A custom-built insulin resistance gene chip

Objectives/Aim—Microarray (gene chip) technology offers a powerful new tool for analyzing the expression of large numbers of genes in many experimental samples. The aim of this study was to design, construct, and use a gene chip to measure the expression levels of key genes in metabolic pathways related to insulin resistance.
Methods—We selected genes that were implicated in the development of insulin resistance, including genes involved in insulin signaling; glucose uptake, oxidation, and storage; fat uptake, oxidation, and storage; cytoskeletal components; and transcription factors. The key regulatory genes in the pathways were identified, along with other recently identified candidate genes such as calpain-10. A total of 242 selected genes (including 32 internal control elements) were sequence-verified, purified, and arrayed on aldehyde-coated slides.
Results—Where more than 1 clone containing the gene of interest was available, we chose those containing the genes in the 5' orientation and an insert size of around 1.5 kb. Of the 262 clones purchased, 56 (21%) were found to contain sequences other than those expected. In addition, 2 (1%) did not grow under standard conditions and were assumed to be nonviable. In these cases, alternate clones containing the gene of interest were chosen as described above. The current version of the Insulin Resistance Gene Chip contains 210 genes of interest, plus 48 control elements. A full list of the genes is available at http://www.hbs.deakin.edu.au/mru/research/gene_chip_tech/genechip_three.htm/.
Conclusions—The human Insulin Resistance Gene Chip that we have constructed will be a very useful tool for investigating variation in the expression of genes relevant to insulin resistance under various experimental conditions. Initially, the gene chip will be used in studies such as exercise interventions, fasting, euglycemic-hyperinsulinemic clamps, and administration of antidiabetic agents

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 × 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 µm, respectively, which provides a relatively large surface area (ca. 3 cm²) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.

Design and simulation of an interdigital-chaotic advection micromixer for lab-on-a-chip applications

This paper presents the design and simulation of a novel passive micromixer. The micromixer consists of two inlet tanks, one mixing channel and two outlet channels. In order to maximise the mixing efficiency, the following considerations are made: (i) The inlet tanks are followed by a series of microchannels, in which the flow is split. The microchannels are arranged in an interdigital manner to maximise the contact area between the two flows. (ii) The microchannels attached to the lower inlet tank have an upward slope while those attached to the upper tank have a downward slope. The higher-density flow is fed to the lower inlet tank and gets an upward velocity before entering the mixing channel. (iii) Two triangular barriers are placed within the mixing channel to impose chaotic advection and perturb the less-mixed flow along the top and bottom surfaces of the channel. (iv) Finally, two outlet channels are incorporated to discard the less-mixed flow. Three-dimensional simulations are carried out to evaluate the performance of the micromixer. Simulations are performed in the absence and presence of the gravitational force to analyse the influence of gravity on the micromixer. Mixing efficiencies of greater than 92% are achieved using water and a 1011'density biological solvent as the mixing fluids.

Design and simulation of an interdigital-chaotic advection micromixer for lab-on-a-chip applications

The paper presents the design and simulation of a novel passive micromixer. The micromixer consists of two inlet tanks, one mixing channel and two outlet channels. In order to maximise the mixing efficiency, the following considerations are made : (i) The inlet tanks are followed by a series of microchannels, in which the flow is split. The microchannels are arranged in an interdigital manner to  maximise the contact area between the two flows. (ii) The microchannels attached to the lower inlet tank have an upward slope while those attached to the upper tank have a downward slope. The higher-density flow is fed to the lower inlet tank and gets an upward velocity before entering the mixing channel. (iii) Two triangular barriers are placed within the mixing channel to impose chaotic advection and perturb the less-mixed flow along the top and bottom surfaces of the channel. (iv) Finally, two outlet channels are incorporated to discard the less-mixed flow. Three-dimensional simulations are carried out to evaluate the performance of the micromixer. Simulations are performed in the absence and presence of the gravitational force to analyse the influence of gravity on the micromixer. Mixing efficiencies of greater than 92 % are achieved using water and a low-density biological solvent as the mixing fluids.

Computational thermal analysis of a continuous flow micro Polymerase Chain Reaction (PCR) chip

The first continuous flow micro PCR introduced in 1998 has attracted considerable attention for the past several years because of its ability to amplify DNA at much faster rate than the conventional PCR and micro chamber PCR method. The amplification is obtained by moving the sample through 3 different fixed temperature zones. In this paper, the thermal behavior of a continuous flow PCR chip is studied using commercially available finite element software. We study the temperature uniformity and temperature gradient on the chip’s top surface, the cover plate and the interface of the two layers. The material for the chip body and cover plate is glass. The duration for the PCR chip to achieve equilibrium temperature is also studied.

A disposable multiplex polymerase chain reaction (PCR) chip and device

Malaysian patent application number PCT/MY2008/000190 Australian application number : 2009203047

Non-optical biosensing in lab-on-a-chip systems

This paper presents a review of the recent trends and developments in non-optical biosensing platforms for lab-on- a-chip systems. This includes design considerations and applications of the non-optical biosensing platforms. The paper first categorizes the non-optical biosensors into four groups. The definition of each group together with a review of the reported works associated with the group are given. A performance analysis of different non-optical detection methods is also presented.

Lab-on-a-chip : a component view

Miniaturization is being increasingly applied to biological and chemical analysis processes. Lab-on-a-chip systems are direct creation of the advancement in the miniaturization of these processes. They offer a host of exciting applications in several areas including clinical diagnostics, food and environmental analysis, and drug discovery and delivery studies. This paper reviews lab-on-a-chip systems from their components perspective. It provides a categorization of the standard functional components found in lab-on-a-chip devices together with an overview of the latest trends and developments related to lab-on-a-chip technologies and their application in nanobiotechnology. The functional components include: injector, transporter, preparator, mixer, reactor, separator, detector, controller, and power supply. The components are represented by appropriate symbols allowing designers to present their lab-on-a-chip products in a standard manner. Definition and role of each functional component are included and complemented with examples of existing work. Through the approach presented in this paper, it is hoped that modularity and technology transfer in lab-on-a-chip systems can be further facilitated and their application in nanobiotechnology be expanded.

Apoptosis goes on a chip : advances in the microfluidic analysis of programmed cell death

Recent years have brought enormous progress in cell-based lab-on-a-chip technologies, allowing dynamic studies of cell death with an unprecedented accuracy. As interest in the microfabricated technologies for cell-based bioassays is rapidly gaining momentum, we highlight the most promising technologies that provide a new outlook for the rapid assessment of programmed and accidental cell death and are applicable in drug discovery, high-content drug screening, and personalized clinical diagnostics.

Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the realtime analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

Global and threshold-free transcriptional regulatory networks reconstruction through integrating ChIP-Chip and expression data

Inferring transcriptional regulatory networks from high-throughput biological data is a major challenge to bioinformatics today. To address this challenge, we developed TReNGO (Transcriptional Regulatory Networks reconstruction based on Global Optimization), a global and threshold-free algorithm with simulated annealing for inferring regulatory networks by the integration of ChIP-chip and expression data. Superior to existing methods, TReNGO was expected to find the optimal structure of transcriptional regulatory networks without any arbitrary thresholds or predetermined number of transcriptional modules (TMs). TReNGO was applied to both synthetic data and real yeast data in the rapamycin response. In these applications, we demonstrated an improved functional coherence of TMs and TF (transcription factor)- target predictions by TReNGO when compared to GRAM, COGRIM or to analyzing ChIP-chip data alone. We also demonstrated the ability of TReNGO to discover unexpected biological processes that TFs may be involved in and to also identify interesting novel combinations of TFs.