Construction method of light addressable potentiometric sensor array for combined detection of diabetes markers

Abstract

The invention discloses a construction method and a detection method of a light addressable potentiometric sensor array for combined detection of diabetes markers. According to the method, the surface of a light-addressable potentiometric sensor array (LAPS array) is functionally modified by combining RGO-CS-Fc and specific recognition enzymes (GOD, PROD and FPOX), so that joint detection of glucose (Glu), 1, 5-sorbitan (1, 5-AG) and glycosylated hemoglobin (HbA1c) is realized. And the RGO-CS-Fc has good conductivity and load capacity, so that the detection sensitivity of the sensor can be improved. The LAPS Array is simple in structure and easy to integrate, and the detection limits of the three markers are 0.5 mg / mL, 10 [mu] g / mL and 1.0 [mu] g / mL respectively.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to an optically addressed potential sensor array for the combined detection of diabetes biomarkers. Background Technology

[0002] In current diabetes diagnosis, single-indicator testing methods all have inherent limitations. Glycated hemoglobin (HbA1c) can stably reflect a patient's average blood glucose level over the past 2-3 months, but its results exhibit a delay effect. 1,5-sorbitol (1,5-AG) can reflect blood glucose levels over the past 1-2 weeks, and its results are not affected by short-term factors such as diet and exercise; however, current research is insufficient to support its use as an independent screening or diagnostic indicator for diabetes. While real-time blood glucose testing can sensitively capture minute fluctuations in blood glucose without time delay, it is easily affected by various physiological and environmental factors, resulting in significant fluctuations in results. Combined detection of diabetes biomarkers can greatly improve the efficiency and accuracy of diabetes screening. Currently, diabetes biomarker detection technologies include enzyme-linked immunosorbent assay (ELISA), test strip methods, and high-performance liquid chromatography (HPLC). The invention patent with publication number CN103278574A utilizes liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) to detect HbA1c. This method has good detection accuracy, but requires solid-phase extraction and gradient elution of the sample, leading to a cumbersome testing process and a long detection time. CN111575339A proposes a quantitative detection technique for 1,5-AG based on a multi-enzyme cascade reaction. While this technique achieves enzymatic quantification of 1,5-AG in biological samples, the complex operational procedures involving multiple coenzymes make the experimental steps cumbersome, resulting in limitations in operational convenience and detection efficiency in practical applications. Therefore, developing a simple, economical, and efficient detection technique for the combined detection of diabetes biomarkers has become an urgent technical need in the current field of diabetes diagnosis and treatment. Summary of the Invention

[0003] The technical problem to be solved by this invention is to construct an optically addressed potential sensor array (LAPS array) for the joint detection of multiple diabetes biomarkers and its detection method. This sensor array is based on reduced graphene oxide-chitosan-ferrocene (RGO-CS-Fc) and has four detection regions, which are coupled to glucose oxidase (GOD), pyranose oxidase (PROD), and fructosyl peptide oxidase (FPOX) respectively, enabling the simultaneous detection of glucose (Glu), 1,5-anhydroglucosidase (1,5-AG), and glycated hemoglobin (HbA1c), with detection limits of 0.5 mg / mL, 10 μg / mL, and 1.0 μg / mL, respectively.

[0004] The detection principle of this invention: This patented optically addressed potential sensor array detection system comprises a LAPS array chip consisting of a silicon substrate, a composite insulating layer (SiO2 / Si3N4), and a gold electrode on the back. The chip uses a silicon epitaxial wafer as a substrate, and the array structure is constructed using microfabrication technology. Four detection regions are prepared through heavy doping and isolation. One unmodified detection region serves as a blank group. The other three detection regions are modified with RGO-CS-Fc and specific recognition enzymes (GOD, PROD, FPOX) to construct LAPSArray sensitive units. Finally, the target compounds Glu, 1,5-AG, and P-HbA1c (HbA1c treated with porcine pepsin to obtain fructose-valine-histidine-containing P-HbA1c) are modified on the corresponding detection regions for combined detection. This chip is mounted in a reaction cell consisting of a four-well metal stage and an electrolytic cell. This reaction chamber, along with the data acquisition board and light source array, was then integrated into a 3D-printed system housing, thus constructing a complete detection platform integrating optical addressing, electrochemical reaction, and electronic signal acquisition functions. The RGO-CS-Fc nanocomposite material (RGO-CS-Fc) possesses a large specific surface area, good conductivity, and an excellent bio-microenvironment, which can enhance the sensitivity of the LAPS Array. When the target molecules Glu, 1,5-AG, and P-HbA1c come into contact with the biosensitive membrane on the detection area, a catalytic oxidation reaction occurs on the membrane surface to produce hydrogen peroxide (H2O2). H2O2 oxidizes the Fc in the sensitive membrane RGO-CS-Fc, causing the Fe... 2+ Oxidized to Fe 3+ This disrupts the potential balance on the sensor surface, causing a voltage shift. Different concentrations of analytes induce varying degrees of change in membrane potential, resulting in different degrees of voltage shifts in the IV curve. By detecting changes in voltage shift, quantitative analysis of Glu, 1,5-AG, and HbA1c concentrations can be achieved, enabling the joint detection of these three substances.

