A dual-channel electrochemical sensor for the simultaneous detection of sepsis biomarkers CRP and LPS

Abstract

This invention discloses a dual-channel electrochemical sensor for the simultaneous detection of sepsis biomarkers CRP and LPS. The sensor comprises the following steps: immobilizing capture probes on the surface of a dual-channel electrode to identify CRP and LPS; bridging the biotinylated probe with streptavidin-horseradish peroxidase to amplify the signal; and detecting CRP and LPS using the current signal generated by the catalytic TMB / H2O2 system. The linear range for LPS detection is 0.2 pg / mL to 5 ng / mL; the linear range for CRP detection is 0.1 μg / mL to 20 μg / mL. This invention provides a dual-channel electrochemical sensor for the simultaneous detection of sepsis biomarkers CRP and LPS, which features high specificity, high sensitivity, and simple operation. It is expected to help clinicians accurately, efficiently, and rapidly diagnose sepsis and assess its progression, and may also provide new insights for the detection of biomarkers for other diseases.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, and more specifically, relates to a dual-channel electrochemical sensor for the simultaneous detection of sepsis biomarkers CRP and LPS. The constructed dual-channel multiplexed CRP and LPS detection mode is expected to be used in the fields of early rapid diagnosis of sepsis and guidance of personalized medicine. Background Technology

[0002] Sepsis is a life-threatening multi-organ dysfunction caused by an uncontrolled host response to infection. It affects millions of patients annually, with a mortality rate as high as 56%. Bacteria are the most important immunopathogens in sepsis, making antibiotic intervention essential. However, antibiotic treatment regimens vary greatly depending on bacterial Gram typing, making pre-treatment Gram typing indispensable. While microbial culture is the most effective method for clinically diagnosing the cause of sepsis, it requires a relatively long time of 48–72 hours, limiting the timeliness of diagnosis and often leading to delays in treatment. Furthermore, this method heavily relies on expensive testing equipment, strict laboratory conditions, and skilled operating techniques, making it less suitable for non-laboratory departments, primary care hospitals, or inexperienced physicians. Therefore, there is an urgent need to establish an economical, simple, sensitive, and rapid method for the diagnosis and Gram typing of sepsis to guide the emergency use of antibiotics.

[0003] With the deepening of physiological and clinical research on sepsis, biomarker detection can achieve early and accurate diagnosis of sepsis. C-reactive protein (CRP) is an important biomarker for bacterial infectious inflammation. During the acute phase of sepsis (6 hours), CRP levels rise rapidly until symptoms subside. Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria and is the root cause of Gram-negative bacterial sepsis. Therefore, LPS concentration directly reflects the infection status of Gram-negative bacteria. Several independent CRP and LPS detection methods have been developed, including immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. However, these methods rely on large-scale testing equipment and complex operating techniques, which is not conducive to the technical integration of on-site testing equipment.

[0004] Electrochemical sensing technology has advantages such as ease of operation, high sensitivity, fast response, stability, portability, and ease of integration. However, electrochemical sensors that simultaneously detect multiple biomarkers across concentration gradients are susceptible to crosstalk from various signals, which is detrimental to the development of clinical point-of-care (POC) assays.

[0005] This invention proposes a dual-channel electrochemical sensor for the simultaneous detection of the sepsis biomarkers CRP and LPS, and establishes a strategy for the simultaneous detection of LPS and CRP using this dual-channel electrochemical biosensor. Simultaneous electrochemical detection of LPS and CRP can rapidly diagnose bacterial sepsis and differentiate the Gram types of infecting strains. The constructed biosensor exhibits good linearity and detection limits, conforming to the concentration ranges required for biomarkers in clinical blood samples. The effects of LPS and CRP on interferon in simulated plasma samples were also determined. Therefore, the dual-channel electrochemical sensor combined with the strategy of simultaneous detection of CRP and LPS as dual biomarkers has potential application value for further Gram typing of bacterial sepsis and guidance for personalized medication. Summary of the Invention

[0006] 1. The purpose of this invention is to provide a dual-channel electrochemical sensor for the simultaneous detection of sepsis markers CRP and LPS.

