| Local surface plasmon resonance(LSPR)has been developed as a novel biosensing technology.By comparison with surface plasmon resonance(SPR)biosensing technology,LSPR has more advantages in technical implementation.Meanwhile,it retains the characteristics of high sensitivity,high selectivity and label-free of SPR.Plant hormones play an important role in the process of plant growth.Abscisic acid(ABA)is extensively studied because it can enhance the plant stress resistance.The conventional detection methods such as immunoassay,chromatography,and chromatography-mass spectrometry are frequently used to detect plant hormone ABA.However,these methods mentioned above are time-consuming in the process of sample pretreatments and the expensive instruments are involved.Generally,the lab technicians can only manage these instruments.With the development of cutting-edge disciplines on hormone action and signal transduction,a novel detection method is urgently needed to achieve the quantitative detection with high specificity and sensitivity of ABA.This paper describes the LSPR biosensing technology used for quantitative analysis of the plant hormone ABA.The LSPR spectrum signal acquisition,the novel LSPR measurement system,the recognition element optimization,the spectrum eigenvalues extraction and as well as the quantitative analysis model in comparison with other analysis methods have been investigated deeply from the LSPR character of gold nanoparticles.The ABA aptamers were optimized to be the specific recognition elements in this LSPR biosensing system for the high sensitive detection of plant hormone ABA.The high efficiency of quantitative analysis method for the determination of plant hormones ABA provides a new tool for agricultural scientists.The main findings are listed as follows:(1)Conventional methods for the detection of plant hormone ABA,such as chromatography and chromatography-mass spectrometry require expensive equipment.The pretreatment process is complicated and time-consuming.The lab technicians can only operate these instruments.In order to solve this problem,a new method with a low-cost LSPR biosening system was established to measure the plant hormone ABA using UV-Vis spectrophotometer.Functionalized gold nanoparticles were prepared by the coordination bond between aptamer and gold nanoparticles.The experimental results show that the aggregation degree of the gold nanoparticles was closely related to ABA concentrations in the sample solutions.The effects induced by the NaCl concentration,the aptamer concentration and the incubation time were studied to verify the detection sensitivity.The characteristic absorbance ratio(A620/A520)obtained from the LSPR spectrum was used for building the model to quantitatively analyze the plant hormone ABA.Under the optimized experimental conditions,when the concentration of ABA was presented in a larger range of 5?20 ?M,the absorbance ratio and the logarithm of ABA concentration has a good linear relationship.The linear correlation coefficient of 0.992 was obtained and the detection limit of 0.33 ?M was calculated.The specificity of this method for detecting the plant hormone ABA was investigated in this experiment.The experimental results indicated that this method for detecting ABA had high specificity.In order to further verify the feasibility of this method in practical applications,ABA detection was conducted with rice leaves,and the results were compared with results from ELISA method.The maximum relative error of 7.93%was calculated,indicating that both methods were comparable.LSPR spectral signals were obtained from the UV-Vis spectrophotometer with low cost and facile detection,which laid a foundation for the further construction of a novel LSPR spectral measurement device to realize the rapid and highly sensitive detection of plant hormone ABA.(2)The experimental results show that the speed of the signal acquisition from UV-Vis spectrophotometer is slow due to the wavelength scan.And there are only two channels in this UV-Vis spectrophotometer.So the parallel measurement is a challenge resulting in a long ABA analysis time.In order to improve the speed of the signals acquisition,a novel multi-channel LSPR biosensing system based on the transmission spectrum mode was constructed after studying the conventional LSPR spectral signal acquisition mode.Experimental results show that this novel LSPR biosensing system has good stability,repeatability and consistency among channels.The absorption spectra of spherical AuNPs with different particle sizes were obtained from this novel LSPR biosensing system.The Savitzky-Golay smoothing algorithm was applied to process the original spectral data and established a fitting curve to calculate the peak wavelengths of LSPR.The relationship between the sizes of AuNPs,the refractive index of the dielectric medium and the LSPR resonance wavelengths were studied.The experimental results show that the resonance wavelength had a red shift with the increase of particle sizes.And a good linear relationship between the particle size and the resonance wavelength had been obtained under the same dielectric medium condition.Moreover,the resonance wavelengths were closely related to the refractive index of the dielectric medium under the certain particle size.For AuNPs with the sizes of 25.5 nm and 41 nm,a good linear relationship between the refractive index of the dielectric medium and the resonance wavelength had been obtained.The sensitivities of 59.46 nm/RIU and 70.38 nm/RIU have been calculated,respectively.Therefore,this novel LSPR biosensing system can realize the rapid acquisition of LSPR spectral signals and also can be used to realize high throughput detection,which provides a new LSPR detection system for further quantitative detection of plant hormone ABA.(3)In order to further realize the rapid ABA detection and improve the detection sensitivity,the customized multi-channel LSPR biosensing system was used to obtain the spectral signals.The ABA quantitative analysis was performed using the specific recognition bond between aptamer and ABA under the catalysis of AuNPs.In order to improve the specificity between the nucleic acid aptamer and ABA,the nucleic acid aptamer sequences were optimized in advance.Under the optimized sequence of ABA aptamer,PolyA-tailed aptamer was proposed to be the specific recognition element.The AuNPs modified with PolyA-tailed aptamer was applied to perform the detection of plant hormone ABA.The experimental results show that the quantities of PolyA-tailed aptamer and different exposure area of AuNPs are closely related to the concentration of ABA.In this case,after adding the reducing reagent of NH2OH and the growth agent of HAuCl4,the catalytic growth reaction of AuNPs was initiated.During the growth process,the AuNPs grew into larger spherical particles in different color and morphology due to the degree of bareness of AuNPs.The sensitivity effect produced by the growth time,the amount of HAuCl4,the number and position of base A,and the concentration of polyA-tailed aptamer were validated.The quantitative analysis model was established based on the peak wavelengths of the LSPR spectrum.Under the optimized conditions,ABA could be quantitatively detected in the range of 0.1 nM?1 mM,while a good linear relationship between the ABA concentration and the peak wavelength shift of the LSPR spectrum was obtained in the detection range of 0.1 nM?1 ?M with the linear correlation coefficient of 0.992 and the detection limit of 1 nM.Other plant hormone interferences such as GA3 and IAA have applied to validate the specificity of this ABA detection approach using LSPR biosing system.It showed that only small responses were found from these interferences.The sensitivity for ABA detection was greatly improved comparing with salt-induced AuNPs aggregation method,which can be attributed to the use of polyA-tailed aptamer and the catalytic ability of AuNPs.Moreover,this multi-channel LSPR system requires no wavelength scanning in the process of spectra signal acquisition and can be used for high throughput detection,which greatly reduces the detection time for LSPR biosensing. |
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