[0005] This invention is carried out according to the following steps:

[0006] Step 1: Construction of the Optically Addressed Potential Sensor Array (LAPS Array) Detection System

[0007] 1. Fabrication of LAPS Array chip: A silicon substrate with a resistivity of 1-8 Ω·cm is provided as the substrate. A silicon dioxide (SiO2) layer and a silicon nitride (Si3N4) layer with a total thickness of 1-2 μm are formed on the substrate by thermal oxidation and chemical vapor deposition as a composite insulating layer. Multiple independent sensing areas are formed by dry etching process. A gold (Au) electrode with a thickness of 1000-1500 Å is deposited on the back of the silicon substrate to form ohmic contact gold, thus obtaining LAPS Array;

[0008] 2. Construction of reaction cell and shell: A system shell is prepared by 3D printing technology, and an electrochemical reaction cell is prepared by machining technology. The reaction cell includes a four-hole metal stage for supporting LAPS Array chip and an electrolytic cell.

[0009] 3. Overall system assembly: The fabricated LAPS Array chip is mounted on the metal stage, and the electrolytic cell is connected to the stage by magnetic attraction. Then, the entire reaction cell is integrated into the system housing, which also integrates a data acquisition board and a light source array.

[0010] Step 2: Preparation of reduced graphene oxide-chitosan-ferrocene nanocomposite material (RGO-CS-Fc):

[0011] 1. Preparation of reduced graphene oxide (RGO): Graphene oxide (GO) powder was dissolved, ultrasonically crushed, and ascorbic acid (AA) was added. The mixture was stirred and reduced to obtain RGO stock solution.

[0012] 2. Preparation of chitosan-ferrocene (CS-Fc): Dilute glacial acetic acid and mix it with chitosan (CS), then add a solution of ferrocene carboxylic acid (Fc); then add N-hydroxysuccinimide / 1-ethyl-(3-dimethylaminopropyl) (NHS / EDC, ratio 4:1) at a volume ratio of 10:1, stir the reaction until the solution turns reddish-brown, and centrifuge to obtain CS-Fc solution.

[0013] 3. Preparation of RGO-CS-Fc: RGO was added to the CS-Fc solution, stirred and centrifuged to obtain the RGO-CS-Fc solution, which was then stored for later use.

[0014] Step 3: Construction of LAPS Array Sensitive Units

[0015] 1. Pretreatment of LAPS Array: The LAPS Array chip was cleaned and dried, then immersed in NaOH for activation, cleaned and dried again, and finally 3-mercaptopropyltriethoxysilane (MPTES) solution was added to obtain MPTES / LAPS Array.

[0016] 2. Modification of RGO-CS-Fc: 1.0 μL-3.0 μL of RGO-CS-Fc solution was added to the surface of MPTES / LAPS Array, and the mixture was allowed to stand and dry to obtain RGO-CS-Fc / LAPS Array.

[0017] 3. Glutaraldehyde was added as a cross-linking agent to the diabetes biomarker detection area, and then 0.1 mg / mL-2.0 mg / mL LGOD, PROD, and FPOX were cross-linked into the three detection areas of the LAPS Array (the fourth detection area was used as a blank group) to construct the LAPS Array sensitive unit.

