The Detection Of Nucleic Acid Biomolecules By Surface-enhanced Raman Spectroscopy And CRISPR/Cas

Nucleic acid detection and analysis technologies are essential for the early diagnosis of diseases and the identification of genetically modified crops(GM).Accurate identification of genes can facilitate the early detection of the disease,thus facilitating the development of specialized treatment plans and improving the survival rate of patients.It is also possible to distinguish genetically modified components in actual crops to meet food safety requirements.Therefore,nucleic acid analysis is of great significance in disease diagnosis and food safety.Currently,a variety of highly sensitive nucleic acid detection and analysis technologies have been developed in disease diagnosis and food safety,including polymerase chain reaction(PCR),recombinase polymerase amplification(RPA),gene sequencing(NGS),and clustered,regularly interspaced,and short palindromic repeats/associated proteins(CRISPR/Cas).Each assay has its own unique advantages and scope of application,in which CRISPR/Cas strategies have the advantages of high accuracy in the target nucleic acid identification for nucleic acid detection and analysis,and the cleavage function of the target sequence and the enzymatic activity of nearby single-stranded DNA or RNA.In addition,CRISPR/Cas protein is specific to the target nucleic acid under the guidance of precisely designed sg RNA,and can realize the detection and analysis of single-base mutation.Therefore,CRISPR/Cas technology has attracted great interest in nucleic acid detection and analysis.Since the concentration of target nucleic acid molecules is extremely low in the samples of relevant diseases or genetically modified crops,their detections and analyses require highly sensitive detection methodologies.Surface-enhanced Raman spectroscopy(SERS)is an optical technique that can amplify the Raman signal near the plasmonic or semiconductor substrates,remarkably improving the detection sensitivity.Integrating SERS with CRISPR/Cas technology can fully use the specificity of CRISPR/Cas for nucleic acid analysis and the high sensitivity of SERS technology,which is gradually becoming a powerful tool and next-generation molecular diagnostics techniques.Aiming at the above research background,this thesis proposes four kinds of gene analytical platforms combining SERS with CRISPR/Cas technology.They are Au NPs aggregation-SERS-CRISPR/Cas12 a,SERS-CRISPR/d Cas9,Fe Au G-MB-SERSCRISPR/Cas12 a,and RPA-SERS-CRISPR/d Cas9 platforms for analyzing cervical cancer-related human papillomavirus(HPV)and transgenic crops(35S promoter and nos terminator).This thesis consists of the following four parts:(1)A gene detection platform was developed based on the SERS and CRISPR/Cas12 a systems that produce signals of different intensities depending on the aggregation and dispersion states of gold nanoparticles,and this platform was applied for the detections of the viral genes HPV 16 and HPV 18.First,the SERS probes were prepared by ss DNA and(4-mercaptobenzonitrile)MBN decorated gold nanoparticles.A Linker ss DNA as the trans-cleavage substrate of Cas12 a can bridge two SERS nanoprobes which have been decorated by ss DNA and MBN.If there is no target DNA in the sensing system,the trans-cleavage function of Cas12 a protein will be inactivated.Thus,the Linker ss DNA is intact,which will induce the aggregation phenomenon of SERS probe and consequently result in strong SERS signals.In contrast,when Cas12a/cr RNAs recognize their target DNAs,Cas12 a starts its trans-cleavage activity to degrade Linker ss DNA.Therefore,SERS probes will be relatively dispersed and SERS signals of SERS probes will remain initially weak.This sensing method enables the highly sensitive detection of HPV genes in serum and pseudoviruses and the detection sensitivity can reach the p M level.Due to the flexibility of DNA probe and cr RNA design,this CRISPR/Cas12a-SERS sensing system is a versatile genetic detection tool that can be applied in the field of in vitro diagnostics.