Quanjun Liu

Microarray-in-a-Tube for Detection of Multiple Viruses

Abstract Background: The detection of multiple viruses is important for pathogenic diagnosis and disease control. Microarray detection is a good method, but requires complex procedures for multiple virus detection. Methods: We developed a novel PCR assay, the microarray-in-a-tube system, which integrates multiple PCR processes and DNA microarrays for multiple virus detection. A 5 × 5 oligonucleotide microarray for detecting 4 respiratory tract viruses (severe acute respiratory syndrome–associated coronavirus, influenza A virus, influenza B virus, and enterovirus) with inner controls was arranged on the inner surface of a specially designed Eppendorf cap with a flat, optically transparent window. Results: We were able to perform all detection processes in the encapsulated system without opening the cap. The 4 viruses were successfully amplified by one-step reverse transcription–PCR in the encapsulated tube. After the PCR process, the microarray-in-a-tube was inverted, and the fluorescence-labeled PCR products were directly hybridized on the microarray. Hybridization signals were obtained with an ordinary fluorescent microscope. The sensitivity of the system for virus detection reached 102 copies/μL. With the help of inner controls, the system provided reliable results without false negatives and false positives. Conclusions: The microarray-in-a-tube system is a rapid, labor-saving tool for multiple virus detection with several advantages, such as convenience, prevention of cross-contamination of the PCR products, and potential for multiple-gene detection.

Investigation of DNA-protein Sequence-Specific Interactions with a ds-DNA Array

The sequence specific recognitions between DNAs and proteins play important roles in many biological functions. The use of double-stranded DNA arrays (ds-DNA arrays) for studying sequence specific recognition between DNAs and proteins is a promising method. Here we report the use of a ds-DNA probe with multi operation sites of restriction proteins in the middle sequence to investigate DNA-protein sequence-specific interactions including methylation. We arranged EcoR I site and Rsa I site on the same duplex DNA probe to fabricate ds-DNA arrays. We used the ds-DNA arrays to study DNA-restriction enzyme reactions before and after duplex DNA methylation under different probe concentration and reaction time conditions. Our results indicated that the ds-DNA arrays can be further biochemically modified and made accessible for interactions between DNAs and proteins in complex multi-step gene-regulation processes.

Detection and analysis system for hybridization images of lab-in-a-tube microarray

A lab-in-a-tube microarray system is developed for sample inspection and signal detection by fabricating a flat transparent window cap of the Eppendorf tube. The oligonucleotide microarray is immobilized on the inner surface of the cap. A small vessel is placed in an Eppendorf tube for storing hybridization solutions. With the microarray system, the full biochemical processes, including gene fragment amplification, fluorescence labeling, hybridization, and fluorescence detection, have been performed in the sealed tube without opening the cap. The images are obtained from a fluorescence microscope and captured by a CCD, and the data are transported to a computer through the universal serial bus (USB). After noise reduction, signal intensity is determined from hybridization image and the presence of gene fragments is identified. The final data output includes sample information, process steps, and hybridization results. A lab-in-a-tube microarray system for detecting ten respiratory viruses at a single detection is designed. High detection throughput and accuracy have been demonstrated with the system.

Hydrogen Peroxide Sensing Based on Inner Surfaces Modification of Solid-State Nanopore

There are many techniques for the detection of molecules. But detection of molecules through solid-state nanopore in a solution is one of the promising, high-throughput, and low-cost technology used these days. In the present investigation, a solid-state nanopore platform was fabricated for the detection of hydrogen peroxide (H2O2), which is not only a label free product but also a significant participant in the redox reaction. We have successfully fabricated silicon nitride (Si3N4) nanopores with diameters of ~50 nm by using a focused Ga ion beam, the inner surface of the nanopore has been modified with horseradish peroxidase (HRP) by employing carbodiimide coupling chemistry. The immobilized HRP enzymes have ability to induce redox reactions in a single nanopore channel. Moreover, a real-time single aggregated ABTS•+ molecular translocation events were monitored and investigated. The designed solid-state nanopore biosensor is reversible and can be applied to detect H2O2 multiple times.

Improvement of Microarray Technologies for Detecting Single Nucleotide Mismatch

With the completion of the Human Genome Project and beginning of post genome era, there is an urgent need for a fast, specific, sensitive, reliable and cost-effective method for the genome-wide polymorphism analysis. Microarray technology is one of the most promising approaches to this need. There are two major concerns in the microarray technology; one is target labeling and the other is the reliability of single nucleotide mismatch discrimination. In this chapter, we reported our recent progress in molecular beacon arrays for detecting label-free targets and a microarray based melting-curve analysis method for improving the reliability of single nucleotide mismatch discrimination. Several successful practical applications of these improved microarray technologies have also been illustrated.

Gel-immobilized molecular beacons for label-free DNA hybridization detection

We report gel-immobilized molecular beacon for DNA hybridization detection in this paper. The specially designed molecular beacons were covalently immobilized in polyacrylamide gel pads array. After hybridization, the complementary noncomplementary target can be easily distinguished with gel-immobilized molecular beacon probes. The results indicate the potential applications of this new gel-immobilized molecular beacon array in parallel, cost effective and label-free DNA hybridization detection.