[0018] Step 4: Plotting the Standard Curve

[0019] 1. Add 1.0 μL to 3.0 μL of Glu, 1,5-AG and P-HbA1c to the three detection areas of the LAPS Array sensitive unit in step 2, and incubate at 20℃~30℃ for 20~30 min to prepare a LAPS Array that can jointly detect diabetes biomarkers.

[0020] 2. Place the LAPS Array working electrode into the LAPS Array system, add phosphate buffered saline (PBS) with a pH of 5.7-8.0, insert the Ag / AgCl reference electrode, turn on the power, measure the IV response curve, and record the voltage offset change.

[0021] 3. Standard solutions of Glu (concentrations of 0.1 mg / mL-3.0 mg / mL), 1,5-AG (concentrations of 10 μg / mL-350 μg / mL), and HbA1c (concentrations of 1.0 μg / mL-75 μg / mL) were detected in three isolated detection zones. Three different working curves for the three markers of Glu, 1,5-AG, and HbA1c were plotted to determine the detection limits of the three diabetes markers.

[0022] Step 5: Combined detection of diabetes biomarkers in actual blood samples

[0023] 1. Add actual blood samples to the interface of the LAPS Array sensitive cells, incubate, and prepare a LAPS Array capable of jointly detecting diabetes biomarkers;

[0024] 2. Place the LAPS Array into the LAPS Array system, and the signal acquisition board acquires, analyzes, and processes the response signal; calculate the concentrations of Glu, 1,5-AG, and HbA1c based on the three different standard curves obtained in step 3.

[0025] In step 1, the thickness of the deposited film is preferably 1.5 μm, and the thickness of the deposited gold is 1200 Å.

[0026] Furthermore, the distance between the Ag / AgCl reference electrode and the working electrode in step one is controlled at 2 mm;

[0027] Furthermore, in step two, the amount of GO is 30 mg and the amount of ascorbic acid is 300 mg;

[0028] Furthermore, the concentration of RGO in step two is 1 mg / mL;

[0029] Furthermore, the diluted glacial acetic acid in step two is 1.0%;

[0030] Furthermore, the CS in step two is 100 mg;

[0031] Furthermore, the Fc in step two is 150 mg;

[0032] Furthermore, the concentration of NaOH in step three is 1 mol / L;

[0033] Furthermore, the glutaraldehyde in step three is 2.5% and the dosage is 2.5 μL;

[0034] Furthermore, the MPTES in step three is 1% and the dosage is 2 μL;

[0035] The preferred amount of RGO-CS-Fc used in step three is 2 μL;

[0036] The preferred concentrations of GOD, PROD, and FPOX enzymes in step three are 0.5 mg / mL, 1.0 mg / mL, and 1.0 mg / mL, respectively; the volume for each is 2 μL.

[0037] Preferably, the pH of the PBS in step four is 7.4, and the concentration is 0.2 mol / L.

[0038] In step four, the preferred dosage of Glu, 1,5-AG, and HbA1c is 2 μL each, the concentration of Glu is 0.5 mg / mL, and the concentrations of 1,5-AG and HbA1c are 1.0 mg / mL.

[0039] In step four, the preferred incubation temperatures for Glu and GOD, 1,5-AG and PROD, and P-HbA1c and FPOX are all 25 °C, and the incubation time is 30 min.

[0040] Step one provides an integrated operating platform, including chips, reaction cells, and light sources, creating a fixed and standardized microenvironment for subsequent steps. Step two involves core biochemical modifications on this platform. Step two also provides a biocompatible nanomaterial with good conductivity, large specific surface area, and high biocompatibility for step three. The material prepared in step two has abundant carboxyl groups, providing a favorable microenvironment for the immobilization of GOD, PROD, and FPOX enzymes in step three. Step three, based on step two, constructs a sensing interface capable of jointly detecting diabetes biomarkers. This provides the LAPS Array sensitive unit interface for the construction of the LAPS Array in step four and the joint detection of diabetes biomarkers in step five, making it crucial for steps four and five. The standard curve in step four can be used to calculate the concentrations of Glu, 1,5-AG, and HbA1c in the blood sample in step five. Therefore, these steps are interconnected and interact with each other; only through their combined implementation can a reliable LAPS Array be constructed, enabling the joint detection of diabetes biomarkers in blood samples.

