Study On The Concentration Detection Of Norfloxacin Antibiotic Based On Terahertz Time-domain Spectroscopy

China is a big country of the production,processing and consumption of livestock and poultry products.The current unreasonable use of antibiotics such as norfloxacin in livestock and poultry breeding causes the problem of excessive antibiotic residues in livestock and poultry products,which poses a serious threat to human health and the ecological environment.The existing antibiotic detection methods mainly include physical and chemical detection methods,microbiological detection methods,and immunoassay methods.These methods have certain shortcomings in terms of pretreatment process,time consumption,test cost,sensitivity,antibody preparation,etc.The development of simple,rapid,sensitive qualitative and quantitative detection methods for the content of major antibiotics have become an urgent need in the industry.Terahertz wave is the last segment of the electromagnetic spectrum that has not yet been deeply applied for research.It can obtain terahertz band spectral response information that cannot be obtained by traditional detection methods,and is of great value for studying the physical and chemical properties of materials.Preliminary studies have shown that terahertz international cutting-edge technology is feasible for the qualitative and quantitative detection of antibiotics.This study took norfloxacin antibiotics with more serious residues as the research object,and designed experiments to carry out research on the construction of terahertz quantitative detection models of higher and lower concentrations of residues.The specific contents include:（1）Research on quantitative detection method of full concentration norfloxacin based on terahertz time-domain spectroscopy technology.In order to explore the qualitative and quantitative detection ability of norfloxacin by terahertz spectroscopy,the doping of norfloxacin in fish meal matrix was taken as the research object,0%,1%,3%,5%,10%,15%,20%,25%,30%,35%,40%,45%,50%,100% were selected as the concentration gradient for higher concentration samples preparation.Taking norfloxacin residue in absolute ethanol as the research object,a total of 29 concentration gradients of 0.001-90μg/ml were selected for the preparation of lower concentration samples.The TERA K15 THz-TDS（Menlo,Germany）in point-point transmission mode was used to measure the spectral data of the sample,and then the spectral differences and characteristics of different concentrations of norfloxacin in fish meal were analyzed.The results showed that the amplitude attenuation and time delay would appear in the time-domain spectrum of the norfloxacin samples with different concentrations,and the higher the concentration of norfloxacin in the fish meal,the greater the time delay,and the analysis results of the frequency domain amplitude were consistent with the time-domain spectrum;After the absorption coefficient is preprocessed by Savitzky-Golay（S-G）convolution and smoothing,the frequency band with high signal-to-noise ratio0.3-1.5THz was selected for follow-up research.The results showed that there were obvious characteristic absorption peaks at 0.825 THz and 1.187 THz in the absorption coefficient of norfloxacin.With the increase of the concentration of norfloxacin in fish meal,the terahertz spectral absorption coefficient of the sample also increased.Using sample absorption coefficient data as input,the feature frequencies were selected by successive projection algorithm（SPA）and stepwise regression,respectively.The feature frequencies selected and characteristic absorption frequencies were established prediction model by multiple linear regression（MLR）respectively.Meanwhile,the least-squares support Vector Machine（LS-SVM）and back propagation-artificial neural network（BPNN）were selected to build the model and predict.The results showed that the calculation model based on BPNN had the best performance,and the Rp and RMSEP were0.941 and 8.790%,respectively.This study showed that terahertz time-domain spectroscopy has a good ability to detect norfloxacin in fish meal matrix with higher concentration.Based on the higher concentration detection method,when the lower concentration of norfloxacin was detected,the optimal correlation coefficient of correct set（Rc）and correlation coefficient of prediction set（Rp）were 0.859 and0.728 and the root mean square error of correct set（RMSEC）and root mean square error of prediction set（RMSEP）were 15.18μg/ml and 18.79μg/ml respectively.The detection ability is very weak,and new detection methods need to be found.