Construction And Application Of Novel Bacterial Electrochemical Sensor

Diseases caused by pathogenic microorganisms have become one of the most serious threats to human health.Diseases such as tetanus,typhoid,hemolytic uremic syndrome,pneumonia,tuberculosis are caused by bacterial infection.For exam ple,food borne diseases caused by Escherichia coli(E.coli)pollution resulted in diarrhea in about 1.5 million children every year,about 2%-7%of patients even develop into hemolytic uremic syndrome.Tuberculosis,caused by widely spread pathogen Mycobacterium tuberculosis(M.tuberculosis),is one of the major public health problems in the world,which causing about 1.8 million deaths in 2015.The key to reduce the spread and mortality rate of the diseases is to develop faster and more sensitive approaches for detection of bacteria.Traditional bacterial culture takes up long time to yield the result due to the long incubation periods.In recent years,a variety of new methods have been developed based on molecular biology,immunology and modern analytical instruments.Though rapid and accurate,these methods still face various shortcomings.Therefore,it is necessary to develop a simple,low-cost,fast and sensitive method for detection of bacteria.The 16S rDNA sequence structure consists of the consta nt region and variable region.The constant region is highly conserved and suitable for universal detection of bacteria.The variable region sequences vary with different bacterium,which are suitable for species typing,especially have advantages in isola tion and identification of caustic and slow-growing bacteria.The methods such as quantitative real-time PCR(q PCR),fluorescence and sequencing based on 16S rDNA sequence detection have been used for rapid and sensitive detection of bacteria.However,the se methods are expensive,which restricted their applications in developing countries.Electrical biosensor is particularly attractive for its low cost and easy popularization.The biosensor system enables to detect nucleic acids by converting hybridization events of nucleic acids between electrode pairs into measurable electrical signals.Combined with the compatibility with advanced semiconductor technology,integration,massive reproducible manufacturing as well as miniaturization,it is promising for the development of high-performance nucleic acids detection methods.Recently,MSPQC sensors have received extensive attention in the field of pathogen diagnosis due to the promising advantages of high sensitivity,low cost and easy operation.A series of M.tuberculosis sensors based on MSPQC system were constructed for sensitive detection of M.tuberculosis.However,the target of these methods was culture-based metabolite or antigen CFP10-ESAT6,it still could not overcome the drawbacks of culture and immu noassay.In this study,we developed several low-cost,fast,simple and sensitive new 16S rDNA bacterial sensors based on biological signal amplification strategies like enzyme-catalyzed amplification,target cyclic amplification and nanoparticle-mediated signal amplification using important pathogenic bacteria E.coli and M.tuberculosis 16S rDNA specific sequences as the detection target,using gold nanoparticles and functional nanomaterials Ti₃C₂ Mxenes as transducer elements.The research studies were as follows:(1)A biosensor platform based on nanogap network electrode was constructed.The preparation of nanogap network electrode is simple and low-cost.The nanogap network electrodes possessed larger surface area and showed higher capture ability to target.It significantly lowered the minimum detection limit.Meanwhile,as its gap of electrodes was smaller,the constructed sensor showed a high s ensitivity to conductimetric response and high mismatch sensitivity in the detection.As a potential sensor platform,combined with various biological signal amplification strategies and the application of functional nanomaterials,it provides a new path to develop low-cost,fast,simple and sensitive biosensors for pathogen detection.