Fluorescence Data Analysis on Gel-Based Biochips

Abstract

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.

Biological Microchip for Simultaneous Quantitative Immunoassay of Tumor Markers in Human Serum

“…Fluorescence was measured on a biochip portable fl uorescence analyzer with laser excitation (Institute of Molecular Biology) using 650/750-nm fi lters (excitation/recording) [2]. Fluorescence signals from each well of the biochip and concentrations o f all tumor mar kers were studied by means of ImagelAssay software (Institute of Molecular Biology).…”

Analysis of SNPs and other genomic variations using gel-based chips

“…A combination of up to four labels such as TCPP, FL, TR, and Cy5 can be used on the same chip. At the same time, TR and TCPP increase melting temperatures of perfect duplexes, probably because of their intercalating ability, and therefore, experiments with these labels must include carefully designed controls [Barsky et al, 2002].…”

“…A high-pressure mercury lamp is the source of epiillumination. The microscope works at up to four wavelengths simultaneously [Barsky et al, 2002]. We use routinely fluorescein, TR, and tetramethylrhodamine derivatives.…”

“…A low-noise PIN photodiode is used as a detector. Working distance of a miniature objective lens is 3 mm, long enough to scan wet chips without disassembling the hybridization chamber [Barsky et al, 2002].…”

“…In some cases it becomes a major shortcoming, for instance, in field conditions or for clinical applications in developing countries. To overcome this hindrance, we developed a simple portable fluorescent analyzer [Bavykin et al, 2001;Barsky et al, 2002]. It is powered by batteries and combines a laser-diode with a Polaroid camera or a digital camera and a wide-field high-aperture objective lens at a total cost of under $2,000.…”