 In the D-D test result shown in C of Figure 15 Among the FN test results shown in D of

[0077] Figure 16 The Figure 15 simultaneous detection results were subjected to a correlation analysis study with ELISA results. The results comparison of CRP, SAA, D-D, and FN in the figure is shown in sequence as A - D. The four biomarkers highly match the ELISA results. The correlation coefficient R of CRP detection 2 = 0.863, the correlation coefficient R of SAA detection 2 = 0.957, the correlation coefficient R of D-D detection 2 = 0.884, the correlation coefficient R of FN detection 2 = 0.940. These results indicate that the two methods can provide the same medical diagnostic value.

Claims

1. A 3D printed assembled multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers, characterized in that: The method is prepared by the following steps: (1) Preparation of 3D printed assembled multi-channel electrodes: first design a three-dimensional diagram of a multi-channel electrode module, a reference module, and a detection cell module, and then produce them by fused deposition 3D printing, and further assemble them into a multi-channel electrode; (2) Preparation of 3D printed assembled multi-channel electrochemical immunosensor: through electrochemical gold deposition and specific probe coupling steps, immunosensor interfaces for C-reactive protein (CRP), serum amyloid A (SAA), D-dimer (DD) and fibronectin (FN) are prepared on different channels; (3) Joint and synchronous detection of CRP, SAA, DD and FN: the target in the sample is quickly assembled with CRP, SAA, DD and FN antibodies respectively by a double antibody sandwich one-step assembly method, and further bridged with horseradish peroxidase of streptavidin, and then based on the detection mode of the multi-channel electrochemical immunosensor, joint and synchronous detection is realized, and hydrogen peroxide is catalyzed to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) on the electrode surface to generate a reduction current signal.

2. A 3D printed assembled multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to claim 1, characterized in that: According to the physicochemical properties of CRP, SAA, DD and FN, antibody probe pairs that can simultaneously bind to the target are set. Based on the low interference characteristics of the double antibody sandwich method and the advantages of the one-step assembly method, the simultaneous operation of the multi-channel immunosensor greatly reduces the multiple and repeated operation steps in the detection, thereby realizing the combined and synchronous detection of CRP, SAA, DD and FN in plasma samples.

3. A 3D printed assembled multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to claim 1 or 2, characterized in that: During the detection process, the linear range of CRP was 0.125-5.00 μg / mL, and the detection limit was 0.115 μg / mL. The linear range of SAA was 0.250-10.0 μg / mL, and the detection limit was 0.106 μg / mL. The linear range of DD was 0.125-5.00 μg / mL, and the detection limit was 0.100 μg / mL. The linear range of FN was 5-500 ng / mL, and the detection limit was 3.14 ng / mL, which were in line with the reference value range of the four biomarkers in clinical detection.

4. A method for preparing a 3D printed assembled multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers, comprising: (1) designing a model using drawing software and manufacturing a physical module using a 3D printer, wherein the physical module includes a multi-channel electrode module, a reference module, and a detection cell module; (2) The surface of the multi-channel electrode module was modified with gold nanoparticles by electrochemical reduction to form different 3D printed electrodes. CRP, SAA, DD and FN probes were combined on different 3D printed electrodes to prepare a multi-channel electrochemical immunosensor. (3) The cell body of the detection cell module includes an Ab2 cell, a SA-pHRP cell and a TMB cell. The cell body of the detection cell module is pre-treated with bovine serum albumin (BSA) for blocking. Then, the sample to be tested and the biotin-modified signal antibody (Ab2) are placed in the Ab2 cell, and the multi-channel electrochemical immunosensor prepared in step (2) is assembled into the Ab2 cell for incubation; (4) During the incubation process of step (3), adding streptavidin-poly horseradish peroxidase (SA-pHRP) to the SA-pHRP pool of the detection pool module in step (3), cleaning the multi-channel electrochemical immunosensor after the incubation of step (3), and assembling the cleaned multi-channel electrochemical immunosensor into the SA-pHRP pool for incubation; (5) During the incubation process of step (4), TMB is added to the TMB pool of the detection pool module in step (3), and the reference module prepared in step (1) is assembled with the detection pool module. After step (4) is completed, the multi-channel electrochemical immunosensor is cleaned, and the cleaned multi-channel electrochemical immunosensor is assembled into the TMB pool for electrochemical detection; (6) The electrochemical detection is performed using the current-time curve (IT) method.

