THz-ATR Spectroscopy And THz Metamaterial Biosensor For Common Pathogenic Bacteria Detection

Background:Pathogen infection is a major threat to human health.It is very important to develop rapid and accurate detection methods for common pathogens for the prevention and treatment of infectious diseases.At present,the mainstream detection method of common pathogens is still based on enzymatic characteristics and biochemical reactions after bacterial culture.However,this method is time-consuming and requires much manual operation,so it is not suitable for early and rapid detection of pathogens and large sample size detection.To achieve rapid identification of pathogenic bacteria,some new detection methods have emerged in recent years.Detection methods based on mass spectrometry greatly simplify the detection steps of samples after bacterial culture and have high detection sensitivity,but expensive detection equipment limits their wide application.Molecular-based detection methods（such as PCR）and antigen-antibody-based detection methods（such as ELISA）have been used in the clinical detection of some pathogens.These methods make a major breakthrough in detection time without bacterial culture and can complete bacterial identification in a few hours,but there are some disadvantages,such as complicated operation steps,high reagent composition,and false positives.Compared with other detection devices,biosensors are easier to miniaturize and portable and have high specificity and sensitivity.Although they have not yet entered the clinical microbiology laboratory,much research has been conducted in this field.However,at present,there is no method based on biosensors that can simultaneously meet the actual needs of rapid,effective,specific and sensitive clinical testing.Therefore,there is an urgent need to establish a new biosensor,which is of great significance for the early diagnosis of infectious diseases.Terahertz（THz）wave is currently a research hotspot in the interdisciplinary field of biophysics.Pathogenic bacteria contain all kinds of biological macromolecules,and in the THz band,molecules can show various vibration forms.Therefore,THz spectroscopy is expected to be an innovative diagnostic tool for the rapid detection of pathogenic bacteria.However,there are still the following prominent problems in the practical application of THz technology in bacterial detection:（1）sensitivity of polar compounds,（2）complexity of spectral data,and（3）detection scale mismatch problem.To effectively solve these outstanding problems,various researchers have proposed different solutions,including THz-ATR technology,a combination of advanced metrology and statistical algorithms,and THz metamaterial technology.Terahertz attenuated total reflection（THz-ATR）technology can obtain and extract the optical and dielectric parameters of the sample to be tested close to the surface of the prism through the interaction of the evanescent wave enhancement and the sample to be tested.The penetration depth of the evanescent wave is tens of micrometers.When the biological sample is attached to the prism,the sample information in the effective area is detected,which can effectively avoid the interference of the excess upper material on the THz signal.Compared with traditional THz spectral detection technology,THz-ATR technology has significant advantages in the measurement of high THz absorbed liquid biological samples.Therefore,using THz-ATR technology to solve the problem of"water sensitivity"in bacterial detection is one of the research directions in this paper.THz spectrum information of pathogenic bacteria is rich and complex.How to accurately extract the spectral characteristic parameters and reduce the influence of noise signals is a challenge for THz spectroscopy technology.In recent years,Machine Learning（ML）technology has developed rapidly.Combined with advanced ML technology,it can effectively improve the recognition rate of THz spectra.Therefore,one of the main research directions of this thesis is to combine THz-ATR technology with ML technology to optimize data extraction and establish an automatic pathogen identification model to improve the accuracy of pathogen detection.Metamaterials can detect the target substance by generating a change in the resonance peak position caused by the local electric field enhancement effect.In addition,a number of studies have shown that the integration of metamaterials and nanoparticles can realize the secondary amplification of signals.Therefore,on the basis of THz spectrum detection,integrating THz metamaterials and aptamer-functionalized nanomaterials to solve the problems of low sensitivity and lack of specificity of the THz spectrum in practical applications is an important direction of this thesis.In conclusion,this study uses THz spectroscopy as a detection platform to systematically conduct research on the detection of common pathogens.First,aiming at the problem of"water sensitivity"when THz spectroscopy is used for pathogen detection,the THz-ATR detection platform is established for label-free measurement of pathogens.On this basis,aiming