Detection Of Pathogenic Bacteria Based On Gold Nanoparticles Labeling ICP-MS Measurement And Its Application

Infection caused by pathogenic bacteria is an important reason for human disease.The rapid detection of bacterial pathogens plays a noticeable role in the fields of disease diagnosis,food safety,public health maintenance,environmental monitoring and preventing the spread of disease.Traditional culture methods for the identification of pathogenic bacteria followed by standard biochemical assays are considered the criterion standard,but the whole processing always take 6–7 days and have several limitations such as dependency on enrichment,selective culture and difficulties with quantitative analysis.Immunoassay methods based on antibodies specificity have been applied because of their simplicity and sensitivity,but the specificities of the antibodies are variable and a relatively long time was required for acquiring the antibodies.Various molecular methods based on nucleic acid technologies have been developed in which sequence-specific DNA oligonucleotides(DNA probes)could be used directly in hybridization assays or as primers in amplification reactions.It is worth mentioning the polymerase chain reaction(PCR)shows more advantages in the section of sensitive and specific than that immunological methods because of amplification.But many kinds of disadvantages often have been mentioned,such as the complicated design of primer,the sophisticated synthesis of fluorescent tags,and the spectral overlap problems.Biosensorsarebroadlyusedin identification of pathogenic bacteria.Inductively coupled plasma mass spectrometry(ICP-MS)is regarded as a powerful technique with high sensitivies,low limit of detection and multielement analysis for the most metal elements in periodic table.The quantification technique of element tagging-based in the field of biomolecules,especially in the detection of protein,peptide,nucleic acid,cell and even the bacteria has gained great attention by using ICP-MS.Metal elements tags were labeled with antibodies,oligonucleotides,which can identificate the target molecules by the means of specificity between them.This identification are usually based on the immunoreaction,hybridization and unique binding between aptamers and target molecules.As a result,the final complexes were immobilized to thesolid-phase such as microplates,magnetic bead and excess reagent could bewashed out to ensure the accuracy of later quantifications.ICP-MS can presentthe precise and sensitive signal of the mass-to-charge ratios come from the labeled element of complexes.Two kinds of elements,lanthanide chelates or metal nanoparticles(gold or silver),were usually utilized as tags to labeled the biomolecule and the specific activities of biomolecules can be retained in the modification.Lanthanide elements have very similar properties and can be tagged to biomolecules using the same methods,which favors multiplex analysis.Nanoparticles are another kind of typical elemental tags not only because of being prepared easily but also the enhanced signal resulting from the nanopaticles containing numerous metal atoms.In the dissertation,novel and rapid detection assays based on the ICP-MS and gold nanoparticles labeling for pathogenic bacteria were studied.The main contents of the present dissertation are as follows:1.Process of pretreatment and storage for bioreagent was stated in order to keep their biological reactivity.Streptavidin was coated on the micro-well to capture the biomolecule labeled with biotin and the BSA was used to block the uncoated sites in this study.The gold nanoparticles were prepared and charactered.Some other conditions such as detection media were also optimized.2.A simple and sensitive detection method for target DNA of Shigella flexneri was developed using quantification of gold nanoparticles(AuNPs)by ICP-MS.The target Shigella flexneri DNA was detected by sandwich hybridization assay,in which the AuNPs-labeled reporter probes and the biotinylated capture probes are specific and complementary to the target DNA sequence.After separated with excess reagent and diluted into HNO3(5%,v/v),197 Au signals from the hybridized compounds were detected sensitively using ICP-MS.Under the optimized conditions(concentration of capture DNA and dilution ratio of reporter probes),this method could detect as little as0.39 pM(LOD,3?)of single-stranded target oligonucleotides and 39.17 pM of double-stranded target sequences(LOD,3?).The relative standard deviation(RSD,n =3)for 10 pM target ssDNA was found to be 5.8% and the relative standard deviation(RSD,n = 3)for the simulated target double-stranded DNA(dsDNA)was 5.5%.To verify specificity of hybridization,three mismatched ss-DNA targets were also tested using the same experimental procedure.A higher net intensity of 197 Au from the matched target DNA(sequence3)was observed,which confirmed that the target DNA could be distinguished easily from similar base-mismatched sequences using this method.3.A method of aptamer-conjugated gold nanoparticles for ICP-MS dual-aptamer-based sandwich detection of staphylococcus aureus was developed,which was based on the specificity of DNA aptamers produced using systemic evolution of ligands by exponential enrichment(SELEX)method to staphylococcus aureus.In this bioassay system,a biotinylated capture aptamer was immobilized on streptavidin coated microplates via biotin-streptavidin affinity,which serves as a capture probe.A secondary aptamer was conjugated to gold nanoparticles(Apt-AuNPs)that sensitively reports the detection of the target.In the presence of target bacterium,an Apt/staphylococcus aureus/apt-AuNPs sandwich complex is formed on the microplate surface and the 197 Au signal of AuNPs followed through ICP-MS.Parameters,such as the concentration of primary aptamer as a capture probe and secondary aptamer as a signaling,were optimized to achieve the highest signal-to-noise ratio of 197 Au signal.A low density of immobilized aptamer onto microplates results in a narrow linear range with a diminished sensitivity.On the other hand,aptamers always adopt a secondary structure with a high level of steric hindrance.The proposed sandwich assay benefits from advantageous of a sandwich assay for increased specificity,microplates as carriers of affinity ligands for solution-phase recognition and final washing separation,AuNPs for signal amplification,and ICP-MS read-out as a sensitive detection.The assay shows a dynamic range from 7×103 to 7×107 CFU/mL with a low detection limit of 279CFU/mL(LOD,3?)forthe bacterial strains of Staphylococcus aureus.Furthermore,the specificity of this method using other bacteria(Shigella flexneri)was studied.To assess the general applicability of this aptamer probe,we investigated the quantification of Staphylococcus aureus in simulative water samples.4.Row milk and derived products are liable to be contaminated by bacterial pathogens as we know staphylococcus aureus was a kind of them.Method based on ICP-MS and Real-Time PCR have been developed to identify staphylococcus aureus in samples of purchased milk from supermarket.The results suggest that the ICP-MS assay showed agreement with Real-Time PCR.In addition,it also simplifies the experimental process and saves the experimental time.Recovery tests were performed by measuring staphylococcus aureus in added into sterilized milk samples.It was demonstrated that results from ICP-MS agreed well with those from Real-Time PCR,indicating that the present method could be applied to real samples.