An Ultrasensitive Aptamer-Antibody Sandwich Sensor For The Detection Of Cortisol In Saliva

Objective Cortisol,is a steroid hormone produced by the adrenal gland as a response to stress and is released syntactically by adrenal cortex,which is often referred to as the stress hormone.Cortisol can regulate mood and health and plays an important role in organ metabolism,immune function,inflammatory response and maintenance of connective tissue function.But too much of it can lead to Cushing’s syndrome,which is characterized by obesity,fatigue and osteoporosis.Excess deficiency can lead to Addison’s disease,which is characterized by weight loss,nausea,vomiting and muscle weakness.Cortisol is present in saliva,but at low levels and with a lot of interference.Traditional cortisol detection techniques are mostly based on large laboratory instruments,but their sensitivity and specificity cannot meet the requirements of non-invasive and sensitive detection of cortisol concentration in human saliva,and they have defects such as high operation requirements and slow analysis speed.Electrochemical sensor is a new detection technology with simple operation,fast response and excellent performance.This method can achieve excellent detection performance by modifying signal amplification materials on the surface of glassy carbon electrode or integrating high-affinity recognition elements to capture target analytes,which meets the current requirements of non-invasive and sensitive detection of cortisol in saliva.Of this study was to use the modification of nanomaterials and identify strategies for collaborative capture of components,the aptamer-antibody sandwich cortisol sensor based on double signal amplification was constructed,double signal amplification was acieved and the performance of sensor was improved,and applied to actual cortisol in saliva samples detection.It provides a new idea for analyzing patients’body state and diagnosing diseases in clinical work.Methods In this study,MWCNTs,CMK-3 and Ag NPs were co-mixed to form nanocomposites by ultrasound.Then the Nanocomposites were modified on the surface of glassy carbon electrode（GCE）to achieve the first electrochemical signal amplification;Au NPs were used to conjugate specific cortisol antibodies to amplify the second signal.The sandwich capture pattern of cortisol Aptamer-antibody is used to specifically recognize and bind cortisol.At the same time,all kinds of nanomaterials used and prepared in this study were characterized by electron microscopy,UV-vis absorption spectrum and Raman spectrum.Then,cyclic voltammetry（CV）was used to characterize the aptamer-antibody sandwich sensor.In order to obtain the best detection results,the electrode construction and the important conditions needed in the quantitative detection of the target were optimized and selected.Finally,differential pulse voltammetry（DPV）was used to determine phosphate buffer solution（PBS）and artificial saliva dilution of cortisol concentration gradient of standard solution and college students’actual saliva samples.The potential value was analyzed qualitatively and the current value was analyzed quantitatively to test the practical ability and application value of the sensor.In addition,a series of methodological validation of the constructed sensor was carried out by measuring the stability,selectivity and reproducibility of the limit of detection（LOD）.Results The successful synthesis and structural specificity of nanocomposites were verified by electron microscopy,UV-vis spectrum and Raman spectrum.CV characterization verified the successful preparation of the material modified electrode and the change of electrochemical activity on the electrode surface.The purpose was to verify the double signal amplification effect and the recognition element cooperative capture strategy.Experimental results showed that the optimal modified volume of nano-composite was 8μL,the optimal modified concentration of aptamer was 10μM,the optimal incubation time of aptamer and nano-composite modified electrode was 12h,and the optimal concentration of antibody was 20μg/m L.Under the optimal experimental conditions above,when cortisol concentration was between 10^-10mg/m L and 10^-5mg/m L,DPV was used for electrochemical detection.The peak current value（I_P）of cortisol standard sample and the current value（I₀）of blank sample were measured.It had a good linear relationship between the current response difference and the logarithm of the corresponding concentration.The correlation coefficient（R²）was 0.997,and the corresponding standard curve equation was y（（35）I,μA）=16.485x(lgc_[Corticol],mg/m L)+175.75,the LOD was 0.09 pg/m L.In the specificity test,target cortisol and disruptors ascorbic acid,uric acid,urea and glucose were detected respectively（similar to or higher than the concentration in saliva of healthy people）,and the DPV current difference?I of the disruptor was significantly lower than the target cortisol,indicating that the sensor had good selectivity.When the prepared cortisol sensor was stored in an environment of 4°C,the measured value of the sensor on the 8th day still remained more than 90%of that on the 1st day,indicating good stability.The same batch of three aptamer modified electrodes were used to detect the same concentration of cortisol Standard solution for many times,and the relative standard deviation（RSD）was less than 5%,which proved that the sensor had good reproducibility.In the detection of the working correction curve,there is a good linear relationship between the difference of the measured current response and the logarithm of the corresponding concentration,R²=0.9923,and the recovery rate in the spiked recovery experiment was 96.90%-104.04%,which proved that the sensor had good precision and certain practical application value.Conclusion In this study,through the use of nanometer composite material modification and identify the components together capture strategy,built a dual signal amplification effect of aptamer-antibody sandwich cortisol sensors.The method had been successfully applied to detect cortisol concentrations in actual saliva samples from undergraduate and graduate students.MWCNTs/CMK-3/Ag NPs nanocomposites were fixed on the surface of GCE to achieve the first signal,and the second signal was amplified by coupling cortisol antibody with Au NPs.Meanwhile,aptamer-antibody sandwich capture mode was used to specifically recognize cortisol.The aptamer-antibody sandwich sensor was used for DPV detection of cortisol,the linear range was 10^-10mg/m L-10^-5mg/m L and the LOD was 0.09 pg/m L.Then a series of methodological verification were carried out on the experiment,which successfully proved that the sensor had good detection performance selective stability and reproducibility.In addition,the sensor had been successfully applied to detect cortisol levels in saliva samples of college students,which firstly explored the clinical significance of cortisol detection.Subsequently,it can be used to develop a portable clinical detection device for cortisol,which is expected to be used to assist in the diagnosis of psychological stress-related diseases in clinical work.