[0007] 2. The dual-channel electrochemical sensor for simultaneous detection of CRP and LPS described in this invention uses a gold electrode modified with a specific capture probe and a biotin-modified signal probe as target recognition components, and SA-HRP catalyzed TMB as a bridging electrochemical signal amplification medium, enabling simultaneous detection of CRP and LPS in a sample.

[0008] 3. The dual-channel electrochemical sensor for simultaneous detection of CRP and LPS described in this invention utilizes a dual-path multiplexing electrochemical sensor mode to simultaneously read out the electrochemical detection signals of CRP and LPS in the same sample.

[0009] 4. The dual-channel electrochemical sensor for simultaneous detection of CRP and LPS described in this invention is fabricated by the following steps in sequence:

[0010] (1) Pretreatment of working gold electrode (AuE): After polishing, AuE is cleaned with ethanol and water, then activated in dilute H2SO4, cleaned with ultrapure water, dried with N2, and ready for use.

[0011] (2) Capture probe assembly: The capture probe (antibody or aptamer) was incubated on the AuE surface for 3 h and the electrode was cleaned;

[0012] (3) BSA sealing: The AuE was immersed in a 0.5% BSA solution to seal it in the dark, and the electrode was cleaned.

[0013] (4) Assembly of antigen and signal probe: The test solution with pre-added signal probe (antibody or aptamer) is dropped onto AuE, incubated for 1 h, and the electrode is cleaned.

[0014] (5) Assembly of streptavidin-horseradish peroxidase (SA-HRP): SA-HRP solution was dropped onto AuE, incubated for 30 min, the electrode was cleaned, and electrochemical detection was immediately performed in TMB / H2O2 solution.

[0015] Specifically, the detection method of a dual-channel electrochemical sensor for the simultaneous detection of sepsis markers CRP and LPS, as described in this invention, is characterized by comprising the following steps: (1) LPS is specifically recognized by a DNA aptamer (capture probe, CP), and the signal is amplified by bridging a biotin-modified DNA aptamer (signal probe, SP) with horseradish peroxidase (SA-HRP) modified with streptavidin through a biotin-avidin system, and hydrogen peroxide (H2O) is catalyzed by horseradish peroxidase (HRP). (2) Oxidize 3,3',5,5'-tetramethylbenzidine (TMB) with 2O2 to generate a reduction current signal; (2) Capture CRP by specific recognition between antigen and antibody, drop CRP and a mixture of biotin signal antibody (Ab2) onto a gold electrode to reduce assembly steps, and finally catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with hydrogen peroxide (H2O2) on the electrode by horseradish peroxidase (SA-HRP) modified with streptavidin and avidin to generate a reduction current signal.

[0016] Furthermore, different nanoprobes were set according to the physicochemical properties and clinical reference values ​​of CRP and LPS markers. The dual-path multiplexed electrochemical sensor mode was used to achieve simultaneous readout of electrochemical detection signals of CRP and LPS in the same sample. The dual working electrode mode was used to achieve low crosstalk detection of dual targets of CRP and LPS with huge concentration differences, so as to successfully verify its joint detection in mixed samples containing LPS, CRP and other interfering standards and clinical plasma samples.

[0017] Furthermore, during the testing process, the linear range of LPS detection was 0.5 pg / mL to 1 ng / mL, with a detection limit of 0.343 pg / mL, and the linear range of CRP detection was 0.1 μg / mL to 20 μg / mL, with a detection limit of 0.05 μg / mL, which are in line with the reference ranges for clinical testing of the two biomarkers.

[0018] Furthermore, the preparation method of the dual-channel electrochemical sensor for simultaneous detection of sepsis markers CRP and LPS according to the present invention includes the following steps:

[0019] (1) Pretreatment of working gold electrode (AuE): AuE is polished and then cleaned in ethanol and water, then activated in dilute H2SO4, cleaned with ultrapure water, dried with N2, and ready for use.

