Preparation and application of synchronous detection total hemoglobin and glycosylated hemoglobin ratio sensor

Abstract

The application discloses a preparation method of a ratio type electrochemical sensor for synchronous detection of total hemoglobin (tHb) and glycosylated hemoglobin (HbA 1c ) "double signal opening". The application also discloses a method for synchronous quantitative detection of tHb and HbA 1c by using the ratio type sensor, and application of the method to accurate quantitative determination of blood sugar in a whole blood sample. Experimental results show that the ratio type "double signal opening" electrochemical sensor constructed by the application has high accuracy, high selectivity, high sensitivity and good anti-interference performance, and is suitable for accurate evaluation of tHb and HbA 1c in a clinical blood sample.

Description

Technical Field

[0001] This invention belongs to the field of analytical technology, specifically relating to a method for simultaneously detecting total hemoglobin (tHb) and glycated hemoglobin (HbA1c). 1c Preparation method and application of ratio-type "dual-signal on" electrochemical sensor. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by persistent hyperglycemia. Long-term blood glucose control is closely related to the occurrence of various complications. Glycated hemoglobin (HbA1c) is a key indicator of diabetes. 1c HbA1c is a product formed by the glycosylation reaction of hemoglobin with glucose under non-enzymatic conditions. Its content can reflect a patient's average blood glucose level over the past 2-3 months, and is therefore widely used in the diagnosis and blood glucose management of diabetes. However, measuring HbA1c alone is insufficient for accurate diagnosis. 1c When using absolute concentrations for diagnosis, the results may be affected by individual differences in erythrocyte lifespan and glycosylation kinetics, leading to biases in specific populations. To improve the reliability of test results, HbA1c is commonly used clinically. 1c The proportion of total hemoglobin (tHb, including glycated and non-glycated hemoglobin), i.e., HbA1c 1c Level (HbA) 1c / tHb), as a more stable and clinically meaningful evaluation indicator.

[0003] Currently, high performance liquid chromatography (HPLC) is considered the method for determining HbA1c. 1c A horizontal reference method. This method enables the separation of HbA2 by chromatography. 1c While quantitative detection of tHb offers high analytical accuracy, this method typically relies on large, sophisticated instruments, is relatively time-consuming, complex, and costly, hindering its widespread application in primary healthcare institutions or point-of-care testing scenarios. In recent years, electrochemical sensing technology has gained traction in HbA1c testing due to its advantages such as ease of operation, rapid response, high sensitivity, and suitability for miniaturization and portability. 1c The detection field has received widespread attention. Most existing electrochemical detection methods typically employ a dual-electrode or dual-channel system to separately detect HbA1c. 1c Hb and tHb are measured independently. This type of detection requires constructing two separate detection interfaces, which not only increases the complexity of the system structure but may also introduce systematic errors due to differences in electrode surface conditions and fluctuations in the detection environment, affecting the accuracy of the final ratio calculation. To simplify the detection process, researchers proposed a dual-signal detection strategy based on a single electrode to achieve HbA2 / tHb ... 1cSimultaneous assessment with tHb. However, existing methods for tHb detection rely on a signal attenuation ("signal-off") response mechanism. This type of signal mode is sensitive to background current fluctuations and is easily affected by non-specific adsorption and matrix interference in complex biological sample systems. Furthermore, a single electrochemical signal lacks an internal ratio calibration mechanism, making it difficult to effectively eliminate errors caused by environmental fluctuations in complex sample environments. Therefore, it is necessary to develop a dual-signal enhanced electrochemical sensing system to achieve HbA2 / tHb detection. 1c Accurate quantitative analysis based on ratio with tHb. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a ratiometric electrochemical sensor with "dual signal activation" for tHb and HbA. 1c At the same time, accurate quantitative analysis.

