Biosensor Development For Nucleic Acid Detection Based On Micro And Nanotechnology

Miniaturization and integration have become the major trends for the development of electronic devices nowadays,which are greatly affected by the micro and nanotechnology.Various lithography and etching technologies have promoted the progress of micro-nano fabrication technology,making development of biosensors toward more portable and intelligent way.At present,miniature chemical and biosensors fabricated based on micro-nanofabrication technology have shown great potential in personal medicine,point-of-care testing(POCT)and other fields.In this thesis,a microfluidic chip and a glass conical nanopore biosensor platform have been development for DNA targets detection,respectively.The details are as follows:In chapter 2,we have designed a novel glass microfluidic chip and the ultrasensitive digital DNA detection has been achieved based on the principle that oil and water are immiscible.The fabrication of glass microfluidic chip was based on optical exposure technology and wet-etching technology.The chip was designed with 30,000 well and connected microchannels,and the volume of each well was ~140 pL.After fulfill of solution in well and channels,fluorinated oil has been utilized to repel water out of the microchannels.Hence,each well was isolated as single reaction chamber by fluorinated oil and digital single molecule detection has been realized.By optimizing the chip processing steps,the rate of bonding success of the chips reached close to 100% under the normal laboratory conditions.The round hole has been chosen as the structure of the reaction chamber because volume calculation was more accurate of this structure.And the neck size of the chamber has been optimized to allow the reaction solution to occupy the cavity more easily,avoiding a large dead volume caused by the air in the hole.The feasibility of digital nucleic acid amplification detection has been evaluated by injecting fluorescein solution to the microchannels.It has been proved that the shape of droplets in the chamber has no deformation and no obvious loss of the solution volume has been observed after sampling and heating operation.In addition,the fluorescence intensity in the chamber was much higher than the background fluorescence in the oil-occupied microchannels.Finally,we carried out digital loop-mediated isothermal amplification experiments on the designed glass microfluidic chip.The fluorescent detection of Clostridium Difficile GDH was successfully realized with 33 aM limit of detection.In chapter 3,we have fabricated glass conical nanopore(~ 60 nm)based on the laser pulling technology.Then we made three layers of modifications in the surface of nanopore.The first layer was modified with(3-mercaptopropyl)trimethoxysilane,and then the 13 nm gold nanoparticles was modified to the surface by Au-S bonding.Finally,the-SH labeled DNA probe was modified onto the surface of gold nanoparticle.The target microRNA-21 could be captured onto surface of nanopore due to hybridization with DNA probe,increasing negative charge of the nanopore surface.Since the ionic current was related with charge of the nanopore surface,concentration of target microRNA-21 could be detected by the developed method.