[0020] (2) Capture probe assembly: The capture probe (antibody or aptamer) was incubated on the AuE surface for 3 h and the electrode was cleaned;

[0021] (3) BSA sealing: The AuE was immersed in a 0.5 wt% BSA solution and sealed in the dark, and the electrode was cleaned;

[0022] (4) Assembly of antigen and signal probe: The test solution with pre-added signal probe (antibody or aptamer) is dropped onto AuE, incubated for 1 h, and the electrode is cleaned.

[0023] (5) Assembly of streptavidin-horseradish peroxidase (SA-HRP): SA-HRP solution was dropped onto AuE, incubated for 30 min, the electrode was cleaned, and electrochemical detection was immediately performed in TMB / H2O2 solution.

[0024] Further, electrode preparation for CRP detection: 3 µL of a 100 μg / mL CRP capture antibody (Ab1) solution was drop-coated onto an AuE electrode and incubated at room temperature in the dark for 3 h. The assembled AuE-Ab1 was rinsed with 10 mmol / L PBS to remove unbound Ab1, dried with N2, and then immersed in 100 μL of 0.5 wt% BSA solution and incubated at room temperature in the dark for 1 min. After blocking, it was rinsed with 10 mmol / L PBS and dried with N2. Then, CRP and 20 μg / mL signal antibody (Ab2) were mixed at a volume ratio of 1:1 to obtain a mixed solution CRP-Ab2. 3 μL of the mixed solution was drop-coated onto the AuE-Ab1-BSA sensing interface and incubated at room temperature for 1 h. Finally, the assembled AuE-Ab1-BSA-CRP-Ab2 was rinsed with 10 mmol / L PBS to remove unbound CRP and Ab2, dried with N2, and then immersed in 1 wt% BSA solution. BSA was used to dilute the SA-HRP stock solution 1000 times. 3 μL of the diluted SA-HRP solution was dropped onto the assembled AuE-Ab1-BSA-CRP-Ab2 interface and incubated at room temperature for 30 min. The AuE was then washed with 10 mmol / L PBS solution. The final sensor AuE-Ab1-BSA-CRP-Ab2-HRP was constructed.

[0025] Electrode preparation for LPS detection: The LPS aptamer capture probe (CP) assembly solution was prepared to a concentration of 1 μmol / L and allowed to stand at room temperature in the dark for 30 min for reduction. 3 µL of the capture probe solution was dropped onto AuE and incubated at room temperature in the dark for 5 h. The assembled AuE-CP was rinsed with 10 mmol / L PBS to remove unbound CP and dried with N2. It was then immersed in 100 μL of 0.5 wt% BSA solution and incubated at room temperature in the dark for 1 min to block excess Au-S bond binding sites. After this, it was rinsed with 10 mmol / L PBS and dried with N2. LPS and 1 μmol / L signal aptamer (SP) were then mixed at a volume ratio of 1:1 to obtain a mixture. 3 μL of this mixture was dropped onto the AuE-CP-BSA sensing interface and incubated at room temperature for 50 min. Finally, the assembled AuE-CP-BSA-LPS-SP was rinsed with 10 mmol / L PBS to remove unbound CP. Unbound LPS and signal aptamers (SPs) were rinsed with PBS, dried with N2, and the SA-HRP stock solution was diluted 1000-fold with 1 wt% BSA. 3 μL of the diluted SA-HRP solution was dropped onto the assembled AuE-CP-BSA-LPS-SP interface and incubated at room temperature for 30 min. The AuE was then washed with 10 mmol / L PBS solution. The final sensor AuE-CP-BSA-LPS-SP-SA-HRP was constructed.

[0026] Furthermore, the above-described preparation method of the present invention yields a dual-channel electrochemical sensor for the simultaneous detection of sepsis markers CRP and LPS.

[0027] Advantages of this invention:

[0028] The dual-channel electrochemical sensor for simultaneous detection of CRP and LPS proposed in this invention utilizes an immune sandwich structure to enhance the specificity of the sensor detection. By reducing assembly steps and directly utilizing biological enzymes to improve simplicity and practicality, two dual-channel electrochemical biosensors for rapid simultaneous detection of LPS and CRP have been constructed. These sensors feature high specificity, high sensitivity, and simple operation, and can significantly save time and costs compared to traditional methods. They are expected to help clinicians accurately, efficiently, and rapidly diagnose sepsis and perform Gram typing, thereby improving the accuracy of antibiotic use. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the dual-channel electrochemical sensor for simultaneous detection of CRP and LPS according to the present invention.