[0041] Beneficial effects

[0042] This invention creatively combines RGO-CS-Fc composite materials with LAPS technology to construct an optically addressed potential sensor array for the joint detection of multiple diabetes biomarkers. This array efficiently amplifies enzyme-catalyzed reaction signals, improves detection sensitivity, and enables simultaneous detection of short-, medium-, and long-term diabetes biomarkers. It overcomes the drawbacks of traditional methods requiring multiple devices and repeated detections, achieving rapid and comprehensive single-platform evaluation. Attached Figure Description

[0043] Figure 1 LAPS Array construction and detection principle diagram;

[0044] Figure 2 Scanning electron microscopy (SEM) images of the surface modification process of LAPS Array with uniform modifiers: (A) bare silicon wafer; (B) MPTES / LAPS Array; (C) RGO-CS-Fc / MPTES / LAPS Array.

[0045] Figure 3SEM images of LAPS arrays modified with different modifiers: (A) GOD / RGO-CS-Fc / MPTES / LAPS Array; (B) PROD / RGO-CS-Fc / MPTES / LAPS Array; (C) FPOX / RGO-CS-Fc / MPTES / LAPS Array; (D) Glu / GOD / RGO-CS-Fc / MPTES / LAPS Array; (E) 1,5-AG / PROD / RGO-CS-Fc / MPTES / LAPS Array; (F) P-HbA1c / FPOX / RGO-CS-Fc / MPTES / LAPS Array.

[0046] Figure 4 Normalized IV curves of LAPS Array detection of different concentrations of Glu (A), 1,5-AG (B), and HbA1c standard solution (C), and IV curves of LAPS Array detection of different blood HbA1c contents. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0048] The construction and detection principle of the combined detection of diabetes biomarkers LAPS Array are as follows: Figure 1As shown: First, a silicon substrate is used as the substrate, and thin films of SiO2 / Si3N4 are deposited on both sides of the substrate as insulating layers. Then, the SiO2 / Si3N4 on the front side of the substrate is etched using a dry etching process to form a 2×2 capacitor array structure. Next, phosphorus is heavily doped between the array layers. Then, SiO2 / Si3N4 on the back side of the substrate is etched using HF, and gold is deposited around the four photosensitive areas using magnetron sputtering technology to form ohmic gold, thus obtaining the LAPS Array. The LAPS Array was then washed sequentially with ethanol and ultrapure water. Following activation, it was immersed in NaOH, washed, and dried. MPTES was then added to the LAPS Array for thiol silanization. RGO-CS-Fc, specific recognition enzymes (GOD, PROD, FPOX), and analytes (Glu, 1,5-AG, P-HbA1c) were sequentially modified into the three detection regions of the LAPS Array via electrostatic adsorption and intermolecular forces, forming a LAPS Array capable of jointly detecting diabetes biomarkers. The LAPS Array utilizes the specific reaction between enzyme molecules on the biosensitive units and the corresponding target analytes to generate signal changes, thereby detecting the concentration of the target analytes. When the target molecules Glu, 1,5-AG, and P-HbA1c come into contact with the biosensitive membrane in the detection region, a catalytic oxidation reaction occurs on the membrane surface, resulting in a change in membrane potential. Different concentrations of analytes cause varying degrees of change in membrane potential, which further leads to potential shifts in the IV curve. By detecting changes in voltage offset, quantitative analysis of Glu, 1,5-AG, and HbA1c concentrations can be achieved. This method effectively and reliably enables the combined detection of diabetes biomarkers.

[0049] The specific implementation steps are as follows:

[0050] Step 1: Construction of the Optically Addressed Potential Sensor Array (LAPS Array) Detection System

[0051] 1. Fabrication of LAPS Array chip: A silicon substrate with a resistivity of 1-8 Ω·cm is provided as the substrate. A silicon dioxide (SiO2) layer and a silicon nitride (Si3N4) layer with a total thickness of 1.5 μm are formed on the substrate by thermal oxidation and chemical vapor deposition as a composite insulating layer. Four independent sensing areas are formed by dry etching process. A gold (Au) electrode with a thickness of 1200 Å is deposited on the back of the silicon substrate to form ohmic contact gold, resulting in a LAPSArray with a size of 10*10 mm.

