Study On Nanocomposite Substrate Based SERS Microchip And Its Application In Biochemical Detection

Surface-enhanced Raman scattering(SERS)technology has become one of the important methods for biochemical sample research,because of its advantages,such as high detection sensitivity,high specificity,mild test conditions,small water interference and the ability to give fingerprints of the analyte at the molecular level.However,when the SERS technique was used in the analysis of biological samples,it was prone to produce some problems such as lower SERS sensitivity,instability and poor SERS signal reproducibility.In addition,before and during measurements,the biological samples on the open SERS-active substrate surfaces were vulnerable to be contaminated,which adversely affected spectroscopic analysis.In this study,the nanoparticles technology,electrochemical preparation,SERS spectra were integrated with microfluidic chip analysis.The design and in suit fabrication of SRES substrate in microchannels were studied based on the electrochemical properties of ITO,and then the integrative SERS microchip was developed.Furthermore,the serum and bacteria were successfully tested and identified rapidly by combining the microchips based SERS analysis system and multivariate statistical analysis methods,which provided a new way and method for the analysis of complex biological samples.The related research has important research value and potential application prospect in species identification and clinical examination.The main research works and the results are as follows:Part 1: Preparation of ITO Glass based SERS microchip and nanocomposite enhancement substrate using electrochemical strategy.In this study,a new approach for constructing a novel microfluidic chip SERS was proposed based on ITO glass with ordered nanocomposite enhancement substrate.Three kinds of nanocomposite(NCs)Au/Ag NCs,Ag/Au NCs and rGO/Ag NCs were designed and used as the SERS microchip substrates.The Au/Ag NCs substrate with an average particle size of 130 nm and a mass ratio of Au core to Ag shell of 0.937: 0.063 were prepared by controlling the electrochemical deposition conditions to optimize and adjust the size of Au core and the thickness of Ag shell.The Ag/Au NCs substrate with an average particle size of about 110 nm and a surface Au of about 12 nm were prepared by electrodeposition and galvanic replacement.Based on two step chronoamperometry electrodeposition method,the rGO/Ag NCs enhancement substrate was prepared by controlling the nucleation potential and growth time and other parameters to adjust and optimize the morphology and composition of the composite substrate.Compared with the pure Ag nanoparticles(NPs),the enhancement factor of rGO/Ag NCs was improved 24 times.The three kinds of nanocomposite enhancement substrate have high SERS activity and good reproducibility due to controllable morphology and size,uniform distribution of particles,as well as the formation of uniform distribution of "hot spots" between the components,and good synergistic effect of SERS between the components.A microfluidic chip SERS was fabricated by plasma bonding.The results show that when the thickness of the PDMS cover is 1mm,the channel height is greater than 130 m,the influence of the SERS cover on the SERS signal was the least.Part 2: Study on the identification of serum species by using microarray SERS chip with integrated ITO-Au/Ag NCs.The SERS chip with 25 micropore arrays which integrated ITO-Au/Ag NCs enhancement substrate was designed and fabricated.Using the integrated SERS microchip,the corresponding SERS test mode and data analysis method were established for complex serum samples.The performance of SERS chip was investigated by using small molecule adenine as probe molecule.The results showed that the designed chip had good SERS sensitivity and reproducibility,whose detection limit was as low as 10-9M,the RSD in the same micro hole was less than 4.6%,and the RSD between the different chips of the same chip was less than 6.2%,and the RSD between the different chips was less than 10.9%.53 individuals,40 dogs,43 rabbits and 36 unknown serum samples were tested by the as-prepared microarray SERS microchip.The serum species identification model was established based on PLS-DA statistical analysis of serum SERS spectra detected on the microchip.The correct rate of classification of human serum,rabbit serum and canine serum was 100%,97.6% and 97.5% respectively,and the correct rate of blind classification was 91.6%.This method,which combinated the serum SERS spectroscopy test base on microarray SERS chip and PLS-DA discriminant model analysis,has great application potential in species identification of serum.Part 3: Study on diabetic serum diagnosis based on integrated ITO-Ag/Au NCs microfluidic SERS chipThe microfluidic SERS chip with five parallel microchannels integrated with Ag/Au NCs was designed and fabricated,and a high sensitivity and high throughput serum detection mode and method was established based on the prepared microfluidic SERS chip.The microfluidic SERS chip with good sensitivity and signal reproducibility was used to detect the serum,in which serum sample was directly sucked into the microchannel from the anticoagulant through negative pressure,which could avoid the pollution and damage caused by the exposure of the serum to the air and could improve the SERS signal stability of serum samples.So the stability of serum SERS signal was improved.A new diagnosis method for diabetes mellitus was established by combining serum SERS analysis based on microfluidic SERS chip and PCA-LDA statistical analysis.In the optimized range of 300~1800cm-1 wave,the model has the highest classification accuracy rate of 98.6% and blind prediction rate of 90%.Through the peak attribution could find that the characteristic peaks of 729cm-1,1102cm-1 and 1331cm-1 in SERS spectra of diabetic serum related to the metabolism of glycoproteins and glycolipids were significantly increased.This method,which combinated the serum SERS spectroscopy test base on SERS chip and PCA-LDA discriminant model analysis,has wide application value and prospect in the field of serum SERS spectroscopy for auxiliary diagnosis of disease.Part Ⅳ: Study on bacteria test based on microfluidic SERS chip with integrated ITO-rGO/Ag NCsBased on the chemical enhancement effect,adsorption efficiency and fluorescence purification characteristics of rGO,microfluidic SERS chip integrated with rGO / Ag NCs substrate were designed,prepared and used for highly sensitive and highly stable SRES detection and identification of bacteria.Compared with microfluidic SERS chip integrated with Ag NPs substrate,the SERS signal intensity of E.coli detected in microfluidic SERS chip integrated with rGO/Ag NCs substrate was increased by 1.74 times,and the autofluorescence intensity was reduced by 49%,which indicated that the reduction of graphene oxide and Ag NPs had a good synergistic effect.At the same time,it could solve the problem of the weak adsorption of metal substrate to bacteria,which could improve the reproducibility of bacteria SERS signal.Escherichia coli and Salmonella can be successfully identified by combined baterial SERS analysis based on the microfluidic SERS chip with PCA-LDA statistical analysis.This microfluidic SERS chip integrated with rGO/Ag NPs has good application prospect in the efficient SERS test of bacteria.