Construction Of Novel Fluorescence Probe Detection Systems And Their Application In The Detection Of Biomarkers

MicroRNA(miRNA)and immunoglobulin G(IgG)are very important biomarkers in the diagnosis of diseases.There are many kinds of them,and the levels of the expression of different types of miRNA and IgG are closely related to the occurrence and development of their respective diseases.Therefore,exploring its detection method has important clinical value.At present,the main detection methods of miRNAs are Northern blotting and real-time fluorescence quantitative PCR(qRT-PCR).The detection methods of IgG are mainly radioimmunoassay and enzyme-linked immunosorbent assay(ELISA).Although these methods have high sensitivity,they have the disadvantages of radioactive contamination,long time-consuming,high cost,large sample size,and false positives.Safe,harmless,high sensitivity,low false positives,low cost,and rapid biomarker detection methods are important requirements in medical tests.Fluorescent labeling is a non-radioactive labeling technology that is safe,harmless,low background,high sensitivity and good stability,and can be used to meet the above requirements.Fluorescent probes based on various fluorescent dyes are widely used in the detection of biological macromolecules such as proteins and nucleic acids.Molecular hybridization technology has been widely used in miRNA detection due to its advantages of simple operation and good specificity.The use of rigid locked nucleic acid(LNA)modified fluorescent probes in combination with a sandwich hybridization system can improve the stability of hybridization complexes between miRNAs and probes,reduce false positives in detection results,increase detection sensitivity,and reduce the time of molecular hybridization.The special advantages of rare earth elements such as long fluorescence lifetime and large chemical shift can be used to reduce the background fluorescence,and the rare earth element ligand has considerable modifiable ability.Thus,a novel hyperbranched ligand can be constructed and used in IgG detection to amplify the fluorescence signal.Based on the above theories,we have carried out the following tasks:(1)In order to construct a safe,rapid and visualized fluorescence detection system of miRNA,the dimethyl octadecyl[3-(trimethoxysilyl)propyl]ammonium chloride(DMOAP)is first modified on the surface of the slide to immobilize the capture probe.Both ends of the target gene hsa-miR-223 were paired with the capture probe and TYE563-LNA to form a sandwich structure.The miRNA was quantitatively detected by the intensity of fluorescence signal in the buffer solution.Its limit of detection(LOD)is as low as 85.2 fM.And the entire detection process can be completed in 2.5 h,saving a lot of time.We used this method to detect hsa-miR-223 in the serum of patients with coronary artery disease and healthy people.40 ?L of serum can meet the detection needs,saving a large number of samples,and verified by qRT-PCR.(2)In order to construct a high-sensitivity,low-background,and low-cost IgG fluorescence detection system,the hyperbranched polyethylenimine(PEI)with a molecular weight of 600 and 1800 was used as a molecular skeleton and reacts with chelating agent diethyltriamine pentaacetic acid(DTPA)to form a novel ligand(PEI-DTPA)that can chelate multiple Eu3+.This ligand was then combined with antibody(anti-IgG).Finally,a plurality of rare earth ions Eu3+ were chelated by DTPA to form an anti-IgG-PEI-DTPA-Eu3+complex.The effect of amplified fluorescence signal was verified by ELISA.The results showed that the fluorescence intensity of anti-IgG-PEI600-DTPA-Eu3+ was 12 times higher than that of anti-IgG-DTTA-Eu3+.Further more,we have found that the fluorescence intensity of anti-IgG-PEI1800-DTPA-Eu3+ was 2.4 times than that of anti-IgG-PEI600-DTPA-Eu3+.But it's background fluorescence also increased.It was found that if 20 mg/mL BSA was used to block the anti-IgG-PEI1800-DTPA-Eu3+ before IgG detecting and washed with Tris buffer solution containing 0.15%Tween20,the background fluorescence can be reduced to a lower range.In summary,this paper provides highly sensitive new fluorescent probe detection system for the detection of miRNA and IgG,providing new ideas for the rapid and simple detection of miRNA and the detection of low-level IgG.