[0005] This invention also discloses a method for preparing the above-mentioned "dual-signal activation" ratiometric electrochemical sensor, characterized by comprising the following steps: Step 1: Polish the glassy carbon electrode (GCE) with a diameter of 3 mm to a mirror finish with 0.05 μm alumina (Al2O3) powder, then ultrasonically clean it in ethanol and deionized water, and dry it at room temperature to obtain a clean glassy carbon electrode. Step 2: Drop-coat 5 μL of GO-CNT / AuNPs composite dispersion onto the surface of the glassy carbon electrode obtained in Step 1, and dry it under an infrared lamp to obtain a GCE / GO-CNT / AuNPs modified electrode. The preparation method of the GO-CNT / AuNPs composite dispersion is as follows: Graphene oxide (GO) and carbon nanotubes (CNTs) at a concentration of 0.5-2 mg / mL are dispersed in deionized water and ethanol-water mixed solvent, respectively. After ultrasonic mixing, polyvinylpyrrolidone (PVP) stabilizer and chloroauric acid solution (HAuCl4) at a concentration of 5-30 mM are added. Then, sodium borohydride (NaBH4) is slowly added dropwise to reduce and generate gold nanoparticles (AuNPs). After centrifugation, washing, and drying, the nanoparticles are dispersed in water. The concentration of PVP is 1 mg / mL, and the concentration of NaBH4 is 0.5 mg / mL. Step 3: Place the modified electrode obtained in Step 2 in 0.1 M PBS buffer (pH 7.4) and electrochemically reduce it using electrodeposition to obtain the GCE / ERGO-CNT / AuNPs electrode; the electrodeposition potential is −1.2 V and the electrodeposition time is 50-200 s; Step 4: Immerse the electrode obtained in Step 3 in a 10-100 μM anthraquinone (AQ) solution and incubate at room temperature for 1-5 h to fix AQ molecules through π-π stacking. After washing, the GCE / ERGO-CNT / AuNPs / AQ electrode is obtained. Step 5: The electrode obtained in Step 4 is activated for 1 h in a PBS solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, 10 μL of 2-10 μM anti-hemoglobin antibody solution (Ab) is added, and the electrode is incubated at room temperature to covalently fix the antibody. After washing, the electrode is blocked with bovine serum albumin (0.5% BSA) solution for 10-60 min to obtain the GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA immunosensing electrode. The concentration of EDC / NHS is 0.1-0.5 M, and the incubation time at room temperature is 1-3 h.

[0006] This invention also discloses a ratiometric "dual-signal on" electrochemical sensor prepared using the above method for measuring tHb ​​and HbA. 1c The method for simultaneous quantitative detection is characterized by the following steps: Step 1, combine tHb and HbA 1c Alternatively, whole blood samples to be tested can be hemolyzed to obtain hemoglobin-lysed blood, which can then be diluted to different concentrations with 10mM phosphate-buffered saline (PBS) at pH 7.4. Step 2: The prepared sensor is incubated sequentially with the tHb-containing sample obtained in Step 1, and then incubated with a 2-10 mM ferrocene boric acid (FcBA) solution, i.e., GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA / tHb / FcBA. After washing, it is placed in PBS buffer (pH 7.4) containing thionine (Thi) and 2-10 mM hydrogen peroxide (H2O2). The incubation time for tHb is 10-40 min, the incubation time for ferrocene boric acid is 40 min, and the concentration of thionine is 5-50 μM. Step 3: Differential pulse voltammetry (DPV) measurement was performed using a three-electrode system (sensor as working electrode, Ag / AgCl as reference electrode, and Pt wire as counter electrode) to record the reduction peak currents of AQ, Thi, and FcBA; the parameters were set as follows: scan range, 0.5 V - −0.7 V; amplitude, 0.05 V; pulse period, 0.5 s; potential increment, 4.0 mV. Step 4, based on the current ratio I Thi / I AQ and I FcBA / I AQ Calculate tHb concentration and HbA1c concentration respectively. 1c Concentration and HbA 1c Level (HbA) 1c(Percentage of tHb) to achieve simultaneous quantitative analysis of dual objectives.

[0007] This invention further discloses a ratiometric electrochemical sensor with "dual signal on" prepared by the above method for use in whole blood samples to detect tHb and HbA1c. 1c The application of accurate quantitative analysis shows that the results obtained by electrochemical detection using the ratio-type "dual-signal on" electrochemical sensor prepared by this method are in excellent agreement with the test results obtained by the standard method of high performance liquid chromatography (HPLC), proving the reliability and practicality of this method.

