DNA Biosensors And Their Integration In Microfluidic Lab-on-a-chip

With the development of economy and society, people put forward higherrequirements for health care. Regardless of effective prevention or treatment of disease,there’s an urgent demand for convenient and inexpensive, rapid and sensitive detectionmethods in cinical diagnoses; on the other hand, it brings serious threat to human beings ofenvironmental pollution, food security and terrorist attacks, so it is desirable ofmicro-portable, simple, rapid and sensitive detection and analysis equipment for livereal-time detection of environmental pollutants, food additives, viruses and bacteria.Therefore, portable and convenient, inexpensive low, fast and sensitive biological andchemical detection and analysis tools is a hot demand for medical, environmental, food,anti-terrorism and other social areas.Biosensor is an analysis device based on biological material or its derivatives asmolecular recognition element, with the advantages including high specificity, higsensitivity and facile experimental detection. Microfluidic lab-on-a-chip is a integrationplatform on several square centimeters integrating the laboratorary fuctions includingmixing, separation, reactions, and testing, with the advantages including miniaturization,integration, automation, low consumption of energy and reagents. In this research, thefabrication of DNA biosensor as well as the application in microfluidic lab-on-a-chip wasinvestigated by their unique advantanges to meet the social needs for portable andconvenient, low-consumption and inexpensive, rapid and sensitive detection and analysis ofchemical and biological moleculars.In this paper, it is investigated that two types of DNA biosensors includingnano-biosensors and aptasensors and their integration on microfluidic lab-on-a-chip. Onone hand, the nanosensor was fabricated by nucleotide-specific DNA recognition moleculeas molecular recognition element and the nanomaterial graphene oxide as transducerelements; on this basis, the nanobiosensor was integrated in the microfluidic chip as adetection module to achieve low consumption and rapid detection of viral genes, combiningwith sample injecting, mixing and other modules on the microfluidic chip. On the otherhand, due to DNA molecule recognition based on the three-dimensional structure, theaptamer of bacterial cells were screened through the whole-cell SELEX method in order todetect the bacterial cells with high specificity and affinity; on this basis, the aptamer was immobilized on a microfluidic chip to carry out the selective capture and detection ofbacterial cells.1. The nano-DNA biosensor based on fluorescence resonance energy transfer (FRET)was investigated for a rapid detection of viral genes. The nanosensor was fabricated byfluorescent complementary DNA probes of target genes as molecular recognition elementsand the nanomaterial graphene oxide as transducer elements. Graphene oxide was able tobind fluorescent ssDNA probe strongly and quench the fluorescene due to the effect ofFRET. In the prescence of target gene, the probe was hybridized with the target gene toform dsDNA complex and fell off from graphene oxide, lead to the fluorescence recovery.The relationship between the concentration of graphene oxide and the efficiency offluorescence quenching was studied, and the optimized composition of graphen oxide andfluorescent molecular probe was50μg/mL:20nM. After the fabrication of nanobiosensor,the detection assay of the target gene and the control gene with mismatched bases wascarried out, the results showing that the nanosensor could detect the target gene specificly.The study on the fluorescence signal of gradient concentration of target gene detected bythe nanobiosensor indicated that it is the quantative analysis for gene detection.Furthermore, poly-molecular polyvinylpyrrolidone (PVP) was utilized to optimize thedetection limit of the target gene detetion by the nanobiosensor from62.35nM to1.56nM,nearly50times. In addition, the kinetic study of the gene detection progress by thenanosensor was carried out, suggesting the physical and chemical mechanisms in thedetection of target gene by the nanosensor.2. The nano-biosensor was integrated in the microfluidic lab-on-a-chip for rapiddetection of viral genes. With the advantage of rapid detection of specific viral genes, thenanobiosensor was integrated in the microfluidic lab-on-a-chip as molecular recognitiondetection module for rapid and low-consumption detection of viral genes, combining withsample injecting, mixing and other functional modules.The fabrication technology was studied of multi-layer three-dimensional finestructure based on optical adhesive. Several key technology were investigated includingchrome carrier, contact lithography, photocopying exposure, pressure and UV joint bondingand so on, ensuring the fabrication of multi-dimensional fine structure and efficient andhigh yield manufacture of microfluidic chips. Different functional areas were integrated inthe microfluidic chip including sample injecting, mixing, testing and waste discharging.The passive Zigzag and Chaotic coupling micro-mixer was designed