Construction Of SERS Aptasensors And Their Applications In Detecting Small Molecules

Surface-enhanced Raman scattering(SERS)spectroscopy,as an ultra-sensitive analysis technique that can quickly obtain the "fingerprint information" of molecules,has been widely used in qualitative and trace analysis in food inspection,environmental monitoring,biomedicine and many other fields.Aptamer is an oligonucleotide sequence with high specificity and affinity for a specific target,which is used as a molecular recognition element and can be applied to the construction of various types of aptamer-based biosensors to detect different targets.In this paper,combining the high sensitivity of the SERS technology with the specific recognition ability of the aptamer,using aptamers as specific recognition elements,two kinds of SERS aptasensors were established for quantitative detection of two small molecular targets,sulfadimethoxine(SDM)and adenosine triphosphate(ATP).Gold nanoparticles(Au NPs)was synthesized using trisodium citrate as reducing agent.Raman reporter molecule 4-mercaptopyrimidine(4-MPY)modified Au NPs(Au NPs@4-MPY)was used as color signal probe,gold-silver core-shell nanoparticles(Au@Ag NPs)with good enhancement effect was used as the SERS substrate.Based on the aggregation of nanoparticles caused by the release of hexadecyl trimethyl ammonium bromide(CTAB)after the specific combination of SDM aptamer and SDM,combined with colorimetric/SERS dual-mode readout signal,the detection of SDM was realized.The concentration of CTAB,SDM aptamer,4-MPY,and the volume of Au@Ag NPs solution were optimized.Under optimal experimental conditions,this method achieved colorimetric detection of SDM with a linear range of4.00-200.00 ng/m L and a detection limit of 2.41 ng/m L,while SERS had a linear range of 1.20-120.00 ng/m L and a detection limit of 0.89 ng/m L.Moreover,this method was applied to the determination of sulfadimethoxine in milk and honey samples,the recovery of SDM in colorimetric mode ranged from 100.47% to118.77%,the relative standard deviation(RSD)was below 4.19%,while in SERS mode,the recovery of SDM was 97.27% to 113.45% and the RSD was below 5.96%.Au NPs synthesized by trisodium citrate reduction was used as SERS substrate and crystal violet(CV)was used as Raman reporter molecule.The G-quadruplex structure formed by ATP aptamer-ATP specific binding could not only resist exonuclease I(Exo I)digestion,but also carry CV molecules through electrostatic andπ-π interactions,and further combine with polycationic protamine through electrostatic interaction.Due to lack of free protamine in the system,the aggregation degree of Au NPs and the SERS signal intensity of CV decreased,thus realizing the quantitative detection of adenosine triphosphate.Factors affecting SERS intensity were optimized including protamine concentration,ATP aptamer concentration,Exo I concentration and enzymatic reaction time.Under optimal experimental conditions,the linear response was obtained for ATP in the concentration range of 5.0 n M-1.0 μM,the detection limit was 1.0 n M.The method was applied to the determination of adenosine triphosphate in human serum and urine samples,the recovery of ATP was between 96.6% and 116.3% and the RSD was below 5.2%.