| Background:In recent years,pathogen-related infectious diseases are still a major potential harm to human health worldwide.The world health organization?WHO?has announced the ten threats to global health and six of them belong to infectious diseases and related high-risk behaviors in 2019.Identification of the source of the infection as soon as possible is of great importance to the diagnosis of infectious diseases.Without the rapid and accurate identification of the pathogenic bacterium,the patients with infectious diseases have to receive empiric antimicrobial treatment instead of targeted antibiotic prescribing.The overuse of broad-spectrum antibiotics may lead to a lack of effective therapy,bacterial resistance and possible challenges to the follow-up treatment.Currently,the traditional microbial identification procedures are still the mainstream methods in many medical institutions,especially in the less developed regions of medical resources.However,the complex operation,time-consuming procedures and large amounts of reagents bring many problems to clinical diagnosis.In order to realize the rapid identification of pathogenic bacteria,some molecular biological methods and biosensors have been developed in recent years.Nevertheless,some drawbacks,such as the expensive instrumentation and the undesirable specificity and sensitivity still remain.Mass spectrometry,such as MALDI-TOF-MS,is deem to be a revolutionary microbial detection technology,which has been successfully applied in some clinical laboratories.However,the sensitivity of this technology is relatively low?105-106 CFU?,and the cost of equipment maintenance is high.In additions,MALDI-TOF-MS still have some defects in identifying some pathogenic species,such as the differentiation between Escherichia coli and Shigella.Therefore,it is of great significance to explore a rapid and accurate method for identifying pathogen bacteria with a high sensitivity and specificity.With the rapid development of information science,interdisciplinary technology provides new insights into scientific research from the perspective of multi-disciplinary integration.Terahertz?THz?biomedical applications are at the frontier of the integration of physical optics and biomedicine.THz radiation generally refers to the frequencies from 0.1to 10 THz,which lies between the microwave and infrared regions of the electromagnetic spectrum.THz radiation has many attractive characteristics for biomedical applications:?i?Spectral fingerprint:the energy levels of low-frequency motions and the rotation or translation of the molecular skeleton coincide with photon energies of THz radiation.TH z technology could be used for label-free detection of biomolecules by measuring their characteristic spectral signatures,which are difficult to be detected by other electromagnetic waves.?ii?Noninvasive properties:the photo energies of THz radiation are much lower than those of X-ray and Gamma-ray.THz technology enables noninvasive detection of samples without potential biological hazards for the researchers.?iii?Convenient operation:THz spectroscopy only takes about ten seconds to acquire high resolution spectral signals.The fast and simple operation process is very suitable for developing small and convenient equipment.Nowadays,THz technology has been widely developed.THz technology is an image-spectrum merging modality,which can not only acquire the spectral information representing the essential properties of the sample,but also can construct the THz image according to its intensity and phase information.Owing to its fast,label-free and non-destructive features,these THz techniques have been applied in detecting various biosamples,including nucleic acids,proteins,cells,bacteria and tissues.Previous studies on pathogenic bacteria using THz spectroscopy mainly focused on the simple spectral exploration of some intracellular components,spores and bacterial cells.However,the study on the differentiation between bacterial species,which is more significant for clinical laboratory,has not been involved.THz spectroscopy has been used to differentiate between Escherichia coli and Bacillus subtilis.However,the mechanism of the THz signal response of various bacteria is still unclear.Most of the existing molecula r dynamics simulation results are quite different from the experimental data.Therefore,it is necessary to explore the mechanism of THz response of pathogenic bacteria.In addition,the low specificity and sensitivity are still the bottlenecks before its applications in clinical laboratory.It is urgently needed to couple other approaches with THz technology to develop efficient biosensor with improved specificity and sensitivity.Rolling circle amplification?RCA?is a commonly used isothermal amplification technology for the nucleic acid detection.To improve the specificity,we combined RCA with THz technology by detecting the amplified products of bacterial DNA instead of bacterial cells.The first step of RCA is to identify and capture the target sequence by the designed single-stranded DNA probes.Using the circular DNA as the template,the linear amplification was realized by DNA polymerase in the isothermal condition.RCA is simple,rapid,convenient and does not need expensive instruments.In addition,this user-friendly method can be performed on a solid-phase support,which is suitable to be integrated with other technologies.RCA has been widely used in the rapid detection of various bacterial DNA.Metamaterials are artificial structures consisting of periodically arranged sub-wavelength resonant units,which become an effective method to improve the sensitivity of THz technology.Metamaterials are very sensitive to the dielectric constant changes of samples on the surface.When THz radiation incidents on the surface,the enhanced local electric field will be induced,and the resonance frequency of metamaterial will be changed after the sample loading.In addition,materials with high refractive index,such as nanoparticles,can produce greater signal