Novel ICP-MS Based Virus Detection Methods With Elemental Labeling And Signal Amplification Strategies

Viruses are important culprits that causing human public health pandemics,they are also the pathogens of many animal or plant diseases.The culprit of the Corona Virus Disease 2019（COVID-19）that appeared at the end of 2019 is a member of virus family（SARS-Co V-2）.Rapid and highly sensitive detection of viruses is vital to preventing and controlling the epidemics.Usually,the targets of virus detection can be virus particles,virus proteins,virus nucleic acids,or virus antibodies.The existence of viruse particles,proteins or nucleic acids are the evidences of the virus,and the virus antibodys are important indicators of infection or vaccination.However,problems such as tedious lab operation,small sample amounts,low target concentrations,and complex sample matrix often exist in virus detection.Therefore,it is of great significance to construct novel virus detection methods with simple operation,low sample consumption,high sensitivity and veracity,and strong anti-interference ability.Element labeling strategy combined with inductively coupled plasma mass spectrometry（ICP-MS）detection is a new type of quantitative bio-analysis method developed in recent years.The strategy has high sensitivity,wide linear range,and strong anti-interference ability and multiplex analysis ability.However,just few methods for virus detection using ICP-MS were reported.The purpose of this thesis is to:1)broaden the application area of element labeling strategy with ICP-MS detection in virus detection;2)introduce a variety of signal amplification strategies into the virus analysis with element labeling and ICP-MS detection,improving the sensitivity of the method;3)achieve homogeneous detection of viral nucleic acids by single particle ICP-MS.The research of this thesis includes the following contents:（1）A novel ICP-MS based immunoassay with gold nanoparticles（Au NPs）labeling for the determination of H9N2 virions was proposed.Magnetic-beads modified with anti-influenza A H9N2 hemagglutinin mono-antibody（m Ab-HA）were utilized for the capture of H9N2 virions in complex matrix.Au NPs conjugated with m Ab-HA were employed for the specific labeling of H9N2 virions for subsequent ICP-MS detection.Under the optimized conditions,the proposed method could detect as low as 0.63 ng m L^-1 H9N2 virions with a linear range of 2-400 ng m L^-1.The developed method was applied for the detection of H9N2 virions in chicken dung samples,and the recovery for the spiking expeiments was in the range of 91.4-117%.The proposed method is simple,sensitive,specific and robust with good application potential in complicated samples analysis.（2）In order to improve the sensitivity of the method,a dual-mode detection method based on immuno-rolling circle amplification（immuno-RCA）and quantum dots（QDs）labeling for fluorescence and ICP-MS detection of H9N2 AIV was established.With the immuno-RCA amplification,the sensitivity of the method was increased with two orders of magnitude.The limit of detection of the proposed method was 17 ng L^-1and 61 ng L^-1,and the linear range was 0.05-5 ng m L^-1 and0.1-5 ng m L^-1 with ICP-MS and fluorescence detection,respectively.The proposed method has a high specificity and reproducibility.The QDs labeling realized dual-modes detection,which can improve the flexibility of the method.Moreover,the analytical results obtained by the two detection modes can be verified mutually.The proposed method was applied to the analysis of chicken serum samples,and the application potential was demonstrated.（3）In order to improve the method sensitivity and simplify the operation,a dual amplification strategy method combining isothermal circular strand-displacement polymerization reaction（ICSDPR）and Au NPs amplification for the DNA analysis was established.In this method,Au NPs acted as both the signal tag of ICP-MS detection and the carrier of the ICSDPR,the dual amplification process can be realized in a one-pot isothermal reaction.The limit of detection was 8.9 fmol L^-1,with a linear range of 0.1-10000 pmol L^-1.Compared with the sample volume（100μL）commonly consumed in ICP-MS-based methods for nucleic acid analysis,the sample volume applied in this work was much less（5μL）.Although two amplification strategies were involved,the entire operation process in this work only required about4.5 h.It indicated that the proposed method achieved fast and sensitive detection of trace analytes.The ICP-MS based DNA assay is sensitive,simple,and fast with low sample consumption.（4）In order to obtain ultra-high sensitivity,a hyper-branched rolling circle amplification（HRCA）based method for HBV DNA determination was established.By using ruthenium complex[Ru（bpy）₂dppz]²⁺（bpy=2,2’-bipyridine,dppz=dipyrido[3,2-a:2’,3’-c]phenazine）as the signal probe,the proposed method realized both ICP-MS and fluorescence detection.The initiated HRCA realized high efficiency amplification of HBV DNA with the generation of a mass of ds DNA.[Ru（bpy）₂dppz]²⁺probe intercalated with the generated double-stranded DNA directly without the assist of the specific nucleic acid.ICP-MS and fluorescence detection of target DNA were realized by detecting the signal of ¹⁰¹Ru in[Ru（bpy）₂dppz]²⁺or the recovered fluorescence of[Ru（bpy）₂dppz]²⁺after the ds DNA intercalation,separately.The limit of detection for HBV DNA was at amol L^-1level,and the method was applied to human serum samples analysis.The analytical results of patient samples were in good agreement of real time-PCR,demonstrating a good application potential of the proposed method.It is worth noting that the[Ru（bpy）₂dppz]²⁺probe was used as an element tag for ICP-MS based DNA detection,which does not require nucleic acid modification,has good stability and low cost,and can be directly used for double-stranded DNA labeling.In one word,by combining the high-efficiency amplification of HRCA and the fluorescent properties of[Ru（bpy）₂dppz]²⁺probe,this method achieved ultra-sensitive,dual-mode detection of target HBV DNA.（5）In order to avoid the separation and washing steps,a homogeneous HBV DNA detection method based on rolling circle amplification（RCA）and single particle ICP-MS（SP-ICP-MS）was established.The long-chain DNA with a large number of repeating sequence units generated by the RCA was applied to guide the self-assembly of small-size Au NPs into large particles.With a proper dwell time,small-size Au NPs does not produce a peak-jumping signal in SP-ICP-MS,while it can produce a high peak-jumping signal in SP-ICP-MS after agglomeration,caused by the RCA product and spermidine.Therefore,the homogeneous detection target DNA can be realized by SP-ICP-MS.Under the optimal conditions,the limit of detection for target HBV DNA was 5.1 fmol L^-1,the linear range was 10-2000 fmol L^-1（R²=0.994）,the relative standard deviation was in the range of 3.7-6.8%.The method was applied to the analysis of human serum samples,and recovery of 94.2-108%was obtained for target DNA in the spiked serum.Comparing to conventional ICP-MS-based nucleic acid analysis methods,this method is a homogeneous analysis method,no need of phase separation and washing steps.Comparing to other homogeneous analysis methods,this method can achieve a high signal-to-noise ratio（>100 times）,and has a good multiplex analysis potential.