| As an important disease biomarker,nucleic acids can reflect the genetic and metabolic characteristics of patients.Nucleic acid testing enables early disease diagnosis,guidance for clinical drug use,and personalized treatment,making it of great significance.Currently,the gold standard for nucleic acid biomarker detection in clinical practice is based on Quantitative Real-time Polymerase Chain Reaction(q PCR),which has limitations such as limited target detection,long detection time,and dependence on expensive instruments.Microfluidic technology has shown great potential in nucleic acid biomarker detection,especially in multi-target detection,high sensitivity quantification,and point-of-care testing(POCT),making it better suited to meet clinical testing needs.Based on microfluidic chip development,this paper focuses on the high sensitivity,multi-target,and rapid detection of nucleic acid biomarkers in clinical diseases.The main research work is as follows:(1)Development of a dual digital PCR detection chip and establishment of a copy number variation(CNV)detection method based on this chip and double enzyme cutting.This method enables the absolute quantification detection of CNV of metabolic syndrome-related genes with dual targets.The developed chip consists of a detection area and a reference area,with dual digital PCR detection function.The chip contains a total of 40,448(2×20224)independent reaction units,with a volume of only 0.67 n L per reaction unit.The pre-processing uses a dual restriction endonuclease method,which can achieve a higher signal-to-noise ratio.The established CNV detection method based on the dual digital PCR chip belongs to absolute quantification,does not rely on standard curves,and is not affected by PCR inhibitors.It has high detection sensitivity,with a detection limit as low as 1.4 copies/μL,and provides accurate quantification results in clinical samples.In summary,the developed dual digital PCR chip enables highly sensitive CNV detection,providing important reference value for accurate quantification of low-concentration samples.(2)To meet the clinical demand for multi-target and isothermal detection of microRNAs,a multiple microfluidic detection chip is developed,and a microRNA isothermal and multiple detection method based on this chip and Duplex-Specific Nuclease(DSN)is established.This method enables simultaneous detection of three microRNAs in tumor cells.The chip has four independent detection areas,each containing four duplicate reaction units.The flexible control of fluid flow"on"and"off"between detection areas is achieved through screw microvalves,ensuring independence of reactions between different detection areas.The chip utilizes a negative pressure-based self-suction liquid distribution method,eliminating the need for external power sources.This detection method does not require complex temperature cycling instruments and operates at a constant temperature of 50°C.By employing pre-embedded probes,the operation steps are simplified.Four types of microRNAs can be quantified simultaneously with a single input,and the concentrations of mi R-100,mi R-155,and Let-7a in tumor cell lysate can be successfully detected within one hour.This method achieves extraction-free detection and is considered as an off-the-shelf detection chip.In summary,the developed multiple microfluidic detection chip has great potential in multi-target detection of microRNAs,isothermal detection,and POCT.(3)To address the issues of low sensitivity and long detection time in microRNA detection,a superfast and reverse transcription-free digital CRISPR(Super-RADAR)detection method for microRNAs is developed based on the digital PCR chip described in Chapter 2.This method allows single-molecule detection of microRNAs in just 15 minutes at 37°C without the need for reverse transcription.First,specific probes are paired with microRNAs,and nearby probes are connected under the action of ligases.Then,signal exponential amplification is achieved through a one-step reaction using RPA-Cas13a.The detection limit of this method is as low as10~2 copies/μL,indicating high sensitivity.Combined with the digital PCR chip,the method enables single-molecule detection of microRNAs with high signal-to-noise ratio and sensitivity as low as 3 copies/μL(5 a M).The detection time on the digital microfluidic chip is only 15minutes,demonstrating fast detection speed.In conclusion,Super-RADAR achieves superfast,high-sensitivity,and single-molecule detection of microRNAs without the need for reverse transcription,which has significant implications for the detection of low-concentration microRNAs.In this paper,novel detection methods for different nucleic acid biomarkers are developed based on microfluidic chips to address the limitations of low sensitivity,single target detection,and long detection time in clinical testing,thereby overcoming the shortcomings of traditional nucleic acid detection methods. |
