| Surface enhanced Raman spectroscopy(SERS),which promotes the enhancement of the Raman signal intensity of target molecules through surface plasma resonance and local surface plasma resonance effect(LSPR)of metal materials.Due to the characteristics of SERS high sensitivity and unique fingerprint narrow spectrum of molecules,it has been applied widely currently.The plant hormone Abscisic acid(ABA)is a major substance affecting crop development and plays a major role in maintaining cell survival and inhibiting cell apoptosis.It can help crops resist adversity effectively and promote fruit ripening.Therefore,ABA detection and timely external regulation of crops are important means to help crops cope with harsh environment,and have important significance in promoting the realization of smart agriculture.At present,the classical methods for the determination of ABA in the laboratory are high performance liquid chromatography(HPLC)and enzyme-linked immunosorbent assay(ELISA).Although these methods have high specificity and sensitivity in detection of ABA,the technical complexities,equipment cost and sample processing requirements were involved intensively.Therefore,the development of highly specific and sensitive ABA detection technologies will help to promote the new plant hormone biosensors and further understand the mechanism of action and molecules signaling for agronomist.In this paper,SERS technology was used to construct SERS biosensor based on aptamer(Apt)recognition,and quantitative detection and experiment of plant hormone ABA were carried out.Firstly,the Raman spectra of plant hormone ABA were studied theoretically,and the consistency of theoretical research and experiment was to be proved.Then,on the basis of theoretical research,Au@Ag core shell nanocomplex was synthesized to enhance SERS signal intensity of internal standard,ABA Apt complement sequence was screened for specific recognition of Apt,and a sensor was constructed for ABA detection with the help of magnetic nanoparticle properties.Furthermore,a SERS and fluorescence dual signals output sensor were constructed on the basis of single signal output for the ABA detection.Finally,excessive modification steps during sensor construction were simplified,and a simple aptamer sensor with unmodified gold nanoparticles LSPR and SERS multi signals output was constructed for ABA detection.The main research contents are as follows:(1)Based on the density functional theory(DFT),the Raman spectra of ABA molecules was calculated by Gaussian09 and GaussView5.0 and verified by experiments.ABA molecular structure was constructed and optimized to obtain ABA molecular energy level frontier orbit,Raman spectroscopy,infrared spectroscopy(IR),nuclear magnetic resonance spectroscopy(NMR).Then,the SERS,IR and NMR of ABA were tested by experiments.The calculation results showed that the characteristic Raman peaks of ABA was found at 1635 cm-1,1271 cm-1,1048 cm-1,865 cm-1,and 612 cm-1,which were from the stretching vibration of the carbon-carbon double bond,the stretching vibration of the carbon-carbon single bond,the non-plane oscillations of the methyl group,the external twisting vibration of the carbon-carbon single bond,and the plane and non-plane oscillations of the C-H bond in ABA molecules,and the maximum characteristic peak of ABA Raman spectrum was located at 1635 cm-1.This research laid a theoretical foundation for SERS technique to quantitatively detect ABA in plants.(2)The silver particles coated on gold nanostructures(Au@AgNPs)with the internal standard molecules(4MBA)was synthesized,a stable SERS signal intensity of 4MBA molecule was used to reflect the concentration of ABA molecule.The ABA aptamer and their supplementary aptamers of ABA were screened,and the ABA aptamer sensor based on core shell nanostructure was constructed by using the properties of magnetic nanoparticles.The linear range of ABA detection was obtained in 1fM～10nm,the correlation coefficient R2 value of 0.9875 was calculated together with the theoretical detection limit was 0.67 fM.This method was applied to detect ABA molecules in fresh wheat leaves,and the relative error was 5.43%～8.94%compared with ELISA assay was obtained.(3)Based on the SERS enhancement and fluorescence quenching properties of gold nanorods(AuNRs),a dual function SERS and fluorescence aptamer sensor was constructed for the quantification of plant hormone ABA.Rhodamine isothiocyanate(RBITC),a molecule with Raman and fluorescent dual signal properties,was used to reflected the molecular concentration of ABA.The signal probe was composed of RBITC,ABA aptamer complement chain and AuNRs,while the capture probe was composed of magnetic nanoparticles and aptamer.The specific binding of signal probe and capture probe will be destroyed with the addition of ABA molecules,resulting in the change of SERS signal of sensor solution.Furthermore,when the AuNRs were etched,the fluorescence intensity of RBITC molecules in the sensor solution changed.Using the dual-function biosensor for the ABA detection,a linear relationship between the SERS intensity and ABA concentrations was showed in the logarithmic range of 100fM～0.1nM.The linear regression equation was y=402x+35566 for the ABA detection using SERS method,the correlation coefficient R2=0.96,and the calculated limit of detection(LOD)was 38 fM was obtained.The linear regression equation of y=1962x+29607 for ABA detection using the fluorescence was established with the correlation coefficient R2=0.99,and the calculated LOD of 0.33pM.The recoveries of this method were calculated to be 85%～108%with the maximum relative standard deviation(RSD)of 9.57%compared with ELISA method.(4)In order to solve the time consuming and uncertain factors caused by excessive modification during the construction of the SERS sensors,crystal violet(CV)as Raman signal probe molecule,ABA aptamer(Apt)and unmodified gold nanoparticles(AuNPs)were used to construct LSPR and SERS double spectrum signals output sensor.Apt was a sensitive element to recognize and capture ABA,CV was a Raman signal probe molecule,and AuNPs coated by Apt have certain anti-aggregation ability under the action of salt solution.Under the action of target ABA molecules,Apt was bound to ABA,Apt fell off the surface of AuNPs,and the AuNPs without protection aggregated in the presence of salt,the color and LSPR performance of detect system,the SERS intensities of CV changed accordingly.The biosensor was used for the quantitative detection of ABA molecules,and the results showed that the SERS spectral intensity and absorbance changed had good linear relationship in the range of ABA concentration of 0.04μM～80μM,the correlation coefficient of absorption spectrum detection R2 was 0.9922,and the detection limit was 15nM.The SERS spectrum R2 was 0.99372,the detection limit LOD was 13nM,the linear range and detection limit of the two evaluation methods were consistent.Furthermore,compared with ELISA,the biosensor has the ability to detect the real ABA extracted from common crops,the SERS detection results showed that the maximum error of ABA content was 13.46%,and the LSPR detection results showed that the maximum error of ABA content was 14.01%,the maximum error of the two detection methods was similar.The LSPR/SERS Apt biosensor constructed here was a general sensing method,which does not require chemical modification and can detect a variety of molecules by changing the Apt sequence and has the ability to detect ABA extracted from a variety of crops.Based on SERS technology,a variety of aptamer biosensors were developed and used for quantitative detection of low content of plant hormone ABA.The research of this paper lays a certain theoretical and experimental foundation for the research of plant hormone online detection and wearable plant hormone sensor... |
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