Multiple Fluorescence Detection Of Nucleic Acid Markers Of Major Diseases Based On Microfluidic Chips

Nucleic acids,including deoxyribonucleic acid(DNA)and ribonucleic acid(RNA),are natural biopolymers composed of nucleotides that store,transmit and express genetic information.Overexpression of nucleic acids is associated with many diseases.Since the levels of early disease markers are often low and a disease is often associated with multiple disease markers,a disease marker detection platform with high detection sensitivity,high throughput and low cost features is important for the early diagnosis,prevention and treatment of major diseases.(1)We took advantage of the rapid and efficient characteristics of exponential amplification reaction(EXPAR)and the high-throughput and multiplexed features of microfluidic to achieve simultaneous,highly sensitive and specific detection of three abnormally expressed mi RNAs in breast cancer.Firstly,the EXPAR generates a lot of triggers,which bind to signal probes to generate fluorescent complexes.Then fluorescent complexes bind to capture probes on microfluidic biochip to generate fluorescence signal.This microfluidic biochip provides good practical application in early cancer diagnosis.The simultaneous detection of multiple targets on microfluidics with a sensitivity of up to a M level and its good specificity provides a promising detection platform for clinical diagnosis.The constructed microfluidic biochip has good practical application in early cancer diagnosis through the testing of actual samples.This study provides a new idea for the combination of nucleic acid hybridization and microfluidics,especially for cancer marker detection.(2)Because the microfluidic chip of the previous system can well meet the requirements of multiplexing,small amount of solution,easy manufacturing and low cost,the microfluidic chip of the previous system is still used.In terms of biological processes,further improvements were made for the previous system.First,instead of continuing the EXPAR amplification method,a nanostructure was used in this system.The nanostructure consists of two parts: the aptamer(containing S1 and S2)and the polymerase.When the target is not present,the polymerase activity is in a suppressed state and cannot perform polymerization.When the target is present,the target binds to the S2 of the aptamer,leading to the dissociation of the nanostructure,releasing S1 and polymerase,and the polymerase activity is reactivated,allowing the extension of the product after S1 and the capture probe bind to form a duplex structure.Here,every component of the nanostructure is fully and skillfully utilized,making the nanostructure’s function greatly enhanced,including the role of recognizing the target,providing an extension template,and providing an assisting extension tool,and the whole process is carried out on the chip,greatly improving the integration of the microfluidic chip.And the process can be carried out normally at room temperature of 25 ℃,and the time can be basically controlled within 1 h.(3)In order to improve the loading of the probe on the glass substrate,we synthesized Ni-IRMOF-74-II.The uniform needle-like and regular hexagonal vacancies of this MOF material can play a role in increasing the binding site,improving the probe loading and protecting the DNA strand,and using these advantages to combine with microfluidics can detect the target with high sensitivity.Meanwhile,using the characteristics of MOFs fluorescence quenching,the fluorescence of the FAM-labeled DNA probe will be rapidly quenched when the DNA probe binds to the MOF material.In the presence of the target mi RNA,the FAM-labeled DNA is released from the MOF to achieve a reversible interaction between MOFs and ss DNA.Based on this,a fluorescence quenched-recovery detection chip can be constructedFinally,this paper provides an effective method and innovative application for the simultaneous detection of multiple disease markers based on microfluidic biochip for multiplex fluorescence detection of major diseases,which has a broad prospect in early clinical diagnosis and plays a positive role in timely detection and accurate diagnosis of diseases.