Study On Microfluidic Digital Isothermal Amplification And Its Applications In Detection Of MiRNA In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is known as the "king of cancer",and is one of the most common malignant tumors.In China,the new liver cancer patients account for more than half of the world every year,and its mortality ranks the second of malignant tumors,thus it has seriously threaten people’s lives and health.HCC is a malignancy with insidious onset,and there are no specific symptoms in early stage.However,approximately 80 percents of HCC patients have turned into advanced stage at the first visit.In particularly,the 5-year survival rate of patients with advanced HCC is almost zero,but for early HCC,the 5-year survival rate can reach more than 60%if with treatment.Hence,early diagnosis and treatment is an important means to fight against liver cancer.Currently,Alpha-fetoprotein(AFP)serum marker is a common indicator for diagnosing HCC,but,only about 60 to 70 percents of patients with early HCC have positive AFP levels.Besides,for B-ultrasound or CT examination,they both cannot detect HCC with tumor diameter less than 2 cm or density similar to liver parenchyma.So,the above factors cause high rates of false negative and missed diagnosis in early HCC screening.With the development and progress of science,the technology of liquid biopsy with great features of noninvasion,good repetition and quantification,high sensitivity is emerging,that provides a useful "weapon" for early screening of HCC.Among it,detection of circulating miRNA biomarkers as a new diagnostic approach has been written into the latest Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China(2019 Edition),and been adopted in early screening,diagnosis,and other clinical applications for HCC.Thus,it is of great significance to promote the precise diagnosis and treatment of HCC.However,miRNA is a type of single-stranded RNA with short length and poor stability,and the academic community has not yet determined a suitable miRNA endogenous reference gene.So,it has greatly influenced the accuracy of its quantitative results,and it is also an important factor that restricted wide applications of miRNA markers in early screening of HCC.In recent years,with the rapid development of isothermal amplification technology,it has shown significant advantages in the efficiency and sensitivity of detection,and it is particularly suitable for the detection of small miRNA molecules.In addition,the rise of microfluidic chip technology has greatly promoted the development of the "third generation" gene amplification technology represented by digital nucleic acid detection(dNAD)technology.With the help of micro-nano reaction chambers of high-throughput micro fluidic chip,it can achieve single molecule segmentation and independent amplification,as well as a real sense of absolute quantification,but without using internal reference gene.In this study,we will make full use of miRNA molecular markers as an academic consensus for early screening of HCC,and perform a study on microfluidic digital isothermal amplification and its applications in detection of miRNA in hepatocellular carcinoma,the main contents are as follows:Starting from the principle of strand displacement amplification(SDA),we innovatively designed a series of fluorescent self-inhibition probes based on the specific structure of miRNA molecule.On this basis,a background-free fluorescent probe strand displacement exponential amplification(BF-ESDA)detection system was established.The program can effectively improve the signal-to-noise ratio in miRNA detection,and was particularly suitable for detecting the lower concentration of miRNA molecules in clinical samples.A dynamic detection range of 9 orders of magnitude can be achieved,and the detection limit can reach 0.08 aM within 90 minutes.A self-priming and dispensing high-throughput chip based on the microfluidic chip technology was designed and developed.The chip can meet the testing needs of one sample,two samples,four samples,and eight samples at one assay.Meanwhile,it only takes 2 to 3 minutes to complete a pretreatment process such as sample injection and oil sealing.Through a series of assays,like anti-evaporation,uniformity of liquid distribution,Oil-liquid phase stability,and biological compatibility,we have demonstrated that the chip meets the needs of digital isothermal amplification detection,and it is an effective carrier for absolute quantification of miRNA markers.In order to enrich the database of miRNA molecular markers in the HCC,three miRNAs(hsa-miR-185,hsa-miR-4311,and hsa-miR-1255b)were obtained through analyses of database and bioinformatics,and verification of clinical samples.In particular,the expression of the three miRNAs has extremely significant difference(P＜0.01)in serum between HCC patients and healthy controls.Furthermore,hsa-miR-4644 was proved to be with higher stability,even was better than other known miRNA reference genes.To establish an early warning model,and improve the detection rate and survival rate of HCC patients.The miRNA markers discovered in this study and reported in the literatures,AFP serum marker were combined and analyzed by logistic regressive.And evaluate the diagnostic efficiency of each molecular marker and their combinations for HCC using receiver operating characteristic curve(ROC)analysis.The results showed that the sensitivity and the specificity to judge whether the subject has HCC can respectively reach 95.24%and 80.95%when using the diagnostic model composed of 7 miRNA and AFP markers.Furthermore,the misdiagnosis rate was as low as 7.14%through verification of clinical samples.Consequently,the diagnostic model established can effectively detect and diagnose hepatocellular carcinoma of early-stage.In summary,this study on digital isothermal amplification detection was performed that built based on the detection method of a novel background-free fluorescent probe strand displacement isothermal amplification technology,the carrier of a self-priming and dispensing high-throughput chip,as well as the target of miRNA molecular markers screened and verified.And finally established a diagnostic model for early warning of hepatocellular carcinoma,which providing an effective solution for improving the detection rate and survival rate of patients.