[0030] Figure 2 This is an it signal diagram of CRP electrochemical detection according to the present invention. Figure 2The light-colored line is the blank control using 10 mmol / L PBS, and the dark-colored line is the current-time curve measured using 10 μg / mL CRP standard solution. It can be seen that there is a significant difference between the signal of 10 μg / mL CRP standard and the background signal, indicating that the immunosensor has high sensitivity.

[0031] Figure 3 This is an it signal diagram of LPS electrochemical detection according to the present invention. Figure 3 The light-colored line is the blank control using 20 mmol / L Tris-HCl, and the dark-colored line is the current-time curve measured using 1 ng / mL LPS standard solution. It can be seen that there is a significant difference between the signal of 1 ng / mL LPS standard and the background signal, indicating that the immunosensor has high sensitivity.

[0032] Figure 4 This is an EIS diagram of the CRP electrochemical electrode assembly of the present invention. Figure 4 EIS characterization of the CRP electrochemical electrode assembly showed that the resistance of the bare electrode (AuE) was approximately 150 Ω, indicating good conductivity after AuE pretreatment. When Ab1 self-assembled on AuE, its resistance increased significantly (~2000 Ω), indicating successful assembly of Ab1 on AuE. After blocking non-specific adsorption sites with 0.5% BSA, the conductivity decreased (~4000 Ω); when a mixed solution of CRP and Ab2 was assembled, the conductivity continued to decrease, while the resistance increased (~5700 Ω). Finally, the impedance reached its maximum (~13700 Ω) through the interaction of SA-HRP with biotin and avidin. These results demonstrate the feasibility of our constructed electrochemical biosensing strategy.

[0033] Figure 5 This is an EIS diagram of the LPS electrochemical electrode assembly of the present invention. Figure 5EIS characterization of the LPS electrochemical electrode assembly showed that the resistance of the bare electrode (AuE) was approximately 400 Ω, indicating good conductivity without modification. After CP self-assembly on AuE, its resistance increased significantly (~8500 Ω), indicating successful CP assembly. After blocking non-specific adsorption sites with 0.5% BSA, the resistance increased slightly (~9700 Ω), possibly because at this concentration (1 μmol / L), CP occupied most of the assembled sites on the AuE interface, and before BSA assembly, CP was inverted, but after blocking, it was upright in the gaps between BSA. When a mixed solution of LPS and SP was assembled, the conductivity decreased significantly, and the resistance increased dramatically (~60000 Ω), because the complex formed by LPS binding with CP and SP hindered electron transfer. Finally, SA-HRP was assembled on the sensor via biotin and avidin bridging, achieving the maximum resistance (~94000 Ω). The results above demonstrate that our proposed electrochemical biosensing strategy is feasible.

[0034] Figure 6 This is a linear relationship between the current response value (I) of the present invention and different concentrations of CRP protein. (The concentrations of CRP standards were 0.1, 0.4, 0.8, 1, 5, 10, and 20 µg / mL, respectively). Figure 6 As shown, under optimal experimental conditions, the current response values ​​of different CRP standard concentrations were detected using the immunosensor via the iterative method. A good linear relationship was observed between the current signal response value (I) and the CRP concentration, ranging from 0.1 to 20 µg / mL. The linear equation was I = -2.60Lg[CRP] - 3.60, where R0... 2 =0.992, detection limit is 50 ng / mL.

[0035] Figure 7 This is a linear relationship graph between the current response value (I) of the present invention and different concentrations of LPS. (The concentrations of LPS standards are 0.2, 0.5, 1, 10, 100, 500, 5000, and 10000 pg / mL). Figure 7 As shown, under optimal experimental conditions, the current response value of the immunosensor was detected using the iterative method for different concentrations of LPS standards. A good linear relationship was observed between the current signal response value (I) and the LPS concentration, ranging from 0.2 to 10000 pg / mL, with I = -0.278 Lg [LPS] - 0.0930, where R... 2 =0.990, and the detection limit is 0.183 pg / mL.