(2)A 16S rDNA nanogap network electrochemical sensor based on enzyme-targeting polyaniline deposition was constructed for rapid detection of E.coli.the electrochemical biosensor detected E.coli by using 16S rDNA as target biomarker,and using oligonucleotide probes and nanogap network electrodes as transducer elements.The nanogap network electrodes are prepared by interconnecting of gold nanoparticles through thiolated peptide nucleic acid(PNA)probes on the substrate of Au interdigital electrode.In the presence of specific 16S rDNA fragments,PNA capture probe hybridized to 5?terminal of the fragments,and detection probe modified with horseradish peroxidase(HRP)hybridized to its 3?terminal.HRP attached to the hybridized detection probe catalyzed the polymerization of anili ne along target chain.Conductive connection was caused by polyaniline deposition between the interrupted gold nanoparticles wires and offered a conductimetric response between the network electrodes.Thus,E.coli could be successfully detected with the detection limit of 100 CFU/ml.The detection time was less than 3 h.It would be widely used for rapid detection of E.coli.(3)A new 16S rDNA multichannel series piezoelectric quartz crystal(MSPQC)sensor based on Exonuclease III(Exo III)-aided target recycling has been developed for rapid detection of M.tuberculosis.The specific 16S rDNA fragment of M.tuberculosis was used as biomarker,DNA capture probes complementary to the biomarker were designed and modified on the surface of Au NPs.The Exo III which could recognise hybrid duplexes and selectively digest DNA capture probe was used to assist digestion cycle by digesting DNA capture probe and releasing the intact target fragment.After all DNA probes loading on the surface of Au NPs were removed,the surface of Au NPs was exposed and conductive connection was formed between the nanogap network electrode by self-catalytic growth of exposed Au NPs in the glucose and HAu Cl₄ solution.This resulted in sensitive response of M.tuberculosis sensor and M.tuberculosis was detected by recording this response.The limit of detection(LOD)of the method was 20 CFU/m L and the detection time was less than 3h.It was expected to be widely used in detection methods of M.tuberculosis.(4)A new family of two-dimensional(2D)transition metal carbides and carbonitrides,named MXenes,was discovered by selectively etching of the MAX phases and has received extensive interests.In our study,an electrochemical M.tuberculosis sensor was constructed by using specific fragment of 16S rDNA of M.tuberculosis H37Ra as target biomarker,peptide nucleic acid(PNA)as capture probe and highly conductive two-dimensional Ti₃C₂ MXenes as the signal amplified transduction material.After the hybridization between PNA and the specific fragment of 16S rDNA on the substrate of PNA-Au NPs nanogap network electrode,the target fragments were directly linked with conductive Ti₃C₂ MXenes by strong interactions between zirconium-cross-linked Ti₃C₂ MXenes and phosphate groups of the target fragments.The linking of Ti₃C₂ MXenes to the hybridized target fragments would bridge the gaps of the interrupted Au NPs in the nanogap network electrode and forming the conductive connection to cause the change in conductance between the electrodes.This conductance change could be used for M.tuberculosis detection.The limit of detection(LOD)of proposed method was 20 CFU/m L,and detection time was2 h.Proposed method would find potential application in rapid detection of M.tuberculosis.(5)A new MSPQC sensor based on Au NPs mediated enzyme-assisted target circulation was constructed for rapid detection of M.tuberculosis.Through the hybridization of I DNA probe and II DNA probe,a stem-loop DNA probe was formed with a protruding 3'terminus.When specific 16S rDNA target fragment of M.tuberculosis was exised,the target DNA open the stem-loop structure and hybridized with the DNA probe I,forming a double stranded DNA with a protruding 3'terminus at the target DNA.Because of the selectivity of Exo III,Exo III could recognize the blunt 3'end and selectively digest the DNA probe I.The target DNA and DNA probe II were released,the target DNA hybridized with another stem-loop DNA probe.Therefore,with the assist of Exo III,a large number of DNA probe II were generated through the circulation of target DNA.The DNA probe II was hybridized with the capture probe modified on the surface of Au electrode and the signal probe labeled with Au NPs which placed in the detection cell of MSPQC and Au NPs were closed to the surface of electrode.After adding the solution containing HAu Cl₄ and NADH,the gold ions in the solution were reduced and deposited on the surface of the Au NPs to realize the growth of the Au NPs,forming a conductive connection between the electrodes,thus providing the amplification of the frequency shift response signal of the detection of M.tuberculosis by the MSPQC sensor.The detection limit of the method was 30 CFU/ml,and the detection time was less than 3 h.