[0008] The present invention is described in more detail below: This invention discloses a method for simultaneously detecting tHb ​​and HbA. 1c The method for preparing a ratiometric "dual-signal on" electrochemical sensor includes the following steps: Step 1: Polish the glassy carbon electrode (GCE) to a mirror finish with 0.05 μm alumina powder, then ultrasonically clean it in ethanol and deionized water, and dry it at room temperature to obtain a clean glassy carbon electrode. Step 2: Drop-coat 5 μL of GO-CNT / AuNPs composite dispersion onto the surface of the glassy carbon electrode obtained in Step 1, and dry it under an infrared lamp to obtain a GCE / GO-CNT / AuNPs modified electrode; The preparation method of the GO-CNT / AuNPs composite dispersion is as follows: Graphene oxide (GO) and carbon nanotubes (CNT) are dispersed in deionized water and ethanol-water mixed solvent, respectively. After ultrasonic mixing, polyvinylpyrrolidone (PVP) stabilizer and chloroauric acid solution (HAuCl4) are added, and then sodium borohydride (NaBH4) is slowly added to reduce and generate gold nanoparticles (AuNPs). After centrifugation, washing and drying, the nanoparticles are dispersed in water; Step 3: Place the modified electrode obtained in Step 2 in 0.1 M PBS buffer (pH 7.4) and electrochemically reduce it using electrodeposition to obtain the GCE / ERGO-CNT / AuNPs electrode; Step 4: Immerse the electrode obtained in Step 3 in anthraquinone (AQ) solution and incubate at room temperature. AQ molecules are fixed by π-π stacking. After washing, the GCE / ERGO-CNT / AuNPs / AQ electrode is obtained. Step 5: The electrode obtained in Step 4 was activated in a PBS solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 1 h. Then, 10 μL of anti-hemoglobin antibody solution (Ab) was added and incubated at room temperature to covalently fix the antibody. After washing, it was blocked with bovine serum albumin (BSA) solution to obtain the GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA immunosensing electrode.

[0009] The diameter of the glassy carbon electrode in step 1 is 3 mm; In step 2, the concentrations of GO and CNT are 0.5-2 mg / mL, preferably 1 mg / mL. In step 2, the concentration of PVP is 1 mg / mL and the concentration of NaBH4 is 0.5 mg / mL. In step 2, the concentration of HAuCl4 is 5-30 mM, preferably 5 mM; In step 3, the electrodeposition potential is −1.2 V and the electrodeposition time is 50-200 s, preferably 100 s; In step 4, the AQ concentration is 60 μM, and the incubation time at room temperature is 1-5 h, preferably 1 h. In step 5, the concentration of EDC / NHS is 0.1 M, the concentration of the antihemoglobin antibody solution is 2-10 μM, preferably 5 μM, and the incubation time at room temperature is 1-3 h, preferably 2 h. In step 5, the BSA concentration is 0.5%, and the incubation time is 10-60 min, preferably 30 min. This invention also provides a ratiometric "dual-signal on" electrochemical sensor prepared by the above method for measuring tHb ​​and HbA. 1c The method for simultaneous and accurate quantitative detection includes the following specific steps: Step 1, combine tHb and HbA 1c Alternatively, whole blood samples to be tested can be hemolyzed to obtain hemoglobin-lysed blood, which can then be diluted to different concentrations with 10mM phosphate-buffered saline (PBS) at pH 7.4. Step 2: Incubate the prepared sensor sequentially with the tHb-containing sample obtained in Step 1, and then incubate with 2-10 mM ferrocene boric acid (FcBA) solution, i.e., GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA / tHb / FcBA. After washing, place it in PBS buffer (pH 7.4) containing thionine (Thi) and 2-10 mM hydrogen peroxide (H2O2). Step 3: Electrochemical measurements were performed using a three-electrode system (sensor as working electrode, Ag / AgCl as reference electrode, and Pt wire as counter electrode), and the reduction peak currents of AQ, Thi, and FcBA were recorded. Step 4: Calculate the tHb concentration and HbA based on the current ratio. 1c Concentration and percentage, enabling simultaneous quantitative analysis of dual objectives.

[0010] In step 2, the incubation time between the sensor and tHb is 10-40 min, preferably 20 min; In step 2, the FcBA incubation time is 40 min, and the FcBA concentration is 2-10 mM, preferably 8 mM. In step 2, the Thi concentration is 5-50 μM, preferably 25 μM; In step 2, the concentration of H2O2 is 2-10 mM, preferably 8 mM; In step 3, the electrochemical method is differential pulse voltammetry (DPV), with the following parameters: scan range, 0.5V - −0.7V; amplitude, 0.05V; pulse period, 0.5s; and potential increment, 4.0mV. Among them, the current ratios in step 4 are respectively I Thi / I AQ and I FcBA / I AQ .