at the complex problem of THz spectral data of pathogens,THz-ATR spectroscopy combined with ML technology was used to improve the detection signal-to-noise ratio,and an automatic pathogen identification method based on a multi-classifier joint voting scheme was established,which could realize the species identification of pathogens in one minute after bacterial culture.Subsequently,to improve the sensitivity and specificity of THz spectroscopy,a THz metamaterial biosensor based on a gold coated magnetic-aptamer was constructed.The sensor uses the"metamateric-nanoparticle"signal amplification strategy to improve the detection sensitivity and uses the aptamer as the target recognition element to ensure the detection specificity,thus establishing a simple and rapid new method for pathogen detection.Finally,the constructed THz metamaterial biosensor was used for S.aureus detection to determine the basic methodological performance parameters,such as detection sensitivity,specificity and repeatability.The THz metamaterial biosensor was tested on clinical urine and blood samples to evaluate its clinical utility.This study provides a useful exploration for the application of THz spectroscopy in clinical microbiology laboratories.Methods:1.THz-ATR spectroscopy for label-free detection of pathogenic bacteria:The standard strains of 13 common clinical pathogens were selected,and 387 samples of standard strains were detected by a self-made sample pool.Principal component analysis（PCA）and Least-squares analysis（LS）were used to distinguish gram-positive bacteria,gram-negative bacteria and fungi.Five common clinical pathogens were collected,including 253 samples of E.faecalis,222 samples of E.coli,227 samples of P.aeruginosa,208 samples of C.albicans and 213 samples of C.tropicalis.THz-ATR spectroscopy was used to detect clinical strains.Two analytical methods,PCA and LS,were used for multivariate statistical analysis of the THz-ATR spectra of clinical strains.2.THz-ATR spectroscopy was combined with an automatic identification method based on a multi-classifier joint voting scheme for the identification of common pathogens:k NN,SVM and RF classifiers were used to construct an automatic identification method based on a multi-classifier joint voting scheme.The THz-ATR spectral features of five standard strains,including the refractive index and absorption coefficient,were selected to form a training set and used to construct an automatic identification algorithm.Six sets of results were used in the voting of the multi-classifier prediction model.The Reilef F algorithm was used to analyze the features of THz spectral data and rank them from large to small in order of importance,and then different numbers of attributes were selected to form feature subsets in order.These feature subsets were used for machine learning to achieve fast and automatic identification and classification of clinical pathogens.A total of 1123 clinical samples were used as the validation set,and the results of traditional bacterial culture and identification were used as the control.The performance of the automated method was evaluated by the ROC curve and AUC score.3.Construction of THz metamaterial chips and aptamer-functionalized gold coated magnetic materials:An Fe₃O₄@Au@Cys@Apt nanocomposite was designed and constructed by organic integration of nano-magnetic beads,nano-gold and aptamer.A flow meter was used to detect the binding efficiency of the aptamer to the bacteria.The synthesis efficiency of the composite was characterized by TEM,energy spectrum analysis,zeta potential and DLS.The subwavelength resonant unit of the THz metamaterial was designed and prepared for the detection of pathogenic bacteria,and its effectiveness was verified.The signal amplification effect of the"metamaterial-nanoparticle"system for pathogen detection in the THz band was verified.4.Research on THz metamaterial biosensor based on gold coated magnetic-aptamer for the rapid detection of S.aureus:Combined with THz spectrum technology,THz metamaterial technology and sample separation and enrichment technology based on gold coated magnetic-aptamer,a THz metamaterial biosensor was constructed.The sensor was used for rapid S.aureus detection,and the experimental conditions of the sensor were optimized.SEM was used to verify the effective formation of Fe₃O₄@Au@Cys@Apt-S.aureus complex.To determine the sensitivity,specificity,and repeatability of the sensor for detecting S.aureus,a recovery experiment was conducted to detect S.aureus in clinical samples using this sensor.Results:1.The characteristic parameters of the THz-ATR spectra of standard strains of 13common clinical pathogens were obtained.The absorption coefficient spectral curves of gram-positive bacteria,gram-negative bacteria and fungi were compared,and the differentiation of gram-positive bacteria,gram-negative bacteria and fungi was realized by further multivariate statistical analysis.Five common clinical pathogens were collected,including 253 samples of E.faecalis,222 samples of E.coli,227 samples of P.aeruginosa,208 samples of C.albicans,and 213 samples