High-throughput screening with photonic crystal biosensors

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Identifying small molecules that inhibit or enhance protein-protein interactions is an important part of the drug discovery process. Being able to screen through large libraries of molecules to find those with the strongest ability to do so is therefore highly desirable.

At the University of Illinois at Urbana-Champaign, Brian T. Cunningham, Paul J. Hergenrother and colleagues have developed an assay based on photonic crystal biosensor technology that enables high-throughput identification of these molecules. As reported in the December 2009 issue of the Journal of the American Chemical Society (JACS), the assay can record protein-protein interactions in real time without relying on antibodies or fluorescence measurements.

Photonic crystal biosensors – a technology developed by Cunningham and his group at the university and by SRU Biosystems, a company Cunningham founded in 2000 – consist of subwavelength polymer gratings coated with TiO2, a high-refractive-index material. This surface reflects a single wavelength, which is particularly sensitive to modulations resulting from the adsorption of biomaterials.

The researchers adapted the technology for high-throughput screening by incorporating it into the bottom surface of each well in standard format 384- and 1536-well microplates. The photonic crystal biosensor assay is measured by a special-purpose readout instrument that can scan a 384-well microplate in about 20 s. The biosensor microplates and the readout system used in the study were manufactured by SRU Biosystems.

Researchers have developed an assay for drug development based on photonic crystal biosensor technology that can record protein-protein interactions in real time without relying on antibodies or fluorescence measurements.

In addition, the investigators developed a surface preparation method for the biosensor that allows capture of immobilized proteins with a histidine tag. “His-tagging” is one of the most commonly used methods for purifying proteins from complex mixtures using liquid chromatography, “so the ability to easily use such proteins with the biosensor is very important,” Cunningham said.

For the JACS paper, the researchers performed proof-of-principle experiments using three protein-protein pairs with well-known chemical modulators: XIAP–caspase-9; XIAP–caspase-7; and FKBP12–mTOR (FBR domain). They also performed “needle in the haystack” experiments, using an inhibitor with an easily measurable signal to demonstrate high-throughput capabilities.

They also showed that the technique needs only a small volume of reagents, which are typically very expensive. “We demonstrated that the proteins on the biosensor surface could be reused multiple times, through at least five regeneration cycles,” Cunningham said. “This is important because sensor surface regeneration dramatically reduces the amount of protein required to perform high-throughput screening.”

Having demonstrated its efficacy, the researchers now are working to apply it. In another group, Hergenrother’s team has been using the technique to probe the efficacy of chemical inhibitors for several disease applications and been performing cell-based assays to validate the function of the molecules identified in the screening.

Cunningham and co-workers, in the meantime, continue to explore new materials and methods to use in making label-free biosensors. In addition, they are developing label-free methods for high-resolution imaging of cell attachment to biosensor surfaces as a means to study the effects of drugs on the cell membrane.

Published: February 2010
Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
Label-free refers to a technique or method that does not require the use of additional labels, tags, or markers to detect or identify specific components or entities. In various scientific and technological applications, labeling often involves attaching fluorescent dyes, radioactive isotopes, or other markers to molecules, cells, or structures of interest. However, in label-free approaches, detection or analysis is performed without the need for these additional labels. Common applications...
Acronym for profile resolution obtained by excitation. In its simplest form, probe involves the overlap of two counter-propagating laser pulses of appropriate wavelength, such that one pulse selectively populates a given excited state of the species of interest while the other measures the increase in absorption due to the increase in the degree of excitation.
antibodiesassaybiomaterialsBiophotonicsBioScanbiosensorsBrian T. Cunninghamcell membranedrug discoveryfluorescencefluorescence measurementGary Boashigh-throughput screeninghis-tagginghistidine tagImagingimmobilized proteinsJACSlabel-freeliquid chromatographymicroplatesmoleculesNewsPaul J. Hergenrotherphotonic crystal biosensorpolymer gratingsprobeproteinprotein-protein interactionsreadout instrumentsreadout systemreagentsSensors & DetectorsSRU BiosystemsUniversity of Illinois

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