(2)A SERS assay based on CRISPR/d Cas9 technology and enzyme-catalyzed amplification was designed to achieve simple and rapid detection of low levels of HPV genes.The CRISPR/d Cas9/sg RNA complex is immobilized on the surface of a NiNTA-modified magnetic bead(MB)to obtain a magnetic probe,which facilitates the separation and washing processing.When the target DNA is present,the sg RNAactivated d Cas9(d Cas9/sg RNA)protein will capture the complementary target DNA to form a ternary complex(d Cas9/sg RNA/DNA).A biotin-end molecular probe is inserted into the chain of captured DNA on the MB surface,which allows the immobilization of the streptavidin-treated HRP according to the biotin-streptavidin interaction.For the case without target DNA,the streptavidin-treated HRP will fail to be captured by MB.Otherwise,it will be fixed on MB when the target DNA exists.The HRP-modified conjugate will catalyze the reaction on the substrate(3,3’,5,5’-tetramethylbenzidine,TMB),causing a blue colored oxidation product(ox TMB).The corresponding detection is then accomplished using a SERS substrate(silica-coated gold nanostars).The catalytic action of the enzyme and the SERS action ensure high detection sensitivity.The method allows the detection of target HPV DNA in complex systems,avoiding steps such as DNA purification and isolation.(3)A novel CRISPR-mediated magnetic surface-enhanced resonance Raman scattering(SERRS)biosensing assay was built for the identifications of 35 S promoter and Nos terminator nucleic acid sequences of transgenic crops.This assay mainly combines the gene-specific recognition function of CRISPR,the resonance Raman effect,and the simplicity of magnetic separation in processing gene samples.A multifunctional magnetic probe is designed,in which the ss DNA sequences on the Fe Au G-MB nanoprobe tend to form G-triplexes due to their abundance of G bases,which can load a large number of methylene blue(MB)that gives the resonance Raman effect under 633 nm laser,resulting in strong SERRS signal.During this experiment,the Fe Au G-MB probe was first mixed with CRISPR/cas12 a reaction solution.Cas12 a recognizes the corresponding DNA,forming a ternary Cas12a/cr RNA/DNA complex.The activated Cas12 a protein starts to degrade ss DNA immobilized on the Fe Au G-MB nanoprobe,leading to a release of MB molecules,which reduces the SERRS signal above the Fe Au G-MB probe.In the absence of the target gene,the Fe Au G-MB probe remains intact and produces a strong SERRS signal.The final use of this analytical platform for the detection of real plant samples revealed that only transgenic crops can cause the reduction of SERRS signals,confirming the practical application value of this method.In addition,the probe has multiple functions,such as the SERS-enhancing ability of gold shells,the resonance effect of MB Raman molecules,and the high loading of G-triplex DNA on MB.(4)Based on the combination of RPA-amplified CRISPR/d Cas9 technology and SERS technology,a nucleic acid analytical platform was established to achieve the identification of nucleic acid fragments from the 35 S promoter and Nos terminator in transgenic crops.The d Cas9 protein activated by sg RNA is conjugated to the surface of silver nanoparticles modified with Raman probe molecules to forms a novel SERS probe(Ag NPs@Ra@d Cas9@sg RNA).When the target nucleic acid is present,the streptavidin-modified magnetic beads(MB/SA)can be enriched with biotin-modified RPA amplification products,which then form a sandwich structure with Ag NPs@Ra@d Cas9@sg RNA,producing strong SERS signals after magnetic separation.When no target nucleic acid is present,there is only a feeble SERS signal above the probes.This target DNA-dependent change in SERS signal intensity can be widely used in DNA biosensing assays.The biggest advantage of this method is that two SERS nanoprobes modified with different Raman signalling molecules can be prepared to achieve the simultaneous detection and analysis of multi-target nucleic acids by taking advantage of the narrow spectral peaks of Raman signalling molecules.And finally,the analysis platform can be used to detect the actual plant samples,and it can be found that only the transgenic crops can produce strong SERS signals,which confirms the practical application value of this method.Therefore,the CRISPR/d Cas9-SERS assay is a promising technology for the simultaneous detection of multi-target nucleic acids.In summary,this thesis establishes four kinds of nucleic acid analytical methods,which provide an important platform for assessing the early diagnosis of diseases and the identification of genetically modified crops.