Pranjul Jaykumar Shah

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.

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.

Scanning conductance microscopy investigations on fixed human chromosomes

Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value for the dielectric constant of different human chromosomes.

A microfabricated platform for chromosome separation and analysis

More and more diseases find their cause in malfunctioning genes. There is therefore still need for rapid, low-cost and direct methods to accurately perform genetic analysis. Currently the process takes a long time to complete and is very expensive. We are proposing a system that will be able to isolate white blood cells from blood, lyse them in order to extract the chromosomes and then perform chromosome sorting on chip. As the physical properties of the chromosomes, such as size and dielectric properties, are needed for designing the chip, we have measured them using an AFM microscope.

A novel passive microfluidic device for preprocessing whole blood for point of care diagnostics

A novel strategy to sort the cells of interest (White Blood Cells (leukocytes)) by selectively lysing the Red Blood Cells (erythrocytes) in a miniaturized microfluidic device is presented. Various methods to lyse cells on a chip exist i.e. electrical, mechanical, chemical and thermal but they need integration of electrodes, traps, reservoirs, heaters, etc which is often difficult at microscale [1 – 4]. On the other hand, FACSlyse protocol uses only osmotic pressure to lyse erythrocytes allowing further isolation of leukocytes. This motivated us to develop a novel herringbone based lyser which works on the principle of mixing whole blood with pure water in time controlled manner to lyse erythrocytes osmotically on a chip.

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.