[0011] This invention aims to solve the problem that existing electrochemical detection methods are difficult to achieve tHb and HbA at a single electrode interface. 1c The problem lies in simultaneous and accurate measurement. To address this goal, this invention focuses on verifying the ratiometric self-calibration capability, anti-interference performance, and applicability of the constructed sensor in real-world samples. The key technical challenge in the experiment lies in constructing a "dual-signal on-state" output on the same electrode and introducing a stable internal reference signal to achieve real-time ratio calibration, thereby reducing the impact of non-specific adsorption and background fluctuations in complex biological matrices on the detection results.

[0012] The beneficial effect of this invention lies in disclosing the fabrication and application of a ratiometric electrochemical sensor with "dual signal activation". This sensor specifically captures tHb via antibody and enhances the Thi signal using the catalysis of endogenous heme in hemoglobin; simultaneously, it utilizes FcBA to specifically bind HbA via borate ester bonds. 1c The cis-diol structure at the mid-glycated end enables the realization of tHb and HbA on a single electrode. 1c Simultaneous quantitative detection. Furthermore, AQ, as a stable internal reference molecule, is detected through a signal ratio (...). I Thi / I AQ and I FcBA / I AQThis method performs real-time self-calibration, thereby improving the accuracy, repeatability, and anti-interference ability of the detection. It is simple to operate, highly sensitive, and has a wide linear range, and can be used for tHb and HbA1c in whole blood samples. 1c The rapid determination provides a promising detection strategy for diabetes screening and long-term blood glucose monitoring, and offers a reference for the construction of a multi-index simultaneous electrochemical detection platform. Attached Figure Description

[0013] Figure 1 The following is a DPV curve of the sensor step-by-step construction process in Embodiment 2 of the present invention: where a is the bare GCE, b is GCE / ERGO-CNT / AuNPs, c is GCE / ERGO-CNT / AuNPs / AQ, d is GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA, and e is GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA / tHb / FcBA; Figure 2 The sensor in Embodiment 3 of the present invention measures tHb and HbA. 1c Validation diagram of the identification strategy: Figure A shows the antibody against HbA1c. 1c Verification of affinity with non-glycated Hb, AuNPs+Ab(a), AuNPs+Ab+HbA 1c (b) and UV-Vis spectra of AuNPs+Ab+unsaccharified Hb(c) (where HbA) 1c (Both non-glycated Hb concentrations were 50 nM); Figure B shows HbA. 1c The catalytic activity of non-glycated Hb on Thi, i.e., antibody competition verification, was performed by measuring HbA with the same tHb concentration but different HbA levels. 1c Level samples (in all cases, tHb concentration was 100 nM, while HbA...) 1c The concentrations were 0 nM (a), 0.5 nM (b), 5 nM (c), 50 nM (d), and 500 nM (e). Figure 3 The sensor in Embodiment 4 of the present invention measures tHb and HbA. 1c Analytical performance graph for quantitative detection: where a represents the sensor pair's performance on different concentrations of tHb and HbA in 0.1 M PBS (pH 7.4) containing 25 μM Thi and 8 mM H2O2. 1c The DPV response curve, where b is I Thi / I AQ The linear relationship between tHb concentration and logarithmic value, where c is... I FcBA / I AQWith HbA 1c Linear relationship graph of concentration logarithmic value; Figure 4 The sensor in Embodiment 5 of the present invention measures the target tHb and HbA. 1c Selective test chart; Figure 5 For the stability and repeatability tests of the sensor in Example 6 of this invention: a) a 20-day stability test of the same electrode in 0.1 M PBS (pH 7.4) containing 25 μM Thi and 8 mM H2O2; b) a repeatability test of five different electrodes prepared by the same method in 0.1 M PBS (pH 7.4) containing 25 μM Thi and 8 mM H2O2 (the purple and blue bars in the figure represent the sensor's response to tHb and HbA, respectively). 1c (Detection performance) Figure 6 For the actual clinical sample testing in Example 7 of this invention, the sensor and the standard HPLC method for detecting HbA1c were verified. 1c Consistency: a is Passing-Bablok regression analysis, b is Bland-Altman analysis (solid line: mean difference; dashed line: consistency boundary). Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are only for illustrating the technical solutions of this invention and are not intended to limit it. Any adjustments and optimizations to the process parameters made by those skilled in the art without departing from the spirit and substance of this invention should be included within the scope of protection of this invention. All raw materials and reagents used in this invention are commercially available. Example

