Fully Integrated Loop-mediated Isothermal Amplification Microdevices For Nucleic Acid Detection

Diagnostic assay plays an important role both in health care clinics and biomedical research.The rapid development of molecular diagnostic technique has remarkably promoted the advancement of biomedicine in the post-human-genome-project era.Nucleic Acid Amplification Tests(NAATs)provide rapid,sensitive and specific diagnosis of infectious,inherited and genetic disease.Currently,there is an urgent requirement for easy-to-use and cost-efficient devices to fulfill various demands.However,conventional NAATs still rely on skilled technicians,which are usually time-consuming and labor-intensive.Microfluidic designs have overcome the limitations of traditional detection assays by integrating sample purification,reagents mixing,nucleic acid amplification and detection to enable multiplex detection within a few hours.In this thesis,we have demonstrated loop-mediated isothermal amplification(LAMP)-based point-of-care testing(POCT)microfluidic devices that can specifically detect multiple pathogens and lung adenocarcinoma cells.We firstly proposed a microfluidic in-gel loop-mediated isothermal amplification(gLAMP)with preloaded reagents as a self-contained microdevice for simultaneous detection of multiple pathogenic bacterial DNA in POCT settings.The microchip consisted of multiple channels with reaction chambers accommodating low-melting-point agarose and all LAMP reagents except the DNA samples,which could be kept at 4? for long-term storage.The device enabled simultaneous tests of four different bacterial DNA targets.The detections of foodborne bacteria(Escherichia coli,Proteus hauseri,Vibrio parahaemolyticus,and Salmonella subsp.Enterica)in serum samples have been demonstrated with high selectivity and sensitivity(as low as 3 copies/?L).Next,we integrated DNA extraction and amplification into a single paper-polydimethylsiloxane(PDMS)microfluidic chip to develop an easy-to-use,pump-free system.An integrated CloneSaver membrane was inserted into the device for on-chip cell lysis and DNA capture.LAMP reaction reagents mixed with low-melting-point agarose was preloaded into the reaction chamber.DNA extraction microchamber and reaction microchamber were separated by a thin layer of PDMS.DNA solution could diffuse into the agarose gel with finger-power without any additional operation as the cross channel opened under the pressure.The paper-PDMS chip could be directly used to detect pathogens without any sample preparation.The results suggested that this device could successfully amplify and detect out as low as 1.6 copies/?L of Salmonella DNA.Microfluidic systems imaged by expensive equipment may not be equipped in resource-limited areas.To fulfill the demands of POCT,a lateral flow detection(LFD)test strip was utilized as a detection method for LAMP amplicon.Thus,the detection results can be observed by naked eyes.Additionally,we replace the conventional ThermoPol buffer with Direct PCR buffer to enable direct LAMP reaction from untreated sample,which did not require any sample preparation procedures,like cell lysis,nucleic acid extraction and purification to amplify specific target sequence.Primer Explorer V4 were utilized to design primers for EGFR?745-750.By using this paper chip,we specifically detected EGFR?745-750 in lung adenocarcinoma NCI-H1650 cells,but not in NCI-H1975 cells and A549 cells as the controls that do not carry the mutation.Furthermore,direct PCR buffer simplified the DNA extraction in microdevices,but increased the experimental costs because of its high price.We developed an integrated paperfluidic chip incorporating DNA extraction,amplification and visual detection with lower cost.FTA card assembled in the chip was used for DNA extraction.Purification buffer and TE-1 buffer preloaded in the PDMS chambers was used to wash the Flinders Technology Associates(FTA)card.The paper-based microdevice allowed for sample-to-answer detecting of single nucleotide polymorphisms(SNP)typing of EGFRL858R from lung adenocarcinoma NCI-H1975 cells.In a summary,we have demonstrated fully integrated sample-to-answer microfluidic chips to realize nucleic acid extraction,amplification,and detection assay from pathogens or mammalian cells.The integrated microfluidic chips can serve as new molecular diagnostic platforms for POCT,which is fast,easy-to-use,inexpensive,and sensitive.