The Signal Amplification Mechanism Of Toxin Protein And Its Applications In Pathogenic Detection

Marine pathogenic microorganisms seriously threaten to public health.Therefore,timely and accurate pathogenic detection methods are very important for the rapid diagnosis and the treatment of disease.Despite recent signal reporter-based methods for bacterial detections and analyses have been well progressed,low-cost,ultra-sensitive,accurate,and rapid diagnosis is still a challenge for precise patient care.Toxin proteins from the microbial defense system（MDS）have been expressed and used as signal amplification probes.Three kinds of pathogenic diagnosis sensors were constructed based on functionalization of toxin proteins to provide rapid diagnosis and early warning of marine pathogenic microorganisms.The main research contents are as follows:1.A novel microbial detection method based onα-hemolysin-labelled sandwich assay（HLSA）has been developed.α-hemolysin was used as a new type of electrical signal reporter in this HLSA method.The hemolysin activated nanocomplexes formed by the combination of target,magnetic capture probe and functionalized gold nanoparticle detection probe,and then gold binding peptide（GBP）was added to release hemolysin into solution.The free hemolysin formed nanopores on the phospholipid bilayer that presented the quantitative current signal depending on the target nucleic acid concentration.Compared with traditional DNA nanopore sequencing methods,HLSA could achieve accurate and quantitative readings directly using the e ONE^HS amplifier without ultra-sensitive and expensive instruments.This method was designed as sandwich probes targeting 16S r RNAs of 10 common pathogens and single cell level nucleic acid concentration detection was achieved.HLSA provides a new idea for the diagnosis of pathogens and viruses2.In order to meet the requirements of ultrasensitive microbial detection,trypsin-amplified nanopore amplified sandwich assay（t NASA）using trypsin in situ mediated proaerolysin as bioelectrical signal probe was proposed based on HLSA.t NASA successfully overcame the amount limitation of the toxin protein signal amplification markers that can be loaded on the nanoprobe,increasing the detection sensitivity by nearly100,000 times,which greatly improved the sensitivity of the nanopore technology.Moreover,the t NASA sensor was able to detect 1 a M concentration or single-cell level nucleic acid through a tiny patch clamp current amplifier without complex analytical algorithms.3.A sensitive biosensor strategy depending on botulinum neurotoxin type A light chain（Bo NT/A LC）activated complex assay（BACA）was proposed.Bo NT/A LC is the surrogate of Bo NT/A and can serve as an ultrasensitive signal reporter with high signal-to-noise ratio to avoid common strong background response,poor stability and low intensity of current biosensor methods.A nanoparticle hybridization system with specific binding probes that recognize pathogenic 16S r RNAs or SARS-Co V-2 gene site was developed to measure double stranded target DNA using Fluorescence Resonance Energy Transfer（FRET）-based assay or colorimetric method.The BACA method validated by six different bacteria strains could achieve a single cell or 1 a M nucleic acid sensitivity.This detection strategy offers a solution for general applications and has a great prospect to be a simple tool when facing public health emergency.The main innovation points are as follows:Focusing on how to realize accurate and sensitive pathogenic detection,this paper developed three pathogenic diagnostic technologies based on toxin protein signal amplification system.These methods effectively reduced the false positive rate and significantly improved the detection sensitivity,and they provided a new research direction for the diagnosis of pathogenic microorganisms.