[0015] Fabrication of a ratiometric dual-signal on-off electrochemical sensor A 3 mm diameter glassy carbon electrode (GCE) was polished to a mirror finish with 0.05 μm alumina powder, then ultrasonically cleaned in ethanol and deionized water, and dried at room temperature to obtain a clean glassy carbon electrode. GO and CNT were separately dispersed in an ethanol-water mixture, ultrasonically mixed, and then PVP stabilizer and HAuCl4 were added. NaBH4 was then slowly added dropwise to reduce AuNPs, which were then centrifuged, washed, and dispersed in water to obtain a GO-CNT / AuNPs composite dispersion. 5 μL of the GO-CNT / AuNPs composite dispersion was drop-coated onto the surface of the glassy carbon electrode and dried under an infrared lamp to obtain a GCE / GO-CNT / AuNPs modified electrode. The electrode was placed in 0.1 M PBS buffer (pH 7.4) and electrochemically reduced for 100 s at −1.2 V (vs. Ag / AgCl) to obtain a GCE / ERGO-CNT / AuNPs electrode. The electrode was then immersed in 60 mM AQ solution and incubated at room temperature for 1 hour. h, AQ molecules were fixed by π-π stacking, and after washing, a GCE / ERGO-CNT / AuNPs / AQ electrode was obtained; the obtained electrode was activated in PBS solution containing 0.1 M EDC / NHS for 1 h, and then 10 μL of 50 μM antihemoglobin antibody solution was added, and incubated at room temperature for 2 h to covalently fix the antibody. After washing, it was blocked with 0.5% BSA solution for 30 min to obtain the GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA immunosensing electrode. Example

[0016] Construction and characterization of a ratiometric dual-signal on-off electrochemical sensor The electrochemical response performance of the above sensor was tested using differential pulse voltammetry (DPV) in a 0.1M PBS (pH 7.4) solution containing 25 μM Thi and 8 mM H2O2, with a CHI832D electrochemical workstation. Figure 1As shown, the unmodified glassy carbon electrode exhibits a reduction peak of Thi at approximately −0.268 V (curve a). After modification with ERGO-CNT / AuNPs, the Thi signal is enhanced (curve b), indicating that the modification layer improves the electron transport capability at the electrode interface. After introducing AQ via π-π stacking, a distinct new peak appears near −0.516 V (curve c), which is attributed to the electrochemical reduction signal of AQ. Subsequently, after immobilization of the antibody (Ab) and blocking agent (BSA), the peak currents of both Thi and AQ decrease to some extent due to the formation of a protein layer at the interface hindering electron transport (curve d). When the electrode is further incubated with tHb and FcBA, the Thi peak current significantly increases. This enhancement is attributed to the peroxidase-like catalytic effect of the heme groups in tHb on the electrochemical reaction of Thi. Simultaneously, an electrochemical peak corresponding to Fc appears at approximately 0.236 V (curve e), indicating that FcBA binds to HbA via a borate ester bond. 1c Specific binding occurs between the cis-diol structure and the thi signal. The enhancement of the Thi signal and the appearance of the Fc signal indicate that the constructed system can achieve tHb and HbA binding at a single electrode interface. 1c The synchronous response forms a ratio detection platform with "dual signal activation". Example

[0017] Antibody binding competition and catalytic activity verification First, the binding affinity of the antibody to glycated hemoglobin and non-glycated hemoglobin was evaluated using ultraviolet-visible spectroscopy. After incubating antibody-modified gold nanoparticles with equal concentrations of glycated and non-glycated hemoglobin, the resulting spectra all exhibited a Soret characteristic absorption peak (derived from the heme structure) at a wavelength of 409 nm, and the absorption intensities were essentially consistent. Figure 2 a) indicates that the binding efficiency of the two hemoglobins to the antibodies is comparable. To further investigate the competitive nature of antibody binding and the differences in catalytic activity, four working electrodes modified with the same antibody were placed in test samples with constant total hemoglobin concentration but different glycated hemoglobin ratios for detection. Differential pulse voltammetry (DPV) results showed that the reduction signal of ferrocene boric acid (FcBA) increased linearly with increasing glycated hemoglobin content, while the reduction current of thionine (Thi) remained constant in all samples. Figure 2 (b) This result confirms that glycated hemoglobin and non-glycated hemoglobin have similar catalytic activity for thionine reduction, and that there is no competitive inhibition between them on the binding of the antibody. These results demonstrate that the sensor described in this invention can simultaneously and accurately detect both total hemoglobin and glycated hemoglobin, providing experimental evidence for the reliability of the "dual-signal on" ratiometric electrochemical sensing platform. Example

