Development of nucleic acid testing on the paper diagnostic

Abstract

Nucleic acid testing (NAT) has attracted particular attention in diagnostic techniques due to their substantially higher sensitivity and specificity than other methods. However, time-consuming, labor-intensive, expensive, and instrument dependent characters of NAT are still remained limitation to point-of-care application. With recent improvements of paper technology, paper-based analytical devices have been advanced to integrate most of key steps of NAT into a single paper platform. Several technical barriers such as thermal control during NAT, inhibitory effect of the paper, requirements for optimization of each reagents with porous materials, and storage of NAT reagent in paper platform were overcome in recent progressed achievements. Among these studies, novel approaches and main ideas for realization of paper-based NAT from my researches were introduced in this dissertation. During research process, each developments and complex investigations have focused on realization of simple diagnostic platform. In chapter 2, a handheld lateral flow strip for rapid DNA extraction from complex samples was demonstrated. The lateral flow system was devised based on investigation of basic property for DNA binding and proper arrangements of various porous materials. Engineering structure of the lateral flow strip made by the arrangements of various materials. Result of rapid DNA extraction from the handheld strip was shown similar efficiency to a commercialized silica column. Effective DNA extraction in various types of complex samples (saliva, whole blood, urine, milk, meat, soil, and pond water) was successfully conducted using the handheld strip within 3 minutes. This fabricated strip could be an excellent platform for DNA storage and transportation. In chapter 3, application of handheld lateral flow strip for DNA extraction to identification of meat species in processed products was described. Mitochondrial genomic DNA (mtDNA) in meat sample is a good candidate for reliable identification of meat ingredients. The handheld strip is successfully applied the extraction of mtDNAs from different meat samples, such as pork, chicken, beef were detected in up to 0.1% adultrated animal species by real-time PCR. The established strategy was verified using mixtures of meat samples, and five commercial processed meat products , such as sausages, cutlets, meatballs and nuggets for labeling and quantitative verification. In chapter 4, paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple target DNA was proposed. Optimal chemical components to enable dry conditions for the LAMP without lyophilization or other technique were described. The simple paper structure by sequential stacking of functional layers was successfully devised for multiplex detection of three targets. Newly discovered property of hydroxynaphthol blue fluorescence was combined with paper system to analyze real-time LAMP signals. This proposed device allowed highly sensitive analysis of three different meningitis bacterial DNA samples in a single device with one-step operation. In chapter 5, a strategy for simple colorimetric analysis of polymerase chain reaction (PCR) product was devised. Simultaneous amplification of horseradish peroxidase-mimicking DNAzyme sequence, functional DNA molecule, during the PCR process was performed using a reverse primer containing a complementary DNAzyme sequences. PCR products including DNAzyme sequence was conformed G-quadruplex structure in the presence of hemin, then promotes a peroxidase reaction which produces the blue colored oxidized TMB (3,3’,5,5’ – tetramethylbenzidine) with hydrogen peroxide. This colorimetric method could be much more easily performed than previous detection method, such as agarose gel based electrophoresis. As a result, this system was showing highly specific and sensitive colorimetric analysis of four kinds of pathogenic bacteria. In chapter 6, an ultrasensitive, colorimetric, and homogeneous strategy for aflatoxin B1 detection using a specific DNA aptamer and two split DNAzyme halves was described. Split halves of DNAzymes is combined with aptamer and generate a colorimetric signal with the hemin. In the absence of aflatoxin B1, the split probes have strong peroxidase activity from associated DNAzyme structure and make observable color signal. In the presence of aflatoxin B1, specific recognition by the aptamer leads to structural deformation of G-quadruplex between aptamer and split DNAzymes, and reduction of peroxidase activity. Therefore, a decrease of colorimetric signal is dependent on the concentration of aflatoxin B1 in the 0.1 ng/mL – 1.0 x 104 ng/mL range.