[0036] Figure 8 The electrical signal of the CRP sensor of this invention is used to respond to the current of different proteins. Figure 8 To investigate whether the presence of common inflammatory factors in sepsis, such as serum amyloid A (SAA), procalcitonin (PCT), interleukin-6 (IL-6), and lipopolysaccharide (LPS), would affect the selectivity of the constructed sensor under the same conditions, the current signals of 5 ng / mL PCT, 1 ng / mL LPS, 0.5 ng / mL IL-6, and 50 μg / mL SAA prepared with CRP assembly solution were similar to the background values ​​with no significant difference, but significantly different from the current signal of 10 μg / mL CRP (~20-fold), indicating that the electrochemical biosensor constructed by this method has good specificity.

[0037] Figure 9 The present invention describes the electrical signal response of the LPS sensor to currents in materials with different structural similarities. Figure 9 Substances with similar compositions to LPS, such as glucose, glucan, cholesterol, L-cysteine, and the common inflammatory cytokine C-reactive protein (CRP), were selected as potential interfering agents. All substances were prepared with Tris-HCl buffer. When the concentration of each interfering agent was 50 or 1000 times that of LPS, its signal was similar to the background value, demonstrating the good selectivity of this strategy.

[0038] Figure 10 This is a linear relationship graph showing the difference in current response of the LPS sensor of the present invention (the difference ΔI between the concentration current signal and the background signal) and LPS diluted 10 times with normal human plasma at different concentrations. (LPS standard concentrations are 0.5, 1, 10, 100, and 1000 pg / mL). Figure 10 Plasma was diluted 10-fold with Tris-HCl buffer to prepare different concentrations of LPS. The signal values ​​in the plasma samples differed from those in the buffer system; therefore, the relationship between plasma LPS and the response current I was investigated. The linear range was 0.5 pg / mL to 1 ng / mL, and the linear equation was: I = -136.79 Lg [LPS] - 687.34, R0 2 =0.988, with a detection limit of 0.343 pg / mL, which is lower than the lowest concentration of LPS in the blood of patients with Gram-negative bacterial infections (10 pg / mL), and fully meets the requirements for LPS detection in blood samples. Detailed Implementation

[0039] To make the technical problems, technical solutions and effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0040] The present invention discloses a dual-channel electrochemical sensor for the simultaneous detection of sepsis biomarkers CRP and LPS, used for Gram typing of sepsis. The specific operating steps are as follows:

[0041] (1) Preparation of instruments, reagents and solutions for detection

[0042] All instruments and equipment used in this invention were purchased from the following companies:

[0043]

[0044] The reagents used in this invention are all from the following companies:

[0045]

[0046] The aptamer sequences used in this invention are as follows:

[0047]

[0048] The reagents used in this invention are prepared strictly according to the following formula.

[0049] ① 0.2 mol / L phosphate buffer stock solution (PB): Prepare 0.2 mol / L Na2HPO4 solution and 0.2 mol / L NaH2PO4 solution as stock solutions. Mix the Na2HPO4 and NaH2PO4 solutions at a volume ratio of 81:19 and store at 4 ℃ protected from light.

[0050] ② 10 mmol / L Phosphate Buffered Sodium (PBS): Measure 20 mL of the stock solution, add 380 mL of ultrapure water and 0.1 mol NaCl powder, and finally adjust the pH to 7.4 with HCl and NaOH.

[0051] ③ 20 mmol / L Tris-HCl buffer: Prepare 20 mmol / L Tris-HCl containing 140 mmol / L NaCl, 5 mmol / L KCl, 1 mmol / L CaCl2, and 1 mmol / L MgCl2, and adjust the pH to 7.4 with 10 mmol / L HCl.

[0052] ④ LPS aptamer capture probe (CP) assembly solution: prepared with 20 mmol / L Tris-HCl buffer and 15 mmol / L tris(2-carboxyethyl)phosphine (TCEP), and should be prepared fresh before use.

