ABSTRACT
This work describes a novel microfluidic device using a thin film transistor (TFT) photosensor integrating a microfluidic channel, a DNA chip platform, and a photodetector for the discrimination of single nucleotide polymorphisms (SNPs). A DNA-arrayed TFT photosensor was used as a DNA chip platform and photo detecting device. Chemiluminescence was used for DNA sensing because chemiluminescence provides higher sensitivity and requires simpler instrumentation than fluorescence methods. The SNP of biotinylated target DNA was detected based on chemiluminescence by using horse radish peroxidase-conjugated streptavidin. The lower detection limit for a model biotinylated oligonucleotide (63-mer) was 0.5 nM, much lower than expected DNA concentrations in a practical application of this device. Furthermore, SNP detection in the aldehyde dehydrogenase 2 gene was successfully achieved using DNA-arrayed TFT photosensor without DNA extraction and DNA purification using PCR products. The assay was completed in less than one hour. Our technology will be a promising approach to developing a miniaturized, disposable DNA chip with high sensitivity.
Subject(s)
DNA/blood , Luminescent Measurements , Microfluidic Analytical Techniques/instrumentation , Oligonucleotide Array Sequence Analysis/instrumentation , Polymerase Chain Reaction , Polymorphism, Single Nucleotide/genetics , Transistors, Electronic , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase, Mitochondrial , DNA/genetics , Equipment Design , Genotype , Microfluidic Analytical Techniques/methods , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotide Probes , Optics and Photonics , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A thin film transistor (TFT) photosensor fabricated by semiconductor integrated circuit (IC) technology was applied to DNA chip technology. The surface of the TFT photosensor was coated with TiO2 using a vapor deposition technique for the fabrication of optical filters. The immobilization of thiolated oligonucleotide probes onto a TiO2-coated TFT photosensor using gamma-aminopropyltriethoxysilane (APTES) and N-(gamma-maleimidobutyloxy) sulfosuccinimide ester (GMBS) was optimized. The coverage value of immobilized oligonucleotides reached a plateau at 33.7 pmol/cm2, which was similar to a previous analysis using radioisotope-labeled oligonucleotides. The lowest detection limits were 0.05 pmol/cm2 for quantum dot and 2.1 pmol/cm2 for Alexa Fluor 350. Furthermore, single nucleotide polymorphism (SNP) detection was examined using the oligonucleotide-arrayed TFT photosensor. A SNP present in the aldehyde dehydrogenase 2 (ALDH2) gene was used as a target. The SNPs in ALDH2*1 and ALDH2*2 target DNA were detected successfully using the TFT photosensor. DNA hybridization in the presence of both ALDH2*1 and ALDH2*2 target DNA was observed using both ALDH2*1 and ALDH2*2 detection oligonucleotides-arrayed TFT photosensor. Use of the TFT photosensor will allow the development of a disposable photodetecting device for DNA chip systems.
