Light-addressable Potentiometric Sensor For The Detection Of Glycated Haemoglobin

Diabetes is considered one of the most pressing human health problems of the 21st century and early diagnosis and regular monitoring of blood glucose levels are considered extremely important to prevent complications arising from diabetes.Glycated haemoglobin（HbA1c）is a stable product formed by the non-enzymatic reaction of glucose and haemoglobin（Hb）in human blood,and its test results can reflect the blood glucose level of a person over a longer period of time and improve the accuracy of diabetes diagnosis.This paper explores a low-cost,highly sensitive and convenient assay for the diabetic biomarker glycated haemoglobin.The main studies are as follows:（1）Reduced graphene oxide-polyacrylamide-ferrocene（rGO-PAM-Fc）nanocom-posites,reduced graphene oxide-chitosan-prussian blue（rGO-CS@PB）nanocomposites and gold nanoparticles（AuNPs）were designed and synthesized.We also used Fourier infrared spectroscopy（FT-IR）,ultraviolet–visible spectroscopy（UV-Vis）,X-ray photoelectron spectroscopy（XPS）,transmission electron microscope（TEM）characterisation and particle size potential to characterise the structure,morphology,surface elements,valence states,and functional groups of rGO-PAM-Fc,rGO-CS@PB and AuNPs,and confirmed the successful synthesis of the above nanomaterials.（2）Study of light-addressable potentiometric sensor（LAPS）based on rGO-PAM-Fc/AuNPs and aptamers for HbA1c detection:Glycated haemoglobin aptamers were used as specific recognition molecules and rGO-PAM-Fc/AuNPs were used as sensing substrates to construct sensitive membranes for the response characteristics of the LAPS sensor.A single-factor method was used to optimise the experimental conditions.Under the optimal conditions,the LAPS-based HbA1c biosensor was able to HbA1c in the range of1.0～100.0μg/m L with a high linearity.Y₁=1.2299X₁+95.6603（Y₁ is the voltage shift of the normalized I-V curve for different concentrations of HbA1c compared to the blank control at photocurrent 0.5,X₁ is the concentration of HbA1c）with a linear correlation coefficient R² of 0.98895.sensitivity of 1.2299 m V/μg·m L^-1 and a minimum detection limit of 42.92 ng/m L.In addition,the HbA1c aptamer sensor showed good specificity,stability and reproducibility.When tested on actual human whole blood samples,it showed low relative errors（0.33～5.90%）with RSD values ranging from 1.20～8.81%.These results indicate that the developed HbA1c sensor is expected to have promising applications in medical diagnostics.（3）Study of LAPS based on rGO-CS@PB/AuNPs and aptamers for HbA1c detection:Glycated haemoglobin aptamers were used as specific recognition molecules and rGO-PAM-Fc/AuNPs were used as sensing substrates to construct sensitive membranes for the response characteristics of the LAPS sensor.The optimal sensor response conditions were obtained by optimization using the response surface method.Under the optimal conditions,the concentration of HbA1c showed a good linear relationship with the potential shift values in the range of 0.1～5.0μg/m L and 5.0～100.0μg/m L.The linear regression equation for the range of 0.1～5.0μg/m L was Y₂=22.02058X₂+57.85223 with a linear correlation coefficient R² of 0.98656 and a sensitivity of 22.02058 m V/μg·m L^-1;the regression equation for the range of 5.0～100.0μg/m L was Y₃=1.04485X₃+153.99775with a linear correlation coefficient R² of 0.99548.The sensitivity was 1.04485m V/μg·m L^-1（Y is the voltage shift of the normalized I-V curve for different concentrations of HbA1c compared to the blank at a photocurrent of 0.5,X is the concentration of HbA1c）and the minimum detection limit was 20.52 ng/m L.In addition,the HbA1c aptamer sensor showed good specificity,reproducibility and stability.The HbA1c biosensor’s ability to detect real samples was further investigated by the direct method using clinical human whole blood samples,This was done with relative errors ranging from 0.18～9.30%and RSD values ranging from 0.12～4.07%for human whole blood samples.（4）Comparison of the two sensors:The rGO-PAM-Fc/AuNPs and aptamer-based LAPS sensors,have a wider linear range and lower detection limits.However,the rGO-CS@PB/AuNPs and aptamer-based LAPS sensor detected HbA1c with a better linear range and a significantly better potential shift than the rGO-PAM-Fc/AuNPs sensitive membrane modified HbA1c sensor,demonstrating that the electron transfer performance,loading capacity or biocompatibility of rGO-CS@PB is superior to that of rGO-PAM-Fc.The specificity of the detection of HbA1c and the relatively good stability may be due to the response surface method used to further optimise the experimental conditions and obtain an excellent voltage offset response of the sensor.Both HbA1c aptamer sensors showed good measurements on actual human whole blood samples,indicating that both constructed sensors are expected to provide a new solution for the clinical diagnosis of diabetes.