Quantitative Determination Of Biomolecules Indirectly And In Situ Monitoring Of Chemical Reactions By Surface-Enhanced Raman Scattering

Raman scattering of molecules is an inelastic scattering of photons effect,it can reflect the structure information of molecules,and any tiny changes of molecular structure or chemical bond vibration will be reflected on the Raman scattering spectrum very sensitively.Thus,Raman spectroscopy technique can be used for structure analysis as it is fast,nondestructive,easy operation and high sensitivity.The appearance of surface-enhanced Raman scattering(SERS)technique further promoted the application and development of Raman technique because it overcomes the shortcomings of traditional Raman technique like low susceptibility and disturbed by fluorescence easily.In the past decades,as the basis of SERS technology,highly active SERS substrate preparation has always been a popular SERS research direction.However,simply to develop a new SERS substrate,and continuously improve the substrate enhancement effect cannot meet the demands of the development of our society anymore.In recent years,more researchers have focused on the expansion of SERS applications,and are committed to combine SERS technology with other technologies to release more potential of this highly sensitive,effective and nondestructive approach.In this paper,firstly,based on the Ag/Si substrate with proven technique and excellent enhancement effect,we expands its application in life sciences,mainly realized the indirect quantitative detection of glucose molecules.And on this basis,quantitative detection of another important biological small molecule,ATP,with a difference value method was also achieved.This work has important practical significance for the diagnosis and prevention of disease.Furthermore,a new nanoporous gold(NPG)material by the method of de-alloying corrosion were prepared,and the real-time in situ monitoring of chemical reactions was achieved with SERS technology.Hence,the mechanism of chemical reactions can be analyzed and understood by means of in situ Raman detection,which will help to reasonably determine the reaction conditions,improve the selectivity,product quality and yield of the reactions.This effort have a self-evident significance for the energy crisis and environmental problems faced by current human society.The main contents of the paper are divided into the following four aspects:1.The realization of quantitative detection of two important biomolecules,glucose and ATP.In this study,using the Ag/Si substrate with excellent enhancement effect developed by our laboratory,the indirect quantitative detection of glucose molecules was realized by the Raman detection of pigment CTC,and a low detection limit as 2.5 × 10-7 M was achieved;Compared to the traditional spectrophotometric method,this method is more rapid and more sensitive.Then,on this basis,combined with the process of glucose phosphorylation,we pioneered the concept of detection of ATP by difference value method,successfully realized the quantitative detection of ATP molecules with a detection limit as low as 4.69 × 10-7 M.In this process,the relationship between the concentration of glucose and ATP were also discussed in detail,and our difference method is proved to be not only sensitive,but also very accurate through confirmatory experiments.This work not only opens up a new way for SERS application in detection of biomolecules,but to realize the rapid and accurate detection of these important biomolecules is also important for the diagnosis and prevention of diseases.2.Preparation and Characterization of NPG.In recent years,NPG has become a research hotspot because of its large specific surface area,good chemical stability and strong conductivity.It has a huge application prospects in many fields like bioscience,chemical,optics and Raman scattering.In this part,we employ Au/Ag alloy as raw material,and prepare NPG substrate by the simple and easy-operation de-alloying corrosion method.The front and side scanning electron microscopy images clearly show its porous structure,and the pore size of nanoporous gold can be regulated easily by controlling the etch time.The SERS performance of the substrate was demonstrated by a series of tests on rhodamine 6G(R6G)molecules with different concentrations.In addition,enhancement factor(EF)up to 106 was figured out.This substrate which shows excellent SERS enhancement effect,chemical stability and good conductivity will play a more important role in the in situ monitoring of chemical reactions.3.Study on photocatalytic reduction of p-nitrothiophenol(pNTP)by in situ SERS technique.In this part,considering its SPR light absorption properties,NPG was employed to carry out the photocatalytic reduction experiments of pNTP.The innovation of this study exhibit in three aspects:First,the NPG substrate not only catalyzes the pNTP reduction reaction,but also serves as a good SERS substrate,so that in the process of photocatalytic reduction,we can implement in situ Raman detection simultaneously,achieving the real-time and dynamic monitoring of the whole reaction process;Second,the pNTP molecules were anchored on the surface of NPG substrate through the "Au-S" bond,so that we can not only qualitative,but also quantitative analyze the Raman test results;Third,the laser is not only used for the excitation source of Raman detection,but also the photocatalytic light source.This not only catalysis the redox reaction,but also simplifies the experimental device.The experimental results show that in the absence of platinum,palladium and other metals,there must be a combination of light and reducing agent sodium borohydride in order to achieve the reduction of pNTP,and even so,it can only partially reduced to the intermediate,4,4'-dimercaptoazobenzene(DMAB),instead of the final production,p-aminothiophenol(pATP).4.Study on the electrochemical reduction process of pNTP by in situ SERS technique.Electrochemical reaction has been widely concerned because of its safety,cleanliness,simple process and high energy conversion rate,and it has a great application prospect.In this section,we will take the electrochemical reduction of nitrobenzene as a probe reaction,employ NPG as both SERS substrate and working electrode,to build an in situ SERS detection platform for electrochemical reactions,aiming to study the electrochemical reaction mechanism(including molecular transformation,intermediate product capture,reaction dynamics information,etc.).The results demonstrate that the direct reduction process dominates both in acidic and alkaline condition.Additionally,our innovative potential cutting off operation reveals the different light stability of the reduction intermediates.Finally,combining the density functional theory(DFT)calculations,the different of in situ SERS spectra in acidic and alkaline electrolyte after cutting off the potential were analyzed based on the change of the Gibbs free energy of the reaction,and the theoretical calculation explained the experimental phenomenon well.We believe this electrochemical in situ SERS monitoring platform will promote the mechanism study of electrochemical reactions.