Construction Of Nano-electrochemical Herbal Sensing Interface And Its Application In Accurate Identification Of Saffron(Crocus Sativus L.)

With the continuous development of the traditional Chinese medicine industry,the demand of medicinal plants widely used in pharmaceutical products and traditional Chinese medicine preparations has continued to increase,whereas the authenticity of medicinal plants has restricted their commercial trade and clinical drug safety.The identification of traditional Chinese medicine has gone from the traditional four major identification methods to molecular identification represented by DNA barcodes and related rapid detection techniques,which provide an effective and objective complement to traditional identification technologies.Studies have shown that the ITS2 sequence has high identification efficiency at the species level and can be used as a universal barcode sequence for identification of medicinal plants.In order to obtain results faster,based on the known species sequences,researchers have developed some methods and techniques for rapid identification.Biosensors have the advantages of miniaturization,low-cost,good specificity,high sensitivity,and fast response speed.They have been widely used in medical research,clinical laboratory diagnosis and bed-side detection.This technology can quickly identify medicinal materials at the level of DNA molecules,especially for commercially available disposable portable chip sensors.After batch modification and molecular functionalization of the sensors in advance,out-door field testing is also expected.Therefore,in the field of medicine identification,this kind of portable electrochemical biosensing interface has certain application potential.This article takes the precious herbal medicine Crocus sativus L.as an example,and aims to develop a nano-electrochemical herbal sensor for rapid identification of herbal medicines.DNA barcoding technology was used to identify the authenticity of the collected medicine samples.Based on the ITS2sequence information of the samples,a DNA probe was designed specifically complementary to the authentic product,which served as the specific identification element of the subsequent electrochemical sensor.Then,a high-curvature three-dimensional nano-structuring array of electrochemical herbal sensing interface was designed.The sensor uses a commercially available screen-printed carbon electrode?SPCE?as a substrate to modify the branching gold nano-flower structure on the carbon working electrode.Compared with the two-dimensional planar gold electrode,the sensor can effectively improve the DNA hybridization efficiency and realize the rapid identification of the precious material C.sativus and its adulterants.The specific research content is mainly divided into the following three parts:Part I:DNA identification of C.sativus and its adulterants.A plant genomic DNA kit was used to extract the total DNA of saffron and its common adulterants collected on the market.After universal primers were used for amplification and sequencing,the sequence peaks were corrected and stitched using analysis software,and the 5.8S and 28S segments at both ends were removed to obtain ITS2 sequences.Comparisons were made in the DNA Barcoding System for Herbal Materials and the NCBI database to determine the accuracy of the collected species.The experimental results showed that ITS2 sequences of most samples could be compared to species and a few to genus,which could be well distinguished among different species.ITS2 sequences of C.sativus and adulterants were significantly different.Subsequently,by comparing and analyzing the differences between saffron and its fake ITS2 sequence,a probe sequence specific to saffron was designed as a fixed probe for subsequent sensors.Part II:Construction of high-curvature nano-structuring interface and its hybridization kinetics.In this experiment,a commercially available screen-printed carbon electrode was used as the substrate,and a nano-roughened gold flower structure array was modified on the substrate by photolithograph-free electrodeposition technique.The surface morphology was characterized by scanning electron microscopy.Based on the ITS2 sequence information,three different DNA targets were designed to investigate the hybridization kinetics of different complementary modes.The experimental results showed that,regardless of the hybridization mode,the 3d nanostructuring interface of such a trans-scale?including macro-scale carbon surface and nano-scale gold branches?reduced the time required for hybridization by about 30 min compared with the 2d planar gold electrode interface,improved the efficiency of DNA hybridization,and showed higher electrochemical response signals.The cyclic voltammetry?CV?was used to investigate the effective active gold area of the two interfaces in 0.5 M H₂SO₄.The results showed that the effective active gold area of the three-dimensional nanostructure interface was5.4 times larger than that of the planar gold electrode.This experiment proves that the constructed high-curvature nano-structuring interface has obvious advantages in detecting long-stranded DNA,providing a theoretical basis for the biosensor to identify medicinal materials by DNA molecules.Part?:HCAuNS-based portable electrochemical herbal sensor for rapid identification of saffron.In order to better apply the high-curvature Au nanostructuring?HCAuNS?interface to the rapid identification of saffron,we optimized the concentration of fixed probes,determined the hybridization time from the kinetic experiment,and also discussed the sensitivity,specificity and repeatability of the sensing interface.The experimental results show that Our nanoE-herb sensor achieves a detection limit of 0.18 fM for the 64-mer fragment of saffron ITS2 barcode with mid-hybridization,and shows superior specificity against even single-base mismatch.The sensor also precisely differentiates saffron from other six adulterants by directly detecting unpurified asymmetric PCR amplicons?⁵00 bp?with ITS2 sequences,suggesting its great potential in the field identification of herbal medicinal species and pathogenic bacteria with specific DNA barcodes.