[0053] ⑤ Aptamer stock solution: Synthetically synthesized DNA adheres to the container tube wall in a light, dry film form, which is easily lost when opened. Therefore, before dissolving, the centrifuge tube containing DNA should be centrifuged at 3000 rpm for 10 minutes. Then, slowly open the cap, add sterile water according to the instructions to prepare a stock solution of a certain concentration, cap the tube, shake it up and down for 5 minutes, aliquot and store at -20 ℃ for later use.

[0054] ⑥ BSA solution: Take 0.8 g BSA and add it to 4 mL of 10 mmol / L PBS solution to prepare a 2% (m / V) BSA solution. This solution can be diluted with PBS solution to prepare 1% (m / V), 0.5% (m / V), and 0.25% (m / V) BSA solutions.

[0055] ⑦SA-HRP solution: Different concentrations of SA-HRP were prepared by adding different volumes of 1% BSA solution to horseradish peroxidase (SA-HRP) stock solution (0.5 mg / mL) labeled with streptavidin.

[0056] ⑧ LPS standard solution: Add 1 mL of sterile water for injection to the purchased lipopolysaccharide (LPS) powder to prepare a 1 mg / mL LPS solution, and then dilute it serially with Tris-HCl buffer.

[0057] ⑨ 5 mM ferricyanide couple: Dilute 0.2 mol / L PB to 0.1 mol / L PB, containing 0.1 mol / L KCl, adjust the pH to 7.0, and then add 5 mmol of potassium ferricyanide and potassium ferrocyanide.

[0058] ⑩ CRP capture antibody (Ab1) dilution solution: Dilute 0.2 mol / L PB to 20 mmol / L, containing 5.8 mmol / L sodium citrate (Na-citrate), and adjust the pH to 6.0 with H3PO4 and NaOH.

[0059] ⑪ CRP signaling antibody (Ab2) dilution solution: Dilute 0.2 mol / L PB to 20 mmol / L, containing 150 mmol / L NaCl, 50% glycerol, and adjust the pH to 7.4 with H3PO4 and NaOH.

[0060] ⑫ CRP standard dilution solution: Prepare 20 mmol / L Tris-HCl containing 150 mmol / L NaCl, and adjust the pH to 8.0 with HCl.

[0061] (2) Pretreatment of AuE

[0062] The gold electrode (AuE) was polished on a rotating disk containing a mixture of 0.05 μm Al₂O₃ and water for 2.5 min, rinsed with deionized water, and ultrasonically cleaned with ethanol and distilled water for 1 min each. After completion, the AuE should be polished to a mirror finish. The AuE in the spare tank was removed, cleaned with deionized water, and placed in 0.5 mol / L H₂SO₄. The AuE was scanned for 30 cycles in the potential range of 0 V to 1.5 V using an electrochemical workstation at a scan rate of 1 V / s. After rinsing with ultrapure water and drying with N₂, it was ready for use.

[0063] (3) Preparation of a dual-channel electrochemical sensor for simultaneous detection of CRP and LPS

[0064] Electrode preparation for CRP detection: 3 µL of a 100 μg / mL CRP capture antibody (Ab1) solution purchased from Hangzhou Qitai Biotechnology Co., Ltd. was dropped onto an AuE electrode and incubated at room temperature in the dark for 3 h. The assembled AuE-Ab1 was rinsed with 10 mmol / L PBS to remove unbound Ab1, dried with N2, and then immersed in 100 μL of 0.5 wt% BSA solution for 1 min at room temperature in the dark. After blocking, it was rinsed with 10 mmol / L PBS and dried with N2. CRP and 20 μg / mL signal antibody (Ab2) were then mixed at a 1:1 volume ratio to obtain a CRP-Ab2 mixture. 3 μL of this mixture was dropped onto the AuE-Ab1-BSA sensing interface and incubated at room temperature for 1 h. Finally, the assembled AuE-Ab1-BSA-CRP-Ab2 was rinsed with 10 mmol / L PBS to remove unbound CRP and Ab2, dried with N2, and then immersed in 1 wt% BSA solution. BSA was diluted 1000-fold with SA-HRP stock solution. 3 μL of the diluted SA-HRP solution (preparation method is shown in detection preparation solution ⑦) was dropped onto the assembled AuE-Ab1-BSA-CRP-Ab2 interface and incubated at room temperature for 30 min. AuE was then washed with 10 mmol / L PBS solution. The final sensor AuE-Ab1-BSA-CRP-Ab2-HRP was constructed and ready for testing.