[0052] (1) Construction of reaction cell and shell: A system shell is prepared by 3D printing technology and an electrochemical reaction cell is prepared by machining technology; the reaction cell includes a four-hole metal stage for supporting LAPS Array chip and an electrolytic cell. The electrolytic cell has four micro channels and a physical barrier is constructed at the bottom to control the distance between the reference electrode and the working electrode to 2 mm.

[0053] 2. Overall System Assembly: The fabricated LAPS Array chip is mounted on the metal stage, and the electrolytic cell is connected to the stage via magnetic attraction. The electrolytic cell is designed with four microchannels, and a physical barrier is constructed at the bottom of the electrolytic cell to control the distance between the Ag / AgCl reference electrode and the working electrode to 2 mm. The entire reaction cell is then integrated into the system housing. The four light source modules are fixed on the 3D housing, and a fabricated 3D coupling device is used to couple the light sources and optical fibers. The optical fibers are embedded in the four channels of the metal stage, aligning them with the four light source sensitive areas of the LAPS Array. The LAPS Array is sandwiched between the metal stage and the electrolytic cell of the electrochemical reaction device. An RF socket combined with an RF cable is used for stable signal transmission. The host computer plots IV curves in real time to visualize the four signals.

[0054] Step 2: Preparation of RGO-CS-Fc nanocomposite materials

[0055] 1. Preparation of RGO: 30 mg of GO powder was dissolved in 30 mL of ultrapure water, and the suspension was then sonicated for 120 min. 300 mg of AA was added and stirred to reduce the solution to obtain 1 mg / mL of RGO stock solution.

[0056] 2. Preparation of CS-Fc: 100 mg of CS and 150 mg of Fc were added to 100 mL of 1.0% glacial acetic acid solution (v / v), followed by the addition of NHS / EDC at a mass ratio of 4:1. The mixture was stirred continuously for 24 h until the solution turned reddish-brown. The solution was then centrifuged at 6000 r / min for 15 min to obtain a pure CS-Fc solution.

[0057] 3. Preparation of RGO-CS-Fc: Mix 15 mL of RGO with 10 mL of CS-Fc solution, stir for 4 h, centrifuge at 8000 r / min for 15 min, remove the supernatant, and obtain RGO-CS-Fc with high purity.

[0058] Step 3: Construction of LAPS Array Sensitive Units

[0059] 1. Pretreatment of LAPS Array: First, the LAPS Array was washed sequentially with ethanol and ultrapure water and then dried. The SEM characterization morphology of the clean bare silicon wafer is shown below. Figure 2 As shown in Figure A, the surface of the bare silicon wafer is smooth and flat. Then, the LAPS Array is activated by immersing it in 1.0 mol / L NaOH for 30 min, followed by washing with pure water and drying. Finally, 2 μL of 1% (v / v) MPTES solution is added to the LAPS Array, and the mixture is placed in a 4°C refrigerator for 12 h to react, thus obtaining a thiol-silanized chip (MPTES / LAPS Array). Its SEM morphology is shown below. Figure 2 As shown in Figure B, fine cracks can be seen on the surface of the hydrolyzed silicon wafer, indicating successful silanization.

[0060] 2. Modification of RGO-CS-Fc: 2 μL of RGO-CS-Fc solution was added to the LAPS Array, and the array was allowed to dry to obtain an RGO-CS-Fc / LAPS Array. Its SEM morphology is shown in the figure below. Figure 2 As shown in Figure C, RGO-CS-Fc exhibits a wrinkled, layered surface, indicating that the material has been modified onto the LAPS Array.