[0018] tHb and HbA1c Electrochemical detection Under optimized experimental conditions, different concentrations of tHb and HbA were detected. 1c The analytical performance of the constructed ratiometric "dual-signal on" electrochemical sensor was evaluated. For example... Figure 3 As shown in a, with tHb and HbA 1c With increasing concentration, the peak current intensities of Thi and FcBA gradually increased, while the peak current of AQ remained relatively stable, indicating that the constructed system can achieve ratiometric detection. Figure 3 As shown in b, within the concentration range of 10 pM to 100 μM, I Thi / I AQ The current ratio showed a good linear relationship with the logarithm of tHb concentration, and the regression equation was y = 0.213x + 1.490 (R²). 2 = 0.998). For HbA 1c , I FcBA / I AQ The current ratio showed a linear correlation in the concentration range of 0.5 pM to 5 μM, conforming to the equation y = 0.070x + 0.637 (R0). 2 = 0.996), such as Figure 3 As shown in c. tHb and HbA 1c The detection limits were 1.24 pM and 0.35 pM, respectively. These results demonstrate that the constructed ratiometric "dual-signal on" electrochemical sensor can detect tHb and HbA. 1c Quantitative analysis. Example

[0019] Selectivity testing of sensors To evaluate the selectivity of the constructed sensor in complex sample environments, it was tested by exposing it to solutions of interfering substances that may coexist in human blood samples. These interfering substances included common high-abundance serum proteins such as human serum albumin (HSA), immunoglobulin G (IgG), and lysine (Lys); common small molecule biological substances such as glucose (Glu), fructose (Fru), ascorbic acid (AA), uric acid (UA), and dopamine (DA); and representative inorganic ions (Na₂O₃). + K + Ca 2+ and Mg 2+ ).like Figure 4 As shown, when the sensor is only exposed to various interferences, I Thi / I AQ andI FcBA / I AQ The ratios showed no significant response; however, when the sensor was exposed to substances containing tHb ​​or HbA... 1c In solutions containing both Hb and HbA, regardless of the presence of interfering substances, the two exhibit similar ratio signals. These results indicate that the constructed ratiometric "dual-signal on" electrochemical sensor effectively detects the interaction between tHb and HbA. 1c The detection has good selectivity. Example

[0020] Stability and repeatability testing of the sensor To evaluate the storage stability of the constructed sensor, the prepared electrode was stored at 4 °C, and its electrochemical response signal was recorded every 4 days. The results are as follows: Figure 5 As shown in Figure a, after 20 days of storage, tHb and HbA... 1c The corresponding signal ratios remained at 97.2% and 93.7% of their initial values, respectively, indicating that the sensor can maintain relatively stable detection performance during storage. Furthermore, under the same experimental conditions, five independently prepared electrode pairs tHb and HbA were selected. 1c Testing was conducted to evaluate the repeatability of the sensor. The results are as follows: Figure 5 As shown in b, tHb and HbA 1c The relative standard deviations (RSDs) of the signal ratios were 1.22% and 1.86%, respectively, indicating that the constructed ratiometric "dual-signal on" electrochemical sensor has good repeatability. Example

[0021] Actual clinical sample testing and validation To evaluate the detection performance of the constructed sensor in real samples, it was applied to the HbA1c detection of 100 whole blood samples. 1c Horizontal detection. HbA1c levels in the above samples. 1c The levels were determined using high-performance liquid chromatography (HPLC) as a control method, with sample concentrations covering both healthy and diabetic levels. Figure 6 As shown in Figure a, Passing-Bablok regression was used to analyze the correlation between the detection results of the method of this invention and the HPLC method. The correlation coefficient obtained was 0.995, indicating that the detection results of the two methods have a good correlation, and no obvious systematic or proportional bias was observed. Further Bland-Altman analysis was used to evaluate the consistency between the two methods, such as... Figure 6As shown in b, the 95% consensus threshold ranged from -0.154% to +0.159%. 98% of the data points fell within this threshold. These results demonstrate that the constructed ratiometric dual-signal on / off electrochemical sensor can achieve detection results similar to HPLC in whole blood sample testing, validating its application in HbA1c in diabetes mellitus. 1c Practical potential and reliability in horizontal testing.

