Binding Interaction Between Binding Proteins And Ligands Based On Electrochemical Impedance Biosensors And Terahertz Time-domain Spectroscopy

There consist of two parts in the study.The first one is the interaction between insect olfactory proteins and neonicotinoid insecticides based on electrochemical impedance biosensors.Electrochemical impedance biosensors can recognize biochemical signals and convert them into corresponding electrical signals through bio-sensitive membranes and transducers attached to the electrodes by using biosensor technology combined with electrochemical impedance technology.This type of sensor shows high sensitivity to the interaction of protein and small molecules.Previous studies have found that certain insect olfactory-associated proteins have strong affinity with neonicotinoid insecticides.This principle can be used to design and develop biosensor detection methods for such insecticides.The results exhibited that chemosensory protein 1(CSP1),antennal special protein 2(ASP2),odorant-binding protein 12(OBP12)and their corresponding mutant proteins,namely CSP1-F44Y26 G,CSP1-Q63 G,ASP2-K51 and OBP12-K117 G,all can bind with imidacloprid strongly.The experimental data were analyzed and a normalized impedance model was establish by using the principle of Randles equivalent circuit and Zview fitting software.The results showed that the sensitivity of the mutant protein CSP1-F44Y26 G increased by 84 % when binding with imidacloprid compared with wild-type protein.The sensitivity of CSP1-Q63 G and ASP2-K51 increased by 19 % and 13 %,respectively.However,that of OBP12-K117 G decreased by 26 %.This experiment provided new ideas and theoretical basis for rapid detection of biosensors of neonicotinoid insecticides.The second part is the interaction of serum proteins with ampicillin based on terahertz time-domain spectroscopy.Terahertz(THz)spectroscopy can directly reflect the fingerprint characteristics of the vibration and rotation of protein molecules in the microcosmic environment.As the main protein in human plasma,Human serum albumin(HSA)can combine with small drug molecules and carry drugs to the target site of lesions.Their interaction studies have important theoretical significance for pharmacokinetics.The interaction between HSA and ampicillin was studied by using Terahertz Time Domain Spectroscopy(THz-TDS)in this experiment.The results showed that the HSA sample signal in the time domain spectrum has a certain attenuation amplitude and delay in phase with respect compared to the reference signal.After obtaining the corresponding frequency domain spectrum by Fourier transform,it was found that the terahertz frequency spectrum of the HSA sample decreased with the increase concentration of ampicillin and the amplitude change increased with increasing frequency.HSA samples were quantified using principal component analysis and variance analysis.The fitting model of the previous frequency domain data was established by using partial least squares and Minitab software,which based on the reported terahertz characteristic peaks of the amino acids.It was found that the amino acid residues histidine(His)serine(Ser)tyrosine(Tyr)and tryptophan(Trp)in the interaction between HSA and ampicillin exhibited strong binding properties.Molecular docking predicted that His146 was the key amino acid site of HSA binding with ampicillin,which was consistent with the experimental fitting of terahertz frequency domain data.Based on the combining measurement results of the interaction between HSA and ampicillin by electrochemical impedance biosensor,the advantages and disadvantages were compared between sensors and terahertz detection methods.Due to the low adsorption capacity of nitrocellulose membranes on the electrodes for large proteins such as HSA,sensor technology limits the sensor's ability to detect protein macromolecules,but the quantitative method of biosensor is simple and convenient.In addition,Terahertz time-domain spectroscopy can achieve non-destructive detection of proteins,making up for deficiencies that do not reflect the characteristics of amino acids under microscopic conditions in sensory detection fluorescence spectroscopy and isothermal titration calorimetry.It is of great significance for studying the fingerprint binding characteristics of protein molecules in the microcosmic environment.