[0065] Electrode preparation for LPS detection: The LPS aptamer capture probe (CP) assembly solution (preparation method see detection preparation solution ④) was prepared to a concentration of 1 μmol / L and allowed to stand at room temperature in the dark for 30 min for reduction. 3 µL of the capture probe (CP, 5'-SS-CTTCTGCCCGCCTCCTTCCACCGATCCATCGAGTTTCTGAGAAAGGCCCGGAGAAACCGCGAGAGGAGACGAGATAGGCGGACACT-3') solution was dropped onto AuE and incubated at room temperature in the dark for 5 h. The assembled AuE-CP was rinsed with 10 mmol / L PBS to remove unbound CP and dried with N2. It was then immersed in 100 μL of 0.5 wt% BSA solution and incubated at room temperature in the dark for 1 min to block excess Au-S bond binding sites. After completion, it was rinsed with 10 mmol / L PBS and dried with N2 to obtain AuE-CP-BSA. Then, LPS and 1... A 1:1 volume ratio of μmol / L signal aptamer (also known as signal probe, abbreviated as SP, 5'-biotin-CTTCTGCCCGCCTCCTTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3') was used to prepare a mixture. 3 μL of this mixture was dropped onto the AuE-CP-BSA sensing interface and incubated at room temperature for 50 min. The final assembled AuE-CP-BSA-LPS-SP was then rinsed with 10 mmol / L PBS to remove any unbound LPS and signal aptamer (SP). The mixture was dried with N2. The SA-HRP stock solution was diluted 1000-fold with 1 wt% BSA. 3 μL of the diluted SA-HRP solution (preparation method see detection solution ⑦) was dropped onto the assembled AuE-CP-BSA-LPS-SP interface and incubated at room temperature for 30 min. The mixture was then rinsed with 10 mmol / L PBS. AuE was cleaned with PBS solution, and the final sensor AuE-CP-BSA-LPS-SP-SA-HRP was constructed and is ready for testing.

[0066] (4) Electrochemical detection using assembled dual-channel electrodes

[0067] A CHI 1030C multichannel electrochemical workstation was used. The working electrode, silver-silver chloride reference electrode, and platinum wire counter electrode were immersed in 500 µL of TMB substrate solution containing H2O2. Time-current curves were scanned at room temperature, and the current-time curves were recorded. The initial potential was 0.1 V, and the scan time was 100 s. The current at 100 s was taken as the final experimental electrical signal value. The dual-channel electrochemical sensor for simultaneous detection of CRP and LPS proposed in this invention utilizes an immune sandwich structure to enhance the sensor's specificity. By reducing assembly steps and directly utilizing biological enzymes, its simplicity and practicality are improved. Two electrochemical biosensors for rapid detection of LPS and CRP, respectively, were constructed. These sensors have the characteristics of high specificity, high sensitivity, and simple operation. Compared with traditional methods, they can significantly save time and costs, and are expected to help clinicians accurately and efficiently diagnose sepsis and perform Gram typing. They also improve the accuracy of antibiotic use.

[0068] Sequence list description:

[0069] Serial number 1 (ID): Signal probe (SP): 5'-biotin-CTTCTGCCCGCCTCCTTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3';

[0070] Serial number 2 (ID): Capture probe (CP): 5'-SS-CTTCTGCCCGCCTCCTTCCACCGATCCATCGAGTTTCTGAGAAAGGCCCGGAGAAACCGCGAGAGGAGACGAGATAGGCGGACACT-3'.