[0022] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing a ratiometric "dual-signal on" electrochemical sensor for simultaneous detection of total hemoglobin and glycated hemoglobin, characterized in that... Follow these steps: Step 1: Polish the glassy carbon electrode (GCE) with a diameter of 3 mm to a mirror finish with 0.05 μm alumina powder, then ultrasonically clean it in ethanol and deionized water, and dry it at room temperature to obtain a clean glassy carbon electrode. Step 2: Drop-coat 5 μL of GO-CNT / AuNPs composite dispersion onto the surface of the glassy carbon electrode obtained in Step 1, and dry it under an infrared lamp to obtain a GCE / GO-CNT / AuNPs modified electrode. The preparation method of the GO-CNT / AuNPs composite dispersion is as follows: GO and CNT, with a concentration of 0.5-2 mg / mL, are dispersed in deionized water and ethanol-water mixed solvent, respectively. After ultrasonic mixing, PVP stabilizer and HAuCl4, with a concentration of 5-30 mM, are added. Then, NaBH4 is slowly added dropwise to reduce and generate AuNPs. After centrifugation, washing, and drying, the AuNPs are dispersed in water. The concentration of PVP is 1 mg / mL, and the concentration of NaBH4 is 0.5 mg / mL. Step 3: Place the modified electrode obtained in Step 2 in 0.1 M PBS buffer (pH 7.4) and electrochemically reduce it using electrodeposition to obtain the GCE / ERGO-CNT / AuNPs electrode; the electrodeposition potential is −1.2 V and the electrodeposition time is 50-200 s; Step 4: Immerse the electrode obtained in Step 3 in a 10-100 μM anthraquinone (AQ) solution and incubate at room temperature for 1-5 h to fix AQ molecules through π-π stacking. After washing, the GCE / ERGO-CNT / AuNPs / AQ electrode is obtained. Step 5: Activate the electrode obtained in Step 4 in a PBS solution containing EDC and NHS for 1 h, then add 10 μL of 2-10 μM anti-hemoglobin antibody solution (Ab), incubate at room temperature to covalently fix the antibody, wash, and block with 0.5% BSA solution for 10-60 min to obtain the GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA immunosensing electrode; the concentration of EDC / NHS is 0.1-0.5 M, and the incubation time at room temperature is 1-3 h.

2. The ratiometric "dual-signal on" electrochemical sensor prepared by the method described in claim 1 is used to measure tHb and HbA. 1c The method for simultaneous quantitative detection is characterized by: Follow these steps: Step 1, combine tHb and HbA 1c Alternatively, whole blood samples to be tested can be hemolyzed to obtain hemoglobin-lysed blood, which can then be diluted to different concentrations with 10 mM phosphate-buffered saline (PBS) at pH 7.

4. Step 2: The prepared sensor is incubated sequentially with the tHb-containing sample obtained in Step 1, and then incubated with a 2-10 mM ferrocene boric acid (FcBA) solution, i.e., GCE / ERGO-CNT / AuNPs / AQ / Ab / BSA / tHb / FcBA. After washing, it is placed in PBS buffer (pH 7.4) containing thionine (Thi) and 2-10 mM H2O2. The incubation time for tHb is 10-40 min, the incubation time for ferrocene boric acid is 40 min, and the concentration of thionine is 5-50 μM. Step 3: Differential pulse voltammetry (DPV) measurement was performed using a three-electrode system (sensor as working electrode, Ag / AgCl as reference electrode, and Pt wire as counter electrode) to record the reduction peak currents of AQ, Thi, and FcBA; the parameters were set as follows: scan range, 0.5V - −0.7V; Amplitude: 0.05 V; Pulse period: 0.5 s; Potential increment: 4.0 mV. Step 4, based on the current ratio I Thi / I AQ and I FcBA / I AQ Calculate tHb concentration and HbA1c concentration respectively. 1c Concentration and HbA 1c Level (HbA) 1c (Percentage of tHb) to achieve simultaneous quantitative analysis of dual objectives.

3. The ratiometric "dual-signal on" electrochemical sensor prepared by the method described in claim 1 is used for the analysis of tHb and HbA in whole blood samples. 1c and HbA 1c Applications in accurate horizontal assessment.

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