[0061] 3. Add 2 μL of 2.5% glutaraldehyde as a crosslinking agent to the diabetes biomarker detection area, and wash away excess glutaraldehyde after 15 min. Then, crosslink 2 μL of GOD, PROD, and FPOX to the three detection areas of the LAPS Array, respectively, with the fourth detection area serving as a blank control; construct the LAPS Array sensitive unit. Its SEM morphology is shown below. Figure 3 As shown in Figures A through C, the difference between the original wrinkled, layered surface (as shown in Figures A through C) is evident. The figures also show a change from the unmodified, layered surface (as shown in Figures A through C). Figure 2 Compared to (as shown in C), the surface morphology of the sensor after enzyme immobilization changed significantly. Figure 3 Figures A, 3B, and 3C correspond to the surfaces after GOD, PROD, and FPOX have been immobilized, respectively. All three figures show that a new, denser covering has appeared on the original wrinkled structure of the RGO-CS-Fc nanocomposite material, resulting in a fuller and rougher surface. The original lamellar edges and pore structures have been partially filled. This indicates that biomacromolecules such as GOD, PROD, and FPOX have been successfully immobilized on the sensor surface via glutaraldehyde crosslinking, thus verifying the successful construction of the sensing unit.

[0062] Step 4: Plotting the standard curve

[0063] 1. Two μL of Glu, 1,5-AG, and P-HbA1c were respectively added to the three detection zones of the LAPS Array sensitive unit constructed in step 2. P-HbA1c refers to the product obtained by hydrolyzing natural glycated hemoglobin (HbA1c) with porcine pepsin. This product is a polypeptide containing the characteristic fragment—fructo-valine-histanidine (Fru-Val-His)—which can be specifically recognized by fructosyl peptide oxidase (FPOX). After incubation at 25°C for 30 min, a LAPS Array capable of jointly detecting diabetes biomarkers was prepared. The SEM morphology of the three diabetes biomarker detection zones is shown below. Figure 3 As shown in D~F.

[0064] 2. Place the LAPS Array working electrode into the LAPS Array system, add 250 μL of 0.2 mol / L PBS solution with a pH of 7.4, insert the Ag / AgCl reference electrode, turn on the power, measure the IV response curve, and record the voltage offset change. Take three measurements for each group of experiments and take the average value.

[0065] 3. Combined detection was performed on standard solutions of different concentrations of Glu (0.5 mg / mL–3.0 mg / mL), 1,5-AG (10 μg / mL–350 μg / mL), and HbA1c (1.0–75.0 μg / mL) to obtain the IV curve response for different concentrations (e.g., Figure 4 (A~C) For Glu, within the range of 0.5–3.0 mg / mL, the Glu concentration (X1) showed a linear relationship with the voltage offset (ΔY1): ΔY1 = 59.0058X1 + 73.3371 (R² = 0.9985), with a sensitivity of 59.0058 mV / mg / mL and a limit of detection (LOD) of 0.5 mg / mL. For 1,5-AG, within the range of 10–350 μg / mL, the 1,5-AG concentration (X2) showed a linear relationship with the voltage offset (ΔY2): ΔY2 = 0.7223X2 + 25.8996 (R² = 0.9173), with a sensitivity of 0.7223 mV / μg / mL and a LOD of 10 μg / mL. Within the range of 1.0–75.0 μg / mL, the HbA1c concentration (X3) showed a linear relationship with the voltage offset (ΔY3): ΔY3 = 3.1309X3 + 93.6938 (R² = 0.9806), with a sensitivity of 3.1309 mV / μg / mL and a LOD of 1.0 μg / mL.