response on THz metamaterials.This strategy can realize the secondary signal amplification.Therefore,it is possible to largely improve the sensitivity of THz technology by the integration of a metamaterials-nanoparticle signal amplification system.In conclusion,this study explored the THz characteristics of common clinical pathogenic bacteria and the rapid detection of nucleic acid fragments based on isothermal amplification,by improving and optimizing the existing THz spectroscopy and imaging technology platform.Besides,this study involved three levels of samples,including pathogenic bacteria colonies,bacteria cells and nucleic acid fragments.We systematically studied the interactions between pathogenic bacteria and THz wave.Through THz spect ral characterization and the construction of two-dimensional THz absorption model of four common infectious pathogenic bacteria,we clarified the dominant role of intracellular water content in the THz signals of pathogenic bacteria,and realized bacterial species identification and viability status evaluation.On the basis of mechanism research,the visualized imaging detection of cultured colonies and in situ imaging analysis of cultured colonies on the culture plate were further carried out using transmission THz imaging and THz attenuated total reflection?THz-ATR?technology by taking advantage of being an image-spectrum merging modality.In order to further improve the specificity and sensitivity of THz technology,we integrated RCA reaction and"metamaterial-nanoparticle"signal amplification system,constructed RCA-THz liquid-phase sensing system and developed THz-metamaterial-nano biosensor based on isothermal amplification.We systematically tested the performance of clinical strains at the molecular level,targeting specific DNA fragments of pathogenic bacteria.The key methodological parameters,such as sensitivity and specificity were obtained.Through the above works,this study constructed a set of label-free THz sensing technology for bacteria colonies,bacteria cells and nucleic acids detection,which provided a new research idea for THz biomedical research and pathogenic bacteria detection.Methods:1.THz spectroscopic investigation of pathogenic bacteria:four common infectious pathogenic bacteria,Escherichia coli,Staphylococcus aureus,Acinetobacter baumannii and Pseudomonas aeruginosa,were selected.Samples of living bacteria,dead bacteria and bacterial powder were prepared by bacterial culture,metal bath heating and freeze-drying.A self-designed and prepared sample cell was used to detect pathogenic bacteria of different species and living states in a label-free manner.2.THz transmission imaging of bacteria colonies:A continuous-wave THz transmission imaging system based on optical pumped gas laser was used in this study.We designed and fabricated a high-resistivity silicon microplate for single bacterial colony imaging.The cultured bacterial colonies were detected by THz transmission imaging directly in the microplate.The above method was used to detect different pathogenic bacteria and mixed samples,and to continuously monitor the viability of the same bacterial colony.3.THz-ATR in situ imaging of bacterial colonies:THz-ATR imaging system based on high-resistivity silicon prism was used for in situ imaging of bacterial colonies.A series of image processing algorithms,inlcuding background segmentation and edge thinning based on K-means clustering,were used to optimize and obtain high contrast THz-ATR images.4.Development of RCA-THz liquid-phase sensing system for bacterial DNA detection:a set of specific lock probe and capture probe was designed based on E.coli DNA sequence as the template.The RCA reaction system based on the surface of magnetic beads was established.The effectiveness of the system was validated by agarose gel electrophoresis,zeta potential measurement and confocal microscopy,and methodological evaluation was also carried out.5.THz metamaterials design and processing:two kinds of metamaterial structures based on periodic arrangement of the metal split-ring resonators were designed and manufactured.Finite element simulation analysis and experimental measurement method were used to evaluate the performance of improving the detection sensitivity.6.Construction and optimization of THz-metamaterial-nano biosensor based on isothermal amplification:based on the RCA-THz liquid-phase sensing system,the"RCA products-nanoparticles"complex was measured by THz metamaterials,and to construct THz-metamaterial-nano biosensor based on isothermal amplification.Conditions such as the hybridization temperature and gold nanoparticle concentration were systematically optimized.7.Methodological evaluation of clinical samples:clinical samples of the four pathogenic bacteria were detected by the THz-metamaterial-nano biosensor based on isothermal amplification,and key methodological parameters such as sensitivity,specificity and accuracy were obtained.Results:1.Spectral parameters including absorption coefficients,refractive indexes,real part of dielectric constant and imaginary part of dielectric constant of four common infectious pathogens,E.coli,S.aureus,A.baumannii and P.aeruginosa,were obtained.A two-component model of THz absorption of pathogenic bacteria was constructed to compare the experimental measurements result and estimated result of the absorption coefficients of E.coli.It was proved that the intracellular water molecules have a great role for the overall absorption of samples,which is the theoretical basis of label-free bacterial detection by THz spectroscopy.2.THz transmission imaging was used to analyze four different bacterial species and the mixed colonies.THz images of different species of pathogenic bacteria differed significantly,and the samples mixed at different proportions of pathogenic bacteria could also be distinguished in the THz images.The viability change of S.aureus varying with time were obtained by THz imaging.3.A high contrast THz-ATR image was obtained,which confirmed the feasibility