Single Microbead-based Amplification-free Ultra-high Sensitivity Detection Of RNA

As an important biomarker,RNA is closely related to many diseases threatening human health.It has become a necessary reference standard for disease diagnosis,whether analyzing abnormal expression and mutation of human RNA or detecting RNA of viruses that infect humans and cause diseases.At the end of 2019,the novel coronavirus pneumonia(COVID-19)broke out globally,and nucleic acid detection of SARS-CoV-2 virus RNA has become an effective measure to prevent COVID-19.However,with the COVID-19 pandemic,problems such as lack of testing instruments,tedious and time-consuming testing procedures,and inaccurate results caused by nucleic acid amplification have become increasingly prominent.Research on simple,rapid and sensitive tests is critical to detect COVID-19 in patients before the infection outbreak.However,the RNA of SARS-CoV-2 is a single stranded RNA with a length of nearly 30,000 bases.Its long length,many mutation sites,and complex structure significantly increase the difficulty of studying new methods for its RNA detection.On the contrary,microRNAs(miRNAs)are a class of non-coding small RNAs with a length of about 18～24 nucleotides.They are characterized by short structure,extremely low abundance,and high sequence similarity of family members,which put forward more demanding requirements for single-cell level detection of miRNAs.The short sequence of miRNA makes primers with a similar length to miRNA in conventional amplification methods need to be further shortened,and the thermal stability of hybridization during amplification is seriously affected,thus reducing the amplification efficiency.To achieve accurate diagnosis at the early stage of the disease when very few cells begin to change,it is vital to study a simple and efficient quantitative counting method of miRNA at the single molecular level without nucleic acid amplification.To solve the above scientific problems,this paper focuses on developing a single microbead-based method for ultra-high sensitivity RNA amplification-free detection,and proposes a new way for single-molecule level RNA detection using a single microbead as a signal limiting/enrichment carrier.The detection platform with a single microbead as the carrier can realize the high contrast between local high-concentration and blank signals and avoid the non-statistical deviation of detecting low-concentration samples.Moreover,all signals are limited to a single microbead,which were captured in this small enough space can be completely detected.The work of this paper is divided into three parts,and the main contents are as follows:1.CRISPR/Cas system combined with a single microbead was applied to detect SARS-CoV-2 RNA at the single-molecule level.Simple and ultra-sensitive detection of SARS-CoV-2 RNA is essential for early diagnosis and prevention of the COVID-19 pandemic.Traditional methods based on nucleic acid amplification use a complex template and primer design,many experimental steps,and a long detection time,which are not conducive to the large-scale application of SARSCoV-2 nucleic acid detection.However,current non-amplified detection methods usually lack sufficient sensitivity.In this chapter,a single microbead combined with CRISPR/Cas13a assay method(SMB-Cas13)was used to detect SARS-CoV-2 RNA with high sensitivity and specificity without amplification.In this method,a single microbead with self-quenched fluorescent single-stranded RNA reporting probe functionalization was used as the reactor.After the complex of Cas13a and crRNA recognizes and binds the target RNA,which will activate its trans-cleavage activity,cas13 can cleavage the fluorescence quenching group on the RNA reporter probe on the surface of a single microbead,so that the single microbead can emit fluorescence.The fluorescence signals generated by the reaction are all enriched on the surface of a microbead,which is equivalent to the hyperlocal concentration effect of the generated signals,dramatically improving the sensitivity of SMBCas13 detection.This method can detect 1 aM(about 3 copies in 5 μL)of SARSCov-2 RNA without reverse transcription or any nucleic acid amplification step.We demonstrate that SMB-Cas13 is a reliable and practical method for quantitatively analyzing and detecting SARS-CoV-2 RNA in throat swab samples.This new platform will provide a simple and ultra-sensitive test for nucleic acid detection of SARS-CoV-2 or other disease diagnoses by RNA detection.2.Capturing single-molecule miRNA by a single microbead to achieve digital quantitative analysis.The content of miRNA in the human body is very low.The quantification of miRNA at the single molecule level is of great significance for clinical diagnosis and biomedical research.However,miRNA sequences are short,so transforming single-molecule miRNA measurements into quantitative signals is a big challenge.This chapter proposes a new digital quantitative method(SMSDQ).MiRNA was confined on a single microbead by hybridizing with the DNA capture probe.AuNPs with surface plasmon resonance enhancement effect were used as the signal label and hybridized with miRNA through reporter probe.The two probes were covalent to each other by click reaction,and the signal of miRNA-mediated AuNPs could be confined to the microscopic field of view by a single microbead.By imaging AuNPs with scattered light,the number of miRNAs in the system can be counted individually.We demonstrated the sensitivity of SMSDQ for single molecule detection and high specificity for single base identification.It was successfully applied to quantify miRNA in one-tenth of the cell lysed samples without complicated sample pretreatment steps.In addition,based on design principles,the proposed SMSDQ assay can be easily extended to detect any RNA/DNA biomarker quantitatively.3.Construction of a detection platform for ultra-high sensitivity quantitative analysis of single-molecule level RNA based on a single microbead as a carrier.Based on the general RNA detection method,SMB-Cas13a fluorescence quantitative analysis and SMSDQ digital quantitative analysis,we built a detection platform for ultra-sensitive quantitative analysis of single-molecule level RNA with a single microbead as the carrier.The selection,modification,manipulation,and imaging schemes of magnetic and non-magnetic single microbead with different modifications were systematically studied employing a micromanipulation platform,magnetic separation or centrifugation,laser scanning confocal microscopy and other means to operate single microspheres.This chapter compares the difference between the methods that use a single microbead and a large number of microbeads,compares microbeads with different materials,properties and modifications,and analyzes the selection principles of different kinds of microbeads in designing experiments.The constructed platform only requires 1μL of test sample without any amplification process and can achieve ultrahigh sensitivity single-molecule level detection of target RNA.It solves the problems of the current high sensitivity RNA detection which relies on nucleic acid amplification,such as complex experimental design and reaction process,easy to be affected by reagents and environment,long reaction time,strong sequence dependence of test results,and prone to false positives caused by non-specific amplification.