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Weizmann Researchers Develop In Vivo Pattern-Generating Bioprobe Platform


NEW YORK (GenomeWeb) – Researchers at Israel's Weizmann Institute of Science have developed a pattern-generating probe platform that they said combines the qualities of conventional small molecule probes and cross-reactive sensor arrays like chemical "noses" or "tongues."

Described in a paper published last week in Nature Nanotechnology, the probe system could enable specific and highly multiplexed detection of biomarkers, binding reactions, and other information in samples like blood and urine as well as in living cells, said David Margulies, a professor of organic chemistry at Weizmann and senior author on the study.

The platform uses small molecule probes that combine a component capable of binding specific protein groups with a fluorescent component that produces different emission patterns upon binding of different analytes. In the Nature Nano study, the researchers developed a set of probes that combined aptamers to three different protein groups — glutathione-S-transferases (GSTs), matrix metalloproteases (MMPs), and platelet-derived growth factors (PDGFs) — with four fluorescence dyes designed to produce unique emission patterns based on the characteristics of the molecules to which they are bound.

For instance, the authors noted, the different surface characteristics of different molecules "may change the distances between the dyes or the polarity of their molecular environments, or lead to fluorescence quenching by amino acid side chains," all of which would lead to an emission pattern characteristic of a particular molecule or complex.

The idea for the probe system stemmed from work by Margulies and others on chemical "nose" or "tongue" sensors, he said. Unlike conventional probes using reagents like antibodies, these systems use arrays of sensors to generate patterns characteristic of a given target or targets. Such systems are able to detect a wide variety of targets by analyzing their different binding patterns.

"Suppose you just want to detect a chemical like sugar," Margulies said. "There is a sensor for sugar that you can buy at the pharmacy. A sensor for cholesterol you can also buy at the pharmacy. A pregnancy test also. But the unique thing about the [chemical] nose is that it is one sensor that can detect a lot of different molecules."

A limitation of these and other array-based pattern-generating platforms is that they are typically only suited for in vitro use.

"They can detect a lot of things, Margulies said, "but they can't enter cells."

The pattern-generating probes his group has developed bring the technology down to a size that can be applied in vivo.

"So we have combined the properties of small molecule probes which are very commonly used to detect molecules inside the cell, with the properties of the artificial nose, which can detect a lot of different things and in a lot of different combinations," he said.

He suggested a number of potential applications for the technology, among them multiplexed biomarker detection using a single probe.

Because the probe can distinguish between different combinations of protein types and isoforms, it could allow researchers and clinicians to measure a variety of targets in a single assay, Margulies said.

"You can imagine a system in which you [use the probes] to test the blood or urine or cells of a patient for different combinations of biomolecules," he said. "You won't need to apply different antibodies to the different [targets]. You can just put in the probe and it will give you a signature [based on its binding of the different targets present]."

The researchers made an initial foray into this sort of application in the recent study, using the probes they generated to detect medically relevant GST, MMP and PDGF isoforms spiked in different combinations into urine samples. They were able to differentiate between 20 of 22 samples based on the probes binding patterns.

The platform could also prove useful for drug screening, potentially allowing researchers to look simultaneously at multiple drug-target binding events, Margulies said. They tested this idea in the study by using the probes to look at binding of the proteins MMP-7, GST-A2, PDGF-BB to different combinations of inhibitors, finding that they could determine the specific binding interactions from differences in the resulting emission patterns.

They also demonstrated the ability of the probes to distinguish between protein isoforms in living cells. Expressing four different GST isoforms in different HE293T cells, the researchers found they were able to classify a set of 35 cells based on their GST isoform content. They followed this with an experiment in which they subjected cells to heat and oxidative stress to alter their GST, PDGF, and MMP expression patterns and used the probes to characterize the response of these cells to these conditions.

Margulies said he and his colleagues are applying the probe platform to several research projects currently. One is focused on using the probes to detect protein glycosylation patterns linked to different diseases. While the majority of protein biomarkers in clinical use are glycoproteins, these molecules are difficult to study due to the complexity and diversity of glycosylation modifications.

"We're working on a system where the probe will target a protein [of interest] and generate different signatures for different glycosylation states," Margulies said. "And then we hope to identify disease based on protein glycosylation patterns or changes in glycosylation state."

They have also developed a version of the probe for tracking aggregation of amyloid proteins involved in conditions like Alzheimer's.

"Researchers really want to gain information about the aggregation state of these proteins, and there are several [aggregation] states that people want to investigate, but you don't have a sensor for all these different states," he said. "So to follow the aggregation of amyloids relevant to Alzheimer's is really complicated."

The Weizmann researchers have been able to use their probes to track dynamic changes in amyloid aggregation, Margulies said.

"This is exactly what our nose does," he noted. "You smell one smell, and if something changes, you smell another smell, right? You don't have to change your nose every time. So I think there are quite a few directions [for the research]."