The Fabrication And Preliminary Application Of Dual-signal Amplification-based Electrochemical Aptasensor For The Detection Of Bisphenol A

Objective Bisphenol A(BPA),the scientific name of 2,2-bis(4-hydroxyphenyl)propane,is currently one of the most widely used raw materials in the production of polycarbonate,epoxy resin and other polymer chemical products.Extensive use of BPA has caused it to enter the environment or the human body from packaging materials or industrial waste water.At the same time,BPA is one of the environmental endocrine disruptors(EDC).It disrupts the endocrine system and causes harm by simulating the function of natural hormones in the natural environment and development process or blocking the natural hormone metabolism pathway.Exposure to BPA is associated with a variety of diseases,such as obesity,diabetes,cardiovascular diseases,neurological diseases,and hormone-related cancers in women,and very low levels of BPA exposure can also cause BPA to enter the body and lead to harm.The traditional methods for detecting BPA levels are mostly based on large equipment,and their sensitivity and specificity cannot meet the requirements of detecting BPA levels in the environment and human body fluids at very low concentrations.Electrochemical biosensors,as a simple,fast and efficient novel technology,can achieve good sensitivity and specificity at the same time by modifying the electrode surface or designing signal amplification strategy,which meets the requirements of the current ultra-sensitive trace detection of BPA.This study aims to use nanomaterial modification and "signal switch"strategy to achieve dual-signal amplification,so as to construct an electrochemical aptasensor based on dual-signal amplification.Thus,the new method for the determination of trace BPA in real samples:human serum and lake water was proposed,providing a new method choice for the detection of trace BPA in the environment and body fluid.Methods In this study,multi-walled carbon nano tubes(MWCNT),amino Fe3O4 nanoparticles(NH2-Fe3O4 NPs)and gold nanoparticles(Au NPs)were used to modify the electrodes to achieve the first electrochemical signal amplification.A "signal-switch"was designed by using the affinity difference between single stranded DNA(ssDNA)and double stranded DNA(dsDNA)towards single-walled carbon nanotubes(SWCNT)to achieve the second electrochemical signal amplification.Meanwhile,the nanomaterials used in this study were characterized by electron microscopy and Fourier infrared spectroscopy,and the electrochemical characterization of the electrochemical aptasensor based on dual-signal amplification was performed by cyclic voltammetry(CV)and electrochemical impedance(EIS)methods.In order to achieve the optimal detection state,the important conditions involved in the sensor construction and quantitative detection were optimized,including incubation time of BPA and Apt-CDNA modified electrode,dilution ratio of real samples,modified volume of nanomaterial and optimized aptamer concentration.Finally,differential pulse voltammetry(DPV)was used to determine the BPA in phosphate buffer solution(PBS)and real samples:human serum and lake water.The qualitative potential value and the quantitative current value were used to analyze the use value of the sensor.In addition,a series of methodological validations were performed on the aptasensor:linear range,limit of detection(LOD),specificity,stability,reproducibility,and accuracy.Results The specific structures of the nanomaterials were verified by electron microscopy characterization and Fourier infrared characterization.CV and EIS characterization verified the changes of electrochemical property on the electrode surface during the construction of the sensor,further proving the successful realization of dual-signal amplification.The results showed that the optimal incubation time of BPA and Apt-CDNA modified electrode was 30 min,the optimal dilution ratio of real samples was 4000 times,the optimal modified volume of nanomaterial was 6 μL,and the optimal modification concentration of aptamer was 20 μmol L-1.At the above optimal experimental conditions,the value of peak current ΔI(I-I0,I:peak current in presence,I0:peak current when there is no BPA)was obtained from the DPVs.The resulting standard curve:y(ΔI,μA)=-14.48x(-lgc[BPA],mol L-1)+326.4 exhibits that the variation of peak current ΔI is linearly dependent on the negative logarithm of BPA concentration ranging from 10-19mol L-1 to 10-14 mol L-1 with a correlation coefficient(R2)of 0.9981.When the BPA concentration increased,ΔI increased correspondingly,proving the "signal-on"mechanism of the signal switch.The LOD was calculated to be 0.08 amol L-1 according to IUPAC regulations.In the mixed-standard specificity experiment,when p-aminophenol,nonylphenol,p-nitrophenol and p-chlorophenol were present together with BPA,the difference rate between the detected value of the aptasensor and that of pure BPA was less than 3%,indicating good specificity.When the aptasensor is stored at 4℃,the detection value of the aptasensor for the same concentration of BPA can still reach more than 90%after 10 days and more than 80%after 16 days compared with the first day,showing good stability.The relative standard deviation(RSD)of multiple detection values for the same concentration of BPA in the same batch of electrodes was less than 5%,showing good reproducibility.In real sample measurement,whether it is human serum or lake water,there is a good linear relationship between ΔI and the negative logarithm of the corresponding concentration,and the correlation coefficient R2 is bigger than 0.9.Meanwhile,the recovery rate in the standard recovery experiment is also between 98%-99%,which proves the practical application value of this sensor.Conclusion In this study,an electrochemical aptasensor based on various nanomaterials modification and signal switching strategiy with dual-signal amplification was constructed for providing the preliminary application on the detection of trace BPA in human serum and lake water.With a variety of nanomaterials with good electrochemical characteristics,such as MWCNT,NH2-Fe3O4 NPs,Au NPs modified electrodes,the first signal amplification was achieved.SWCNT-based signal switching strategy was designed to achieve the second signal amplification due to the different affinity between ssDNA and dsDNA.The electrochemical biosensor was used for DPV detection of BPA,and the linear range was 10-19 mol L-1-10-14 mol L-1,and the LOD was 0.08 amol L-1.Through a series of methodological verification,it is proved that the electrochemical biosensor has good specificity,stability,reproducibility and accuracy.Most importantly,its successful application in human serum and lake water demonstrates the potential of the electrochemical biosensor in the field of trace detection of BPA in real samples,providing new ideas for the prevention and diagnosis of diseases,environmental monitoring and other fields.