Indumathi Vedarethinam

Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization

Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3 5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies.

Fabrication and Characterization of 3D Micro- and Nanoelectrodes for Neuron Recordings

In this paper we discuss the fabrication and characterization of three dimensional (3D) micro- and nanoelectrodes with the goal of using them for extra- and intracellular studies. Two different types of electrodes will be described: high aspect ratio microelectrodes for studying the communication between cells and ultimately for brain slice recordings and small nanoelectrodes for highly localized measurements and ultimately for intracellular studies. Electrical and electrochemical characterization of these electrodes as well as the results of PC12 cell differentiation on chip will be presented and discussed.

Conducting Polymer 3D Microelectrodes

Conducting polymer 3D microelectrodes have been fabricated for possible future neurological applications. A combination of micro-fabrication techniques and chemical polymerization methods has been used to create pillar electrodes in polyaniline and polypyrrole. The thin polymer films obtained showed uniformity and good adhesion to both horizontal and vertical surfaces. Electrodes in combination with metal/conducting polymer materials have been characterized by cyclic voltammetry and the presence of the conducting polymer film has shown to increase the electrochemical activity when compared with electrodes coated with only metal. An electrochemical characterization of gold/polypyrrole electrodes showed exceptional electrochemical behavior and activity. PC12 cells were finally cultured on the investigated materials as a preliminary biocompatibility assessment. These results show that the described electrodes are possibly suitable for future in-vitro neurological measurements.

Advanced microtechnologies for detection of chromosome abnormalities by fluorescent in situ hybridization

Cytogenetic and molecular cytogenetic analyses, which aim to detect chromosome abnormalities, are routinely performed in cytogenetic laboratories all over the world. Traditional cytogenetic studies are performed by analyzing the banding pattern of chromosomes, and are complemented by molecular cytogenetic techniques such as fluorescent in situ hybridization (FISH). To improve FISH application in cytogenetic analysis the issues with long experimental time, high volumes of expensive reagents and requirement for trained technicians need to be addressed. The protocol has recently evolved towards on chip detection of chromosome abnormalities with the development of microsystems for FISH analysis. The challenges addressed by the developed microsystems are mainly the automation of the assay performance, reduction in probe volume, as well as reduction of assay time. The recent focus on the development of automated systems for performing FISH on chip is summarized in this review.

A Semi-Closed Device for Chromosome Spreading for Cytogenetic Analysis

Metaphase chromosome spreading is the most crucial step required for successful karyotyping and FISH analysis. These two techniques are routinely used in cytogenetics to assess the chromosome abnormalities. The spreading process has been studied for years but it is still considered an art more than a science. The chromosome spreading greatly depends on the environmental conditions such as humidity and temperature, which govern the evaporation of fixative, in which the cells are suspended. The spreading is normally performed manually in ambient conditions on glass slides, which are hydrophilic, and thus allow for better quality spreads. Further cytogenetic analysis depends on the quality of the spreads, which is dependent on the skills of the personnel and is thus limited to laboratory settings. Here, we present a semi-closed microfluidic chip for preparation of the metaphase spreads on a glass and a Topasr substrate rendered more hydrophilic by oxygen plasma treatment coupled with photografting. The device consists of a microfluidic chamber with perfusion holes that facilitate the evaporation of fixative and reliable formation of the spreads. The usability of the chromosome spreads formed on the glass and the Topasr slide is tested by performing FISH analysis.

Microtechnologies Enable Cytogenetics

Cytogenetic analysis is an important tool in preand postnatal diagnosis as well as cancer detection. In a traditional cytogenetic technique known as karyotyping the metaphase chromosome spreads are prepared on a glass slide and stained with a Giemsa stain. The stain reveals a specific banding pattern for each chromosome – a chromosome bar code. Karyotyping is often supplemented by the molecular cytogenetic technique Fluorescent In Situ hybridization (FISH), which requires the use of fluorescently labeled DNA probes to target a specific chromosome region. In FISH the chromosome preparations (metaphase spreads or interphase nuclei) are heat denatured, followed by application of the probe and hybridization at 37 °C. FISH can be performed on interphase nuclei on non-cultured cells in less than 24 hrs, but the chromosome structure cannot be visualized. On the other hand, metaphase FISH has the advantage of visualizing the entire karyotype at once and can detect potential abnormalities at a high resolution. But, the long analysis time and culturing required for metaphase FISH are important disadvantages.