[0066] Step 5: Combined detection of diabetes biomarkers in actual blood samples

[0067] 1. 2 μL of blood sample was sequentially added to the sensitive units of the three detection areas of the LAPS Array and incubated in a 25°C incubator for 30 min. Four clinical samples were selected, with the clinical parameter being the percentage of HbA1c. The HbA1c values ​​were 4.8% (sample 1), 5.4% (sample 2), 8.3% (sample 3), and 11.1% (sample 4), respectively. According to the conversion formula between average blood glucose and glycated hemoglobin (average blood glucose (mmol / L) = glycated hemoglobin content × 1.6 - 2.6), the corresponding blood glucose values ​​were 0.9144 mg / mL (sample 1), 1.0872 mg / mL (sample 2), 1.9224 mg / mL (sample 3), and 2.7288 mg / mL (sample 4). In the detection of Glu, the direct method was used, and 2 μL of supernatant was directly dropped onto the sensitive unit. In the detection of 1,5-AG, a spiking method was used. 50 μL of each supernatant sample was mixed with 1.0 mg / mL of 1,5-AG standard at a ratio of 19:1 and then modified onto the corresponding sensitive unit, resulting in a spiked concentration of 50 μg / mL. In the detection of HbA1c, since HbA1c cannot be directly used for enzymatic digestion, blood samples required pretreatment. Before detection, the blood sample was centrifuged at 8000 rpm for 15 min. 25 μL of the precipitate (red blood cell fraction) was taken, and 10 μL of 500 kU / L porcine pepsin was added. The mixture was incubated at 37℃ for 3 min to decompose HbA1c into a solution containing Fru-ValHis, which was then modified onto the HbA1c detection area. The voltage offset obtained from different blood samples was substituted into the corresponding standard curve to obtain the corresponding concentration value, and the relative error between the detected value and the actual concentration was calculated. Before combined detection, six blood samples with HbA1c contents (data measured by high-performance liquid chromatography) of 4.8%, 5.4%, 6.7%, 7.6%, 8.3%, and 11.1% were selected for testing. The corresponding voltage offsets were obtained, and a curve showing the relationship between voltage offset and HbA1c content was plotted. The normalized IV curves obtained from blood samples with different HbA1c contents are shown below. Figure 4 As shown in Figure A, with increasing HbA1c content, the IV curve skews to the right, and the voltage offset corresponding to the blank group increases. Within the range of 4.8% to 11.1%, the potential offset (ΔY4) shows a good linear relationship with the HbA1c content (X4), with the linear equation ΔY4 = 31.0350X4 - 28.3084 (R² = 0.9745), and the detection sensitivity is 31.0350 mV / s.

[0068] 2. Place the LAPS Array in the LAPS Array system. The signal acquisition board collects, analyzes, and processes the response signal. Normalize the detected data, using the normalized photocurrent value at 0.5 as the working point. At the working point, calculate the voltage offset between the detection areas modified with different diabetes biomarkers and the blank group (naked detection area). Substitute the voltage offset obtained for each detection area into the standard curves in step 3: Glu standard curve: ΔY1=59.0058X1+73.3371; 1,5-AG standard curve: ΔY2=0.7223X2+25.8996; HbA1c standard curve: standard solution curve: ΔY3=3.1309X3+93.6938 and actual sample standard curve: ΔY4=31.0350X4-28.3084. Calculate the concentrations of Glu, 1,5-AG, and HbA1c in the blood sample. The detection results are shown in Tables 1, 2, and 3.

[0069] Table 1 Results of LAPS Array detection of Glu in blood samples

[0070]

[0071] Sample 1: Normal blood glucose; Sample 2: Normal blood glucose; Sample 3: Mild hyperglycemia; Sample 4: Severe hyperglycemia

[0072] Table 2 Results of LAPS Array detection of 1,5-AG in blood samples

[0073]

[0074] Table 3 Results of LAPS Array detection of HbA1c in blood samples

[0075]

