Single-cell enzyme monitoring?
SERRS-based technique sensitive enough to measure reactions from as few as
500 molecules

By Charles Q Choi

A new technique to rapidly detect enzyme activity published online August 8
in Nature Biotechnology is sensitive enough to identify reactions from as
few as 500 molecules, according to researchers at the University of
Strathclyde, Glasgow, who say their method could potentially detect multiple
enzyme activities simultaneously and at levels found within single cells.

"We think we can probably apply the technology to most enzyme classes,"
researcher Barry Moore said of the method, which employs surface-enhanced
resonance Raman scattering (SERRS). In SERRS, the target compound is
adsorbed onto a roughened metal surface, producing an enhanced vibrational
spectrum of the target, characterized by multiple sharp peaks, that serves
as a fingerprint. The research team used a suspension of citrate-reduced
silver particles roughly 40 nanometers in diameter as their metal surface.

The key to detecting enzyme activities at ultra-low levels is a newly
devised class of substrates covered by a University of Strathclyde patent.
Each consists of three components-an enzyme recognition site, a
benzotriazole azo dye, and an enzyme-cleavable linker that stably joins the
other components. When free, the dye has a strong penchant for adsorbing to
silver nanoparticles by displacing their citrate surface layers, and when
this happens it can generate an increase of up to 10 to 14 times in the
SERRS signal intensity, enough that near single-molecule detection levels of
such dyes are observed.

The substrates proved susceptible to hydrolysis by a wide range of
hydrolases, including lipases, esterases, and proteases. In experiments, the
substrates acted rapidly, screening for activity and enantioselectivity for
14 enzymes in less than 30 seconds. Extrapolating this productivity gives a
potential throughput of roughly 100,000 enzymes per day per instrument,
comparing favorably with other screening techniques, the investigators said.

"You can measure SERRS on any standard Raman spectrometer," researcher
Duncan Graham said. He noted no high-throughput SERRS techniques are
currently employed, although his group has suggested high-throughput SERRS
techniques previously for genomics.

The technique detected 0.025 micrograms/milliliter of lipase from
Pseudomonas cepacia after reaction for 10 minutes, corresponding to at most
0.8 picomoles of enzyme in the 1-milliliter sample volume. Given the
microscope lens SERRS uses only interrogates a small portion of the sample,
a conservative estimate puts the actual sample volume at picoliters, whereas
more realistically it is closer to femtoliters, the researchers wrote in
their report. This suggests they likely sampled reactions arising from only
500 or so molecules of enzyme at most, they add. "In the long term, you
could think of measurements directly in vivo, where you would get the
nanoparticles into the cells," Moore told The Scientst.

The investigators are currently synthesizing new substrates to monitor other
major enzyme classes, such as phosphatases and p450s. They suggest the
system could be optimized so that SERRS can be used to monitor single-enzyme
kinetics, as has already been done with fluorescence. Also, so far, standard
Raman optics have been used, but the authors note there is great scope for
miniaturization. Graham noted his group was developing a microfluidics
device to monitor enzyme selectivity and activity.

Because each dye produces a characteristic SERRS spectrum-akin to a
fingerprint-that can easily be identified and quantified in a mixture,
synthesizing substrates with different enzyme recognition sites coupled with
different dyes could make it possible to monitor the action of multiple
enzymes simultaneously, the investigators suggest. They note that it is very
difficult to achieve by alternate techniques and is expected to provide a
pathway to a wide range of new bioanalytical assays.

"One application of this technology is in the development of new diagnostic
methods, such as in detecting specific enzyme biomarkers of disease states.
For example, enzymes such as metalloproteinases are known to be involved in
cancer initiation and progression," Moore said.

"It's ingenious work," said Chad Mirkin of Northwestern University in
Evanston, Ill., who did not participate in this research. "It's a technique
that's going to allow you to look at a variety of molecular biotechnology
processes with high sensitivity with a technique that looks like it's fairly
straightforward to implement.

But Mirkin said that calibration would be "one of the big issues with
this."

"One of the main problems with SERRS is changing SERRS responses, since
every surface is slightly different, with hot spots and cold spots, and
calibration is often a difficult issue to deal with," Mirkin said.

Links for this article
B.D. Moore et al., "Rapid and ultra-sensitive determination of enzyme
activities using surface-enhanced resonance Raman scattering," Nature
Biotechnology, DOI:10.1038/nbt1003, August 8, 2004.
http://www.nature.com/nbt/

Barry D. Moore
http://www.chem.strath.ac.uk/people.php?id=cbas115

Raman spectroscopy
http://en.wikipedia.org/wiki/Raman_spectroscopy

Duncan Graham
http://homepages.strath.ac.uk/~bas96104/

Duncan Graham: Publications
http://homepages.strath.ac.uk/~bas96104/publications.htm

"First person: Chad Mirkin," The Scientist, 17:9, January 27, 2003.
http://www.the-scientist.com/yr2003/jan/upfront4_030127.html

Chad Mirkin
http://www.chem.northwestern.edu/~mkngrp/

>From The Scientist.com
http://www.biomedcentral.com/news/20040809/01

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