of in-situ measurement.The ATR attenuation values of A.baumannii and E.coli were 74.65±0.56%and 71.55±0.49%,respectively?p<0.05?.THz-ATR technology can be used for in situ measurement of pathogenic bacterial colonies.The standard deviation was significantly smaller than that of transmission imaging,which shows the better repeatability of the method.4.The electrophoretic results proved that the RCA reaction produced high molecular weight single-stranded DNA products.Zeta potential measurements showed that the zeta potential?42.9±0.90 mV?of RCA beads was significantly lower than that of blank beads?29.7±0.15 mV?and capture probe beads?28.6±0.25 mV?,indicating that the coverage of amplified nucleic acid products on the surface of RCA-MB.Fluorescence dyeing and confocal microscopy showed that the amplified nucleic acid products were dyed green by SYBR Green II,but no obvious color appeared on the captured probe beads.The above results confirmed the effectiveness of the RCA reaction system on the surface of the beads and the RCA-THz liquid-phase sensing system was successfully constructed.The detection limits of synthetic bacterial DNA and clinical samples were 0.6×10-1010 M and 0.05 ng?L-1,respectively.The difference of average THz absorption coefficient between E.coli and the reference(39.4±4.0cm-1)was significantly higher than that of P.aeruginosa samples(5.2±1.3cm-1)and A.baumannii samples(5.0±1.1cm-1)?p<0.05?,demonstrating the good specificity of the proposed method.5.Finite element analysis showed that the resonance shifts of four pathogenic bacteria,E.coli,S.aureus,A.baumannii and P.aeruginosa were different.E.coli and S.aureus could produce different frequency shifts on THz metamaterials,and the resonance shifts between living and dead E.coli were also different.6.The construction of THz-metamaterial-nano biosensor based on isothermal amplification was verified.The frequency shift of E.coli RCA product was 45.78 GHz compared to the blank THz metamaterials,which was significantly larger than that of negative control?15.21 GHz?and blank control?7.61 GHz?.The frequency shift?61.04GHz?of the THz metamaterials in the presence of gold nanoparticles was significantly larger than that of the pure RCA product?45.78 GHz?.The detection conditions of THz-metamaterial-nano biosensor based on isothermal amplification were optimized:the temperature of hybridization reaction between PLP and target nucleic acid sequence was60?,the amount of E.coli DNA ligase needed was 5U,and BSA should be used in the conjunction reaction.After reaction,excess PLP could be eliminated using exonuclease I and exonuclease III,and the optimum amount of CP-MBs was 1?M.The Phi 29 DNA polymerase was 10U,the concentration of dNTPs mixture was 10mM,the optimum amplification time was 40 minutes,and the"RCA product-nanoparticle"complex obtained at the concentration of 1nM of AuNP-MP solution had the largest frequency shift on THz metamaterials.7.RCA reaction and THz metamaterials-nanoparticle signal amplification system can significantly improve the detection specificity and sensitivity.The frequency shifts of the RCA product-nanoparticle complex on the THz metamaterials were linearly related to the logarithm of the concentration of the target nucleic acid sequence?y=16.23x+28.66,R2=0.9465?.The minimum detection limit for clinical samples was 0.1 pg/?L-1.Compared with traditional detection methods,the sensitivity,specificity and accuracy of this biosensor for the detection of four clinical pathogens were:E.coli?100%,95.8%and 98.0%?,S.aureus?100%,100%and 100%?,P.aeruginosa?90.9%,97.4%and 96%?and A.baumannii?100%,97.6%and 98.0%?.The above indexes were analyzed by Kappa test,and the results of the two methods were consistent?p<0.05,Kappa>0.75?.Conclusion:1.The mechanism of signal response and main influencing factors were clarified,which provided theoretical support for the rapid and label-free detection of pathogenic bacteria by THz technology.A new method for rapid and label-free detection of pathogenic bacteria based on intracellular water content has been developed,which can be used to identify pathogenic bacteria species and evaluate their living status in a few minutes.2.The visual imaging and in-situ detection of pathogenic bacteria colonies after culture were carried out using THz transmission imaging and THz-ATR technology.The practical value of THz imaging technology at the bacteria colony level was verified.By transforming complex THz imaging results into simple color changes in the three-dimensional color display model,bacterial species detection and the target in mixed samples could be identified without complex data analysis.A rapid method for direct identification of the target bacterial colonies was developed for emergencies.3.RCA-THz liquid-phase sensing system was constructed by combining nucleic acid isothermal amplification with THz spectroscopy,which solved the issue of lack of specificity,and realized the effective detection of bacteria DNA in clinical samples.The synthetic target nucleic acid fragments and genomic DNA of clinical strains of E.coli were detected using the developed RCA-THz liquid-phase sensing system.A series of important methodological parameters,such as the sensitivity,repeatability and specificity,were obtained,which provided a general scheme for the highly sensitive detection of nucleic acids using THz technology.4.A highly sensitive and specific THz-metamaterial-nano biosensor based on isothermal amplification was developed to detect clinical samples at the nucleic acid level.This biosensor was composed of RCA reaction and THz metamaterial-nanoparticle signal amplification system.Compared with the existing clinical microbial detection technologies,it has several advantages,such as fast,accurate,highly sensitive and specific,and is has certain clinical application value.It provides a new label-free sensing technology for laboratory medicine from the perspective of frontier interdisciplinary. |
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