Quantitative Detection Of NADH Based On Surface-enhanced Raman Scattering

Raman scattering is a kind of special optical phenomenon that the incident light irradiates the media surface and interacts with molecules leading to the change of optical frequency after scattered by molecules.The frequency variation between scattering light and incident light is determined by the molecular.Different molecular can produce individual frequency variation,therefore,each material has its unique Raman spectra.It is for this reason that Raman spectra is usually referred to "fingerprint spectrum".Raman spectroscopy can provide abundant information of samples such as molecular structure and natural frequency of the molecular vibrations and has been widely used in the scientific research.However,Raman scattering technology also has its own limitation.Raman scattering cross section of general molecule is so tiny(eg.10-21?10-30 cm2·sr-1)that the Raman intensity is weak.And it the test result may not meet the testability requirement in actual testing,especially in the field of trace matter detection.Free electrons on the surface of rough metal produce resonance under the excitation of the laser,making the intensity of electric field increase,and resulting Surface Enhanced Raman Scattering(SERS).SERS technology has rapid detecting speed,high sensitivity and is a kind of nondestructive testing which overcomes the disadvantages of conventional Raman technique.Due to these characteristics,SERS can be used for trace analyte detection and has been widely applied in chemical,biological,medical and many other fields.In recent years,the level of research on the nanotechnology advances by leaps and bounds,and offers many helps for the preparation of SERS substrate.The precious metal materials,such as Au,Ag and Cu,possess less interband transition,indicating that the energy of incident light will not be converted to thermal energy if we choose the appropriate excitation wavelength,and realizing efficient surface plasmon resonance.Therefore,Au,Ag and Cu are often used to prepare the SERS substrate.In this paper,nanoporous gold was used as SERS substrate which was prepared by electrochemical method and the morphology of substrate can be controlled by changing reaction temperature,scanning cycle and scanning rate.Whereafter,a series of substrates with different aperture were characterized systematically to screen the one with the optimal SERS performance.Nanoporous gold substrate with dense and homogeneous structure was prepared through the control of reaction conditions.Crosslinkings exist among the holes and form the multi-channel structure which providing abundant SERS "hot spots".In this thesis,the reduced form of nicotinamide-adenine dinucleotide(NADH)was detected indirectly using a novel enzyme-assisted method based on nanoporous gold substrate.And the test resulting can achieve the aim of quantitative and trace detection.The main content of this thesis are combed as follows:Firstly,preparation,structural characterization and performance optimization of the nanoporous gold.Cover glass was cleaned and modified by a layer of Au film with the thickness of 100 nm(cover glass played a supporting role).Electrochemical method was adopted in this experiment to prepare nanoporous gold using a three-electrode system through cyclic voltammetry,and the Au film worked as the working electrode.In the circulation process,zinc(Zn)atoms in electrolyte deposited to and separated from the Au electrode continually,alloy-dealloy reaction occurring on the surface of Au electrode contemporarily.Au electrode became rough gradually by repeating this cyclic process and formatting porous structure.To obtain the optimal substrate,the experiment conditions,such as reaction temperature,scanning cycle and scanning rate were changed.The experimental results showed that porous morphology began to appear when the temperature was higher than 50? and the aperture of the holes increased gradually with the increasing of scanning cycle and scanning rate.A series of substrates with different aperture were characterized systematically to screen the one with the optimal SERS performance.Rhodamine 6G(R6G)was chosen as the analyte in SERS test because of its obvious and strong Raman characteristic peaks.Series concentration of R6G were detected on the nanoporous gold substrates to consider the performance of enhancement.By contrast,the limit of detection(LOD)of the substrate prepared under the condition of 50?,20 cycles and 5 mV/s was as low as 10-7 M.Additionally,it is this substrate that performed best repeatability in the test.Therefore,these reaction conditions were used in the subsequent experiments as optimal conditions.Secondly,establishing a novel method for the detection of NADH based on the nanoporous gold——indirect method.NADH is a kind of indispensably important molecule in cell metabolism and the concentration of it can directly affect the metabolism of living cells.Quantitative detection of NADH allows for understanding of the overall cellular energy metabolism and monitoring of fermentation processes.In the earlier experiments,we detected NADH based on Au/Ag-based SERS substrates directly,but the molecules reacted with Au or Ag leading to degeneration according to the SERS spectra.In this paper,NADH was detected indirectly using a novel enzyme-assisted method based on nanoporous gold substrate.NADH generated H2O2 with the action of NADH oxidase in the first step reaction,and the H2O2 reacted with a chromogen generating a pigment with the catalysis by horseradish peroxidase(HRP)in the second step reaction.NADH can be detected through testing the SERS signal of the pigment.Eight kinds of chromogens were screened,and o-tolidine(OT)was chosen for the subsequent experiments.OT diimine produced from reaction of OT and H2O2 had obvious Raman peaks which were different from those of OT,and the LOD of OT diimine could reach the level of practical application.Additionally,the concentration of OT was optimized to 2 × 1 0-3 M for the widest linear range between the logarithmic integrated SERS intensity of the peak centered(IgS 1448)and the logarithmic H2O2 concentration(1gC(H2O2)).Relationship cure between 1gS 1448 and 1gC(H2O2)was built under this concentration.Two groups of experiments using different concentrations of NADH were done to verify the accuracy of the relationship curve.Final results showed that the logarithmic relative errors were less than 3%,which proved that this method was accurate enough to using in the actual detection.