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

Abstract

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.

A Semi-Closed Device for Chromosome Spreading for Cytogenetic Analysis

“…There has been few reports on performing cytogenetic analysis using microfabricated devices [6][7][8][9][10][11][12][13][14][15]. They offer a reduction in the reagents volume with great focus on minimizing the amount of the DNA probe used.…”

“…For the development of a polymer based device for cytogenetic analysis, the proper polymer needs to be selected. The selection criteria are based on the resistance to chemicals used in the fixative, optical properties and ease of structuring [6]. Some of the commonly used polymers such as polycarbonate (PC) or polymethylmethacrylate (PMMA) are good candidates for microfluidic devices, however not for this particular application, as PC autofluoresces and PMMA is not resistant to ethanol.…”

Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell

“…15,17 In one study of first metaphase chromosomes in which the X-chromosome in a peripheral blood lymphocyte was detected by FISH, the consumed reagent volume was only 14 mL using the microfluidic device, as compared to 327 mL required in traditional FISH analysis. 18 Sequential injections with the microdevice also facilitated the procedure. 17 There have been a few reports on the integration of padlock/ RCA with a microdevice [20][21][22][23] extracted DNA solution was used as the sample.…”

“…[15][16][17][18][19] Microfluidic-based analyses require 0.5 to 1 μL of probe solution, as compared to the 10 μL required for conventional FISH; this reduces the cost by 90 -95%. 15,17 In one study of first metaphase chromosomes in which the X-chromosome in a peripheral blood lymphocyte was detected by FISH, the consumed reagent volume was only 14 mL using the microfluidic device, as compared to 327 mL required in traditional FISH analysis.…”

“…Although attempts have been made to reduce the reagents used in microfluidic in situ DNA analysis by FISH [16][17][18] and an automated microfluidic system for FISH has been developed, 17 this is the first report of an automated microfluidic system for in situ padlock/RCA.…”