of C.tropicalis.The characteristic parameters of THz-ATR spectra were obtained successfully,and different multivariate statistical algorithms were used to distinguish clinical pathogens.This study provides ideas for exploring the application of THz-ATR spectroscopy in label-free detection of bacteria in the future.2.We construct an automatic pathogen identification method based on THz-ATR spectroscopy combined with a multi-classifier joint voting scheme.The method achieved an average accuracy of 80.77%in detecting five common pathogens and 99.60%in detecting E.faecalis.The AUC score of the automatic identification method based on the joint voting scheme was 0.8664,which was significantly higher than that of the single classifier scheme,indicating that the established method improved the accuracy of THz-ATR spectroscopy for pathogen detection.3.The Fe₃O₄@Au@Cys@Apt composite material was constructed,and the flow fluorescence detection results showed that the synthesized aptamer could effectively capture the target bacteria.The Fe₃O₄@Au putamen structure was characterized by TEM and energy spectrum analysis,and the Fe₃O₄@Au@Cys@Apt composite was successfully synthesized by zeta potential and DLS.The effectiveness of metamaterial chips and nanocomposites for detecting pathogenic bacteria has been demonstrated.The△f of S.aureus was 6.0 GHz,the△f of P.aeruginosa was 6.5 GHz,and the△f of the blank material was about 8.0 GHz.When the pathogen combined with the composite material to form a complex,the△f was 27 GHz,which was significantly higher than that of pure bacteria and the blank material,indicating that the constructed"metamaterial-nanoparticle"strategy sensor has an excellent signal amplification effect.4.The optimized conditions of the constructed THz metamaterial biosensor for S.aureus detection based on a gold coated magnetic-aptamer were as follows:the optimal incubation time was 40 min,and the optimal volume ratio of the nanomaterial to the bacterial solution was 1:1.There is a good linear relationship between the concentration of the S.aureus bacterial solution and its signal response on the THz metamaterial.The linear regression equation was=3.07log C+0.93（R²=0.99289）,and the linear range was 1×10³ CFU/ml to1×107 CFU/ml.The limit of detection（LOD）was 4.78×102 CFU/ml.The△f of S.aureus was significantly higher than that of S.pneumoniae,P.aeruginosa,E.coli and Mix1（S.pneumoniae+P.aeruginosa+E.coli）,and the DI values of the three interfering bacteria and Mix1 were less than 10%.The RSD of the intra-assay,intra-day and inter-assay repeatability tests of S.aureus were 2.69%,4.44%and 10.17%,respectively.The above results show that the constructed sensor has good sensitivity,specificity and repeatability.The biosensor was used to detect urine samples and blood samples of S.aureus at 1.0×10³CFU/ml,1.0×10⁵CFU/ml and 1.0×10⁷ CFU/ml.The RSD of the prepared samples ranged from 2.3%to 7.9%,and the recovery rate ranged from 82.4%to 109.6%.These results indicate that the constructed biosensor has the potential for rapid detection of S.aureus in clinical samples.Conclusion:1.THz-ATR spectroscopy has the potential to be used for label-free detection of pathogens.However,due to the diversity of clinical samples and patient sources,the genotype and phenotype of the same microorganism from different sample sources may be slightly different,so clinical pathogens show greater overall heterogeneity than standard strains.It will be helpful for the application of THz-ATR spectroscopy to clinical sample detection by combining advanced learning algorithms and mining meaningful spectral data information.2.Combining ML technology with THz-ATR spectroscopy technology,k NN,SVM and RF classifiers were used to develop and build an automatic identification method based on a multi-classifier joint voting scheme,which solved the problem of poor performance of conventional algorithms in analyzing THz-ATR spectral data of clinical pathogens and significantly improved the accuracy of spectral analysis.This strategy is label-free and reagent-free and enables pathogen identification within 1 minute after bacterial culture.3.By integrating magnetic nanoparticles,gold nanoparticles and aptamers,a novel functionalized aptamer magnetic nanoparticle composite was constructed.Based on the aptamer highly specific recognition strategy and magnetic separation strategy,the problem of insufficient specificity of traditional THz spectral technology for clinical sample detection was solved.Meanwhile,combined with a THz metamaterial chip,a signal amplifying system of"metamaterial-nanoparticle"is formed,which effectively improves the detection sensitivity.4.A THz metamaterial biosensor based on a gold coated magnetic-aptamer was established and used for S.aureus detection.The critical detection parameters of the sensor,including sensitivity,and its practical application potential were verified in a clinical sample analysis experiment.With the advantages of being simple,fast,high throughput,high sensitivity and high specificity,the biosensor is expected to be a new tool for clinical pathogen detection.