High throughput screening by multichannel glass fiber fluorimetry

Abstract

As a tool for screening large numbers of biological samples by means of amplification ͑e.g., Q␤ or PCR͒ we have constructed a thermocycler that includes optionally a 96-channel or 960-channel glass fiber fluorimeter ͑combined with a cooled charge-coupled-device camera͒. We briefly describe the system integration of all components like liquid handling, thermostats, an x,y,z robot arm, and the glass fiber fluorimeter. The integrated glass fiber fluorimeter allows sensitive on-line measurements in 960 channels within 5 s. Two different screening procedures were carried out. In a first experiment PCR reactions were done in the presence of the known PCR inhibitor hematin and its suppressor transferrin. The system was used to titrate the suppressor with the inhibitor hematin in order to determine the maximum inhibitor concentration tolerated at a given suppressor concentration. We processed 96 PCR samples in parallel with 11 different concentration steps. In a second experiment the 960-channel glass fiber fluorimeter was used to monitor on line the amplification of the Q␤ system in the presence or absence of an inhibitor ͑heparin͒. Since the doubling time of Q␤ RNA variants is about 20 s, on-line detection is crucial for the experimental setup. The evolution of new RNA species adapted to high inhibitor concentrations could be proved by comparison of the fluorimetric signal and electrophoresis.

Background Suppression in Frequency-Domain Fluorometry

“…For a single exponential decay the long lifetime can be obtained using Eqs. [8] and [9]. Alternatively, the resulting data can be directly used in currently available frequency-domain software to recover the multiple decay times without any modification.…”

“…Fluorescent detection is being used throughout the biosciences for numerous applications including clinical chemistry, DNA sequencing, FISH, flow cytometry, high-throughput screening, and cellular imaging (1)(2)(3)(4)(5)(6)(7)(8). In many instances the sensitivity is limited by interfering autofluorescence from the sample rather than detectability of the emission.…”

Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity

“…In recent years, major efforts have been undertaken in miniaturization, parallelization, and automation of screening technologies, as well as in the development of new homogeneous assays and the integration of highly sensitive and rapid detection devices. Among the many alternative principles of detection, fluorescence-based techniques are widely used (7,8); examples are fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarization, time-resolved fluorescence, and fluorescence correlation spectroscopy (FCS) (9)(10)(11)(12).…”

PCR‐linked in vitro expression: a novel system for high‐throughput construction and screening of protein libraries

“…Significantly, it is speedy and completely suits automation. With a precise liquid handling system and an array technology [17–20], this strategy could be a powerful tool for high‐throughput construction and screening of various protein libraries.…”