[0071] The DNA sequences in the sequence listing provided by this invention are known. The "5'-biotin-" and "-3'" are omitted in the signal probe (SP); the "5'-SS-" and "-3'" are omitted in the capture probe (CP). The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a dual-channel electrochemical sensor for the simultaneous detection of sepsis markers CRP and LPS, characterized in that, Includes the following steps: (1) Pretreatment of working gold electrode AuE: AuE is polished and then cleaned in ethanol and water, then activated in dilute H2SO4, cleaned with ultrapure water, dried with N2, and set aside. (2) Capture probe assembly: The capture probe was incubated on the AuE surface for 3 h and the electrode was cleaned; (3) BSA sealing: The AuE was immersed in a 0.5 wt% BSA solution and sealed in the dark, and the electrode was cleaned. (4) Assembly of antigen and signal probe: The test solution with the signal probe added in advance was dropped onto AuE, incubated for 1 h, and the electrode was cleaned; (5) Assembly of streptavidin-horseradish peroxidase SA-HRP: SA-HRP solution was dropped onto AuE, incubated for 30 min, the electrode was cleaned, and electrochemical detection was immediately performed in TMB / H2O2 solution. (6) Electrode preparation for CRP detection: 3 µL of a 100 μg / mL CRP capture antibody Ab1 solution was dropped onto AuE and incubated at room temperature in the dark for 3 h. The assembled AuE-Ab1 was rinsed with 10 mmol / L PBS to remove unbound Ab1, dried with N2, and then immersed in 100 μL of 0.5 wt% BSA solution and incubated at room temperature in the dark for 1 min. After blocking, it was rinsed with 10 mmol / L PBS and dried with N2. Then, CRP and 20 μg / mL signal antibody Ab2 were mixed at a volume ratio of 1:1 to obtain a mixed solution CRP-Ab2. 3 μL of the mixed solution was dropped onto the AuE-Ab1-BSA sensing interface and incubated at room temperature for 1 h. Finally, the assembled AuE-Ab1-BSA-CRP-Ab2 was rinsed with 10 mmol / L PBS to remove unbound CRP and Ab2, dried with N2, and then rinsed with 1 wt% BSA solution. BSA was used to dilute the SA-HRP stock solution 1000 times. 3 μL of the diluted SA-HRP solution was dropped onto the assembled AuE-Ab1-BSA-CRP-Ab2 interface and incubated at room temperature for 30 min. The AuE was then washed with 10 mmol / L PBS solution. The final sensor AuE-Ab1-BSA-CRP-Ab2-HRP was constructed. (7) Electrode preparation for LPS detection: The LPS aptamer capture probe CP assembly solution was prepared to a concentration of 1 μmol / L and allowed to stand at room temperature in the dark for 30 min for reduction. 3 µL of the capture probe solution was dropped onto AuE and incubated at room temperature in the dark for 5 h. The assembled AuE-CP was rinsed with 10 mmol / L PBS to remove unbound CP and dried with N2. Then, it was immersed in 100 μL of 0.5 wt% BSA solution and incubated at room temperature in the dark for 1 min to block excess Au-S bond binding sites. After that, it was rinsed with 10 mmol / L PBS and dried with N2. Then, LPS and 1 μmol / L signal aptamer SP were mixed at a volume ratio of 1:1 to obtain the mixed solution LPS-SP. 3 μL of the mixed solution was dropped onto the AuE-CP-BSA sensing interface and incubated at room temperature for 50 min. Finally, the assembled AuE-CP-BSA-LPS-SP was rinsed with 10 mmol / L PBS to remove unbound CP. Unbound LPS and signal aptamer SP were rinsed with PBS, dried with N2, and the SA-HRP stock solution was diluted 1000-fold with 1 wt% BSA. 3 μL of the diluted SA-HRP solution was dropped onto the assembled AuE-CP-BSA-LPS-SP interface and incubated at room temperature for 30 min. The AuE was then washed with 10 mmol / L PBS solution. The final sensor AuE-CP-BSA-LPS-SP-SA-HRP was constructed.

2. The preparation method according to claim 1 yields a dual-channel electrochemical sensor for the simultaneous detection of sepsis markers CRP and LPS.

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