Microfluidic System Based Nucleic Acid Aptamer Screening Methods

In view of the complex,lengthy,intelligence,and low automation of current aptamer screening methods,this paper aims to develop an automated,integrated,and intelligent microfluidic screening system based on microfluidic chips.The protein microarray microfluidic screening system(PMM-SELEX)has been designed and fabricated including dual-microfluidic amplification system(Dual-MAS)for aptamer screening process monitoring and PCR library amplification system,and microfluidic bio-bead ssDNA regeneration system(ssDNA-MRS).The above three units were used to systematically study the three important processes(separation,amplification,and library regeneration)of the aptamer screening.In addition,the establishment of the screening chip,amplification chip,and library regeneration can make the aptamer screening process to be automation,intelligence,and standardization.In addition,simultaneous detection of two protein biomarkers was achieved by high-efficiency microfluidic chips and high-specificity aptamer probes.The main research contents of this paper are as follows:1.Selection of aptamers based on protein microarrays integrated with microfluidic chipsAn efficient and fast method based on protein microarrays integrated with microfluidic chips for the process of SELEX(systematic evolution of ligands by exponential enrichment)has beens developed.Lactoferrin from bovine milk was used as target protein,while bovine serum albumin(BSA),?-Lactalbumin,?-Lactoglobulin and Casein as negative proteins.They were separately dotted and immobilized to prepare the protein microarray and the resulting microarray was further integrated into microfluidic chip for SELEX(PMM-SELEX)process.The interaction between aptamer candidates and the targets could be monitored by the fluorescent microarray scanner.Surface plasmon resonance(SPR)was used to calculate the dissociation constants(Kd).The aptamer Lac-6a was then used for detection of Lactoferrin by fluorescence polarization.2.Dual-microfluidic amplified systems for aptamer screening monitoring and optimal PCR amplificationA Dual-microfluidic amplified system(Dual-MAS)based on the qPCR fluorescence detection chip has been developed including high-throughput PCR chip for multi-information aptamer screening monitoring and accurate amplified the enrichment sequences.The multi-functional Dual-MAS could simultaneously detect the amounts of the enriched sequences,provide library diversity characterization and the optimal PCR round numbers.Firstly,the Dual-MAS allows the quantitative detection the concentration of the nucleic acid library.Secondly,the qPCR curve of Dual-MAS could be used for monitoring the diversity of the libraries,providing a critical reference for determined the number of rounds required for successful aptamer screening.Thirdly,according to the qPCR amplification curve in Dual-MAS,the optimal PCR round numbers for amplified the enrichment sequence could be obtained.Finally,in order to verify the reliability of Dual-MAS used for aptamer screening,we integrated the Dual-MAS with PMM-SELEX for rapid screening of aptamers.In addition,pollution contamination and fragment loss caused in the open environment can be completely avoided because the separation,monitoring,and amplification process are completely carried out in the closed microfluidic chip.3.Microfluidic bio-bead chips for for rapid generating single-stranded DNA in SELEXA microfluidic ssDNA regeneration chip based on bio-microbeads was designed and fabricated.The preparation of the single-stranded DNA from the PCR product could quickly be acomplished via the microfluidic bio-bead chips.The chip employed SA-modified glass beads as the capture unit,the highly efficient covalent reaction between the 5-terminal biotin-labeled PCR product completes and avidin in microfluidic channel could be carried out.After washing with the buffer,the fluid was replaced with NaOH solution to complete the double-stranded melting process.the flow rate of the microfluidic biological microbead chip and the dilution fold of the PCR product were optimized.Under optimal conditions,the dsDNA capture efficiency in the PCR system was 93.2%.In addition,the optimal ssDNA generation rate and the smallest dsDNA exfoliation rate were achieved through optimization of NaOH solution concentration.Compared with other reported methods,the microfluidic ssDNA regeneration method has significantly improved the recovery rate and significantly reduced time consumption.In addition,the entire operation process has a high degree of automation,less human intervention,and high repeatability,which solves the cumbersome and time-consuming steps of single-stranded DNA regeneration in the current aptamer screening process.Therefore,the microfluidic single-chain regeneration chip is of great value in the development of a fast,efficient,and highly reproducible aptamer screening system.4.Multivalent aptasensor arrays and silver aggregated amplification for multiplex detection in microfluidic devicesA rapid and sensitive aptamers-based sandwich assay in microfluidic devices based on multivalent aptasensor array(MAA)chip and silver aggregated amplification(SAA)strategy for detection of two biomarkers were developed.Firstly,aptamers-modified silver nanoparticles were dotted in the array to form MAA chip.Then PDMS was used to form a microfluidic device.Finally,the target proteins and two kinds of aptamer-modified silver nanoparticles(Tag-A and Tag-B)were rapidly injected into the microfluidic device.The aptamers on MAA chip recognized target,and the resulting targets would further bind with Tag-A and Tag-B.As a result,the aggregation reaction between each other could significantly amplify fluorescence signal.Ultimately,the presented method was used to simultaneously detect PDGF-BB and VEGF-165,and the results showed good specificity and sensitivity.5.Screening of lomefloxacin aptamers based on polydopamine nanospheresScreening aptamers using nano-materials(such as graphene oxide,gold nanoparticle,carbon nano-tube,etc.)which could quench the fluorescence and absorb the single stranded DNA(via.hydrogen bond,?-? bond,charge transfer,and other non-covalent approaches without the conformational DNA),it can excellently separate specific aptamers from non-specific ones.In this case,we can shorten the cycles,enhance the success rate,and reduce the labour intensity of systematic evolution of ligands by exponential enrichment(SELEX).In this experiment,polydopamine nanospheres(MNPs@PDAs)was used to screen the Lomefloxacin.Also,the magnetic separation technique was used to screen small molecular targets rapidly.The interaction between aptamer candidates and the targets could be monitored by recovery ratio of ssDNA and the whole MNPs@PDAs-SELEX process through seven-round selection.As a result,we successfully obtained the aptamer AF-3,which recognized the lomefloxacin with high affinity specificity.The presented screening method showed that MNPs@PDAs would be a promising reagent in the efficient aptamers selection of other targets.