Claims

1. A method for constructing an optically addressed potential sensor array for the joint detection of diabetes biomarkers, characterized in that, Includes the following steps: Step 1: Construction of the Optically Addressed Potential Sensor Array (LAPS Array) Detection System (1) Fabrication of LAPS Array chip: A silicon substrate with a resistivity of 1-8 Ω·cm is provided as the substrate. A silicon dioxide SiO2 layer and a silicon nitride Si3N4 layer with a total thickness of 1-2 μm are formed on the substrate by thermal oxidation and chemical vapor deposition as a composite insulating layer. Multiple independent sensing areas are formed by dry etching process. A gold Au electrode with a thickness of 1000-1500 Å is deposited on the back of the silicon substrate to form ohmic contact gold, thus obtaining LAPS Array; (1) Construction of reaction cell and shell: A system shell is prepared by 3D printing technology and an electrochemical reaction cell is prepared by machining technology; the reaction cell includes a four-hole metal stage for supporting LAPS Array chip and an electrolytic cell. The electrolytic cell has four micro channels and a physical barrier is constructed at the bottom to control the distance between the reference electrode and the working electrode to 2 mm. (2) Overall system assembly: The fabricated LAPS Array chip is loaded onto the metal stage, and the electrolytic cell is connected to the stage by magnetic attraction. Then, the reaction cell is integrated into the system housing. The housing also integrates a data acquisition board and a light source array. Step 2: Preparation of reduced graphene oxide-chitosan-ferrocene composite material RGO-CS-Fc (1) Preparation of reduced graphene oxide (RGO): Graphene oxide (GO) powder was ultrasonically dispersed, ascorbic acid (AA) was added, and the powder was reduced to obtain RGO stock solution; (2) Preparation of chitosan-ferrocene CS-Fc: dilute glacial acetic acid, add chitosan CS and ferrocene carboxylic acid Fc, then add N-hydroxysuccinimide / 1-ethyl-(3-dimethylaminopropyl)NHS / EDC, wherein the mass ratio of NHS / EDC is 4:1, stir and centrifuge to obtain a pure CS-Fc solution; (3) Preparation of reduced graphene oxide-chitosan-ferrocene nanocomposite RGO-CS-Fc: RGO was added to the CS-Fc solution; the mixture was stirred and centrifuged to remove the supernatant; the RGO-CS-Fc solution was obtained. Step 3: Construction of Sensitive Units (1) Preprocessing of optically addressed potential sensor array LAPS Array: The LAPS Array chip is cleaned and dried, then immersed in sodium hydroxide (NaOH) for activation, cleaned and dried, and 3-mercaptopropyltriethoxysilane (MPTES) solution is added to obtain MPTES / LAPSArray; (2) Modification of RGO-CS-Fc: RGO-CS-Fc is added to the surface of MPTES / LAPS Array and dried to obtain RGO-CS-Fc / LAPS Array; (3) Glutaraldehyde was added as a cross-linking agent to the detection area of ​​diabetes markers, and then glucose oxidase GOD, pyranose oxidase PROD, and fructosyl peptide oxidase FPOX were cross-linked to the three detection areas of the LAPS Array respectively. The fourth detection area was used as a blank group to construct a sensitive LAPS Array unit with three detection areas. Step 4: Plotting the standard curve (1) Glucose Glu, 1,5-sorbitol 1,5-AG and pretreated-glycated hemoglobin P-HbA1c were respectively added to the three detection areas of the LAPS Array sensitive unit in step 2 and incubated to prepare a LAPS Array that can jointly detect diabetes biomarkers; (2) Place the LAPS Array working electrode into the LAPS Array system, add phosphate buffer PBS, insert the Ag / AgCl reference electrode, turn on the power, measure the IV response curve, and record the voltage offset change; (3) Detection of Glu, 1,5-AG and HbA1c standard solutions of different concentrations, plotting working curves, and obtaining the detection limits of the three diabetes biomarkers; Step 5: Combined detection of diabetes biomarkers in actual blood samples (1) Add actual blood samples to the interface of the sensitive unit of the LAPS Array, incubate, and prepare a LAPS Array that can jointly detect diabetes biomarkers; (2) Place the LAPS Array into the LAPS Array system, and the signal acquisition board collects and analyzes the response signal; calculate the concentrations of Glu, 1,5-AG and HbA1c according to the standard curve obtained in step 3.

2. The method according to claim 1, characterized in that: The GO in step 2 is 30 mg; the ascorbic acid is 300 mg; and the RGO concentration is 1 mg / mL.

3. The method according to claim 1, characterized in that: The volume ratio of the diluted glacial acetic acid in step 2 is 1.0%; the CS is 100 mg; the Fc is 150 mg; and the volume ratio of glacial acetic acid solution to NHS / EDC is 10:

1.

4. The method according to claim 1, characterized in that: In step 3, the concentration of NaOH is 1 mol / L; the volume ratio of MPTES is 1%, and the amount used is 2 μL; the amount of RGO-CS-Fc used is 2 μL; the concentrations of GOD, PROD, and FPOX enzymes are 0.5 mg / mL, 1.0 mg / mL, and 1.0 mg / mL, respectively; and the amount used for each is 2 μL.

5. The method according to claim 1, characterized in that: In step 4, the pH of the PBS is 7.4 and the concentration is 0.2 mol / L; the amount of Glu, 1,5-AG and HbA1c used is 2 μL; the incubation temperature of the enzymes GOD, PROD, FPOX and the diabetes markers Glu, 1,5-AG and HbA1c is 25°C and the incubation time is 30 min.

6. The optically addressed potential sensor array for the combined detection of diabetes biomarkers obtained by the method according to any one of claims 1-5.

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