and fabricated in the mixing region in order to mix the reagents automatically by the microfluidic chip. The areaof the microfluidic chip was small to5.40cm2, but there were a plurality of parallel unitsable to complete the detection of multiple samples simultaneously.The sample and the test solution were added into the microfluidic chip simultaneously.Following experimental procedures such as sample injecting, mixing and testing, the viralgene was detected rapidly. The fluorescence quenching efficiency depended on grapheneoxide concentration in the microfluidic chip was studied, and the optimized composition ofgraphene oxide and the fluorescent molecular probe was300μg/mL:1μM. Thesimultaneous detection of target gene, mismatched gene and control gene showed that themicrofluidic chip was able to detect target gene specifically. The detection of target genesin the gradinent concentration showed that the microfluidic chip was able to detect targetgene quantitatively.3. By establishing Whole-cell SELEX method, the aptamer to live bacterial cells wasscreened and characterized. In whole-cell SELEX, living cells in their natural state werethe target. Through multiple rounds of evolutionary selection, the aptamers targeted to cellsurface molecules were enriched from the oligonucleotide library.In Whole-cell SELEX, the evolutionary pressure in the aptamer screening process wasimproved constantly through the regulation of the number of competing molecules, so thatthe specifitivity of the aptamers were enhanced. At the same time The binding assay ofpotential aptamer candidates targeted to E. coli was monitored by flow cytometry followingeach round of selection. The whole-cell SELEX of eight rounds were performed to screenthe aptamer potentially binding to the target. With increasing round of SELEX, thefluorescence intensity of cells with fluorescent aptamer increased to a maximum value of64.7%at the sixth round for aptamer pools.The highly enriched aptamer pools were cloned and sequenced and a total of40sequences were obtained. The sequence and structure of aptamers obtained were analizedby molecular simulation method. The result showed that aptamer sequences form thestructures of stem-loop and G-quadruplex, indicating that the complex of stem-loop andG-quadruplex played a part in the specific recognition and binding of aptamers to thebacteria. Specificity and affinity of aptamer were performed by using flow cytometry. It isshowed that the aptamers generally displayed strong binding affinity for bacteria, themaximum reaching to62.3%. Furthermore, the aptamer sequences showed preferentialbinding to E. coli ATCC11775compared to the other types of bacteria. It is inferred that the aptamers are able to bind to E. coli11775specifically. The apparent dissociationconstants Kdof EA1P was estimated to be24.8±2.7nM based on the fit of the nonlinearregression curve.4. The aptamer was integrated into microfluidic lab-on-a-chip for selective capture anddetection of bacterial cells. The aptamer was immobiled on the microfluidic chip asrecognition and capture elements with the advantages of recognizing and bingding thebacteria specifically to capture and dectect bacterial cell selectively.The fabrication technology of PDMS-Glass hybrid microfluidic chip was studied,especially focusing on the effects of the time of plasma oxidation treatment and themicro-channel width on chip bonding. The method of aptamer immobilization on themicrofluidic chip was studied via biotin-avidin strong binding and the effectiveness of themethod was confirmed. The results indicated that the number of immobilized aptamers wasrelated to aptamer concentration. The aptamer based device was built by combiningmicrofluidic chip, injection devices and detection devices. The performance test of thedevice was carried out by the detecting patterns protein through the aptamer targeted to theprotein in order to verify the feasibility of the device. Finally, the microfluidic chip basedon the bacteria aptamer carried out the assay for selective capture and detection of bacterialcells. The results showed that the number of target cells captured in the microfluidic deviceis2orders higher than that of the control cells. It is indicated that the aptamer-basedmicrofluidic device that can specifically detect bacterial E. coli. The gradient concentrationof target bacteria was detected, indicating that the microfluidic chip was able to detectbacteria quantitatively, and quantitative mathematical formulas was obtaind.In conclusion, the construction of FRET-based nano-biosensor and selection ofaptamer targeted to E.coli will be helpful with rapid, sensitive and specific detection both ingenetic level and the cellular level. With the advantages of microfluidic chip inminiaturization, integration, automation and low consumptions, the integration ofnano-biosensors or aptasensors with microfluidic lab technology will achieve the goal ofportable, convenient, inexpensive low-power, rapid and sensitive biological and chemicaldetection and analysis tools, They are benifiting for kinds of fields including medicaldiagnostics, environmental protection, food security, anti-terrorism detection.