ANALYTICAL CURRENTS Antibody nanoarrays Screening for anthrax

It is hard enough to attach functional biomolecules to a microfabricated sur- face, but now David Klenerman and col- leagues at the University of Camb...
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ANALYTICAL CURRENTS Antibody nanoarrays It is hard enough to attach functional biomolecules to a microfabricated surface, but now David Klenerman and colleagues at the University of Cambridge and Imperial College London (both in the U.K.) have gone orders of magnitude smaller and attached antibodies to a nanoscale surface. The approach offers a unique way to create antibody nanoarrays that can address specific features on ultrasmall surfaces. To make the arrays, the researchers fabricated a nanosurface by using a gallium focus ion beam microsope. The surface contained regularly spaced holes within a thin film (50 nm) of gold. The

an electrode in a nanopipette and an ionic solution. The sign and magnitude of the applied voltage were used to control the number of molecules that were delivered. To address each hole individually, the researchers added anti-IgG to the holes containing IgG. They observed green fluorescence from the IgG and red fluorescence from the anti-IgG simultaneously, providing evidence that the antibody–antigen interactions were specific and that a particular feature on a nanosurface can be addressed by adding a second biomolecule. (J. Am. Chem. Soc. 2004, 126, 6508– 6509)

gold film allowed the position of a nanopipette, which was used to deliver biomolecules into the holes, to be viewed by optical microscopy. To resist nonspecific adsorption, the gold surface was coated with a self-assembled monolayer (SAM) that was terminated with hexa(ethylene glycol) groups. The holes were functionalized with a SAM of 3-mercaptopropionic acid, which facilitates the immobilization of IgG antibodies through electrostatic interactions. The researchers delivered IgG antibodies into the holes using a technique in which an ion current flows between

Screening for anthrax inhibitors

(a) 100

Conventional techniques for screening large

to SAMs on a gold-coated

libraries of small compounds use fluorescent

plate. Solutions of anthrax

labels that can interfere with the activities of

lethal factor plus 8 library com-

substrate molecules. High background noise

pounds were spotted onto each

and false positives also can be problems

of the 100 wells on the SAMDI

with fluorescent labeling methods. Milan

plate. When small molecules in

Mrksich and colleagues at the University of

the solution had no effect on

Chicago have abandoned fluorescent labels

the lethal factor, two MS peaks

in favor of MS-based detection. In their ap-

were observed at low m/z val-

proach, self-assembled monolayers (SAMs)

ues, which indicated that the

bound to substrate molecules are adhered

peptide was cleaved. When a

to a MALDI plate, and cleavage of the sub-

small molecule in the spotted

strate is monitored by MALDI TOFMS. The

solution inhibited the cleavage

researchers identified an inhibitor of an-

activity of the lethal factor, two

thrax lethal factor toxin with this SAMs-for-

peaks were observed at high m/z.

MALDI (SAMDI) method.

50 0 1000

2000

3000

4000

Lethal factor

Relative intensity (%)

(b) 100 50 0 1000

2000

3000

4000

m/z

Spectra resulting from (a) inhibition of cleavage and (b) cleavage by anthrax lethal factor. (Adapted with permission. Copyright 2004 Macmillan Publishing Ltd.) centration-dependent manner. The molecule

Using the SAMDI method, Mrksich and

also inhibited cleavage in a solution-based

In the assay, a peptide that can be

colleagues discovered one molecule that

assay and in human cells. (Nat. Biotechnol.

cleaved by anthrax lethal factor is attached

repressed lethal factor cleavage in a con-

2004, 22, 717–723)

© 2004 AMERICAN CHEMICAL SOCIETY

A U G U S T 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y

255 A