5. The method for manufacturing a 3D printed assembled multi-channel electrochemical immunosensor for combined simultaneous detection of sepsis biomarkers according to claim 4, characterized in that: The specific steps are as follows: (1) Design and production of 3D printed assembled multi-channel electrochemical immunosensor: First, use 123D software to design the three-dimensional model of each component and save it in stl format; then slice the three-dimensional model layer by layer, use JGcreat 2.5.0 to establish a printing sequence, and save the generated program in gcode format; then upload the gcode file to the 3D printer for automatic printing; finally, assemble the manufactured components according to the design plan; the 3D printed multi-channel electrochemical immunosensor includes 3D printed electrode components, convex components, concave components, reference module support components and detection cell components, which are assembled into multi-channel electrode modules, reference modules and detection cell modules after printing; (2) Interface assembly of 3D printed assembled electrochemical immunosensor for simultaneous detection of CRP, SAA, DD and FN: The interface assembly method of SAA, DD and FN electrochemical immunosensor was consistent with that of CRP electrochemical immunosensor. The interface assembly process of CRP electrochemical immunosensor was as follows: the 3D printed electrode was ground and polished on coarse sandpaper and fine sandpaper with aluminum oxide (Al2O3) suspension, and then ultrasonicated in ethanol and ultrapure water for 30 s, and then washed with ultrapure water; the 3D printed electrode was immersed in 2.8 mmol / L chloroauric acid (HAuCl4) solution, and nanogold was deposited using the current-time curve (it) method with a scanning voltage of −0.2 V and a scanning time of 500 s; the 3D printed electrode was washed with ultrapure water and scanned using CV in 0.5 mol / L sulfuric acid (H2SO4) solution with a scanning potential of 0-1.5 V and a scanning rate of 1. V / s, the scanning segment number is 60; the electrode is cleaned with ultrapure water and blown dry with nitrogen, 3 μL of 10 mmol / L phosphate buffered saline (PBS) containing 100 μg / mL CRP capture antibody (CRPAb1) is added to the electrode surface, and sealed and incubated at room temperature for 16 h in a dark place; the 3D printed electrode is cleaned with 10 mmol / L PBS, and the 3D printed electrode is immersed in 100 μL 10 mg / mL BSA solution and incubated at room temperature for 30 min, and cleaned with 10 mmol / L PBS to form a CRP electrode for use; the SAA, DD and FN electrochemical immunosensor interfaces are assembled in the same way as the CRP electrochemical immunosensor interface, using SAA capture antibody (SAA Ab1), DD capture antibody (D-DAb1) and FN capture antibody (FN Ab1) to form SAA electrode, DD electrode and FN electrode for use; (3) Combined simultaneous detection of CRP, SAA, DD and FN: The combined simultaneous detection method of SAA, DD and FN is the same as that of CRP. The CRP detection method is as follows: soak all areas of the detection cell module with 10 mg / mL BSA for 30 min and blow dry with nitrogen; take 5 μL of sample and mix it with 5 μL of 10 mg / mL BSA solution containing 20 μg / mL CRP biotin-modified signal antibody (CRP Ab2), and add it to the Ab2 pool for CRP detection in the detection cell module; align the multi-channel electrochemical immunosensor with the position of the Ab2 pool and assemble it into the Ab2 pool in the detection cell module, incubate at room temperature for 50 min, remove the multi-channel electrochemical immunosensor, wash it with 10 mmol / L PBS solution, and blow dry with nitrogen; take 10 μL of 10 mg / mL BSA solution containing 0.5 μg / mL SA-pHRP BSA solution was added to the SA-pHRP pool, and the cleaned multi-channel electrochemical immunosensor was aligned with the position of the SA-pHRP pool and assembled into the SA-pHRP pool in the detection pool module. The mixture was incubated at room temperature for 30 min, and the multi-channel electrochemical immunosensor was removed and cleaned with 10 mmol / L PBS solution and dried with nitrogen. 400 μL of enhanced TMB solution was added to the TMB pool, and the cleaned multi-channel electrochemical immunosensor was aligned with the position of the TMB pool and assembled into the TMB pool in the detection pool module, so that the CRP electrode obtained in step (2) was immersed in the solution of the TMB pool. At the same time, the reference module was also assembled into the detection pool module, and the CHI1030C multi-channel constant potentiostat was used for detection. The SAA, DD and FN combined synchronous detection method was the same as the CRP combined synchronous detection method, using the biotin-modified signal antibody of SAA (SAA Ab2), the biotin-modified signal antibody of DD (D-DAb2) and the biotin-modified signal antibody of FN (FN Ab2) respectively. (4) Electrochemical detection parameters: CRP, SAA, DD, and FN were detected simultaneously by the multi-channel IT method. The detection voltage was −0.2 V and the detection time was 100 s. The IT results were collected and the current value at 100 s was recorded.

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