（2）Research on quantitative detection method of low concentration norfloxacin based on terahertz metamaterial technology.Taking a low-level concentration of norfloxacin solution in absolute ethanol as the research object,choosing 0,0.001,0.01,0.1,1,10,100μg/ml as the concentration gradient for sample preparation,and trying a new type of terahertz metamaterial structure was used for signal enhancement research.Firstly,the transmission spectrum of the metamaterial simulated by Micro Studio CST software was compared with the actual measured transmission spectrum to find the appropriate resonant frequency to simulate the photoresist with different thickness on the metamaterial surface.The simulation results showed that with the increase of dielectric thickness,the frequency of metamaterial transmission peak is red-shifted,and the corresponding sensitivity is 3.1 GHz/10μm,which theoretically demonstrates the feasibility of metamaterial detection of norfloxacin solutions with different concentrations.The TERA K15 terahertz time-domain spectrometer produced by Menlo,Germany,was used to actually measure the transmission spectra of norfloxacin films with different concentrations on the surface of the metamaterial.It was found that when the concentration of norfloxacin was 0.001-0.1μg/ml,the amplitude of the transmission peak was increases with the increase of concentration and the transmission peak appeared red-shift.When the concentration was greater than 0.1μg/ml,with the increase of concentration,the transmission peak frequency appeared red shift and the amplitude value decreased.Norfloxacin concentration and red-shift value satisfy the functional relationship: y=0.0049997×x0.2052;the terahertz transmission spectra of norfloxacin films with different concentrations on the surface of the metamaterial were further analyzed by Principal component analysis（PCA）cluster analysis.It was found that the metamaterial structure could detect the minimum concentration of norfloxacin in anhydrous ethanol as0.01μg/ m L,which met the residue limit of fluoroquinolones antibacterial drugs in animal-derived food specified by the national standard（GB 31650-2019）.This study showed that the terahertz spectroscopy combined with metamaterial structure technology can realize the quantitative detection of norfloxacin at low concentration.（3）Research on visualization and quantitative detection of norfloxacin content based on terahertz spectral-imaging fusion technology.Norfloxacin with different concentrations in fish meal matrix was used as the research object.Sample preparation was carried out with 0%,1%,3%,5%,10%,15%,20%,25%,30%,35%,40%,45%,50% and 100% as concentration gradient.Terahertz imaging data of all samples were measured using TERA K15 imaging mode produced by Menlo Company in Germany.The following four methods were used to select the characteristic frequencies.The results were as follows: the maximum frequency of amplitude difference between pure norfloxacin and fish meal was 0.356THz;the characteristic frequencies of stepwise regression and SPA were 0.975 THz,1.163 THz,1.294 THz and 0.963 THz,1.144 THz,respectively.The characteristic absorption frequencies were 0.825 THz and 1.187 THz.Visualized imaging of different concentrations of norfloxacin in fish meal with different characteristic frequencies was researched.Morphological calculations were used to process the image to remove boundary noise,and BPNN was used to establish an imaging evaluation model to evaluate the imaging effects of different characteristic frequencies.The imaging effect of norfloxacin at 0.825 THz of characteristic absorption peak was the best,and the evaluation parameters were the best.Precision and Sensitivity were 0.939 and 0.933,respectively.This showed that the terahertz map-spectrum technology can realize the visual imaging of norfloxacin in fish meal matrix;Further analysis found that at 0.825 THz,the average gray value and concentration of the region of interest of norfloxacin in fish meal at different concentrations met the relationship: y=0.00442×x^1.9933+3.016,the coefficient of determination R² is 0.979.This study showed that the terahertz spectral-imaging fusion technology in visual and quantitative detection of norfloxacin concentration had good potential.The above research further expands the scope of research and exploration of the application of new terahertz spectroscopy technology in the agricultural field,and the results will provide theoretical support for the development of rapid inspection methods for norfloxacin antibiotics,which will help ensure the quality and safety of feed and livestock products in my country.The protection of human health and ecological safety has important practical significance and scientific value.