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Attaching single biomolecules selectively to the apex of AFM tips for measuring specific interactions.

ABSTRACT: We present a general approach for preparing well-defined AFM tips for probing single target molecules. We demonstrated that carboxylic acid groups could be generated by electrochemical oxidation selectively at the apex of an AFM tip that is coated with a monolayer of oligo(ethylene glycol) derivatives for resisting nonspecific interactions. These carboxylic acid groups were used as handles to tether only one ligand molecule, such as biotin, to the tip apex for measurement of specific interactions with biomolecules.

SUBMITTER: Gu J

PROVIDER: S-EPMC1366859 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Attaching single biomolecules selectively to the apex of AFM tips for measuring specific interactions.

Gu Jianhua J Xiao Zhongdang Z Yam Chi-Ming CM Qin Guoting G Deluge Maxence M Boutet Sabine S Cai Chengzhi C

Biophysical journal 20050930 5

We present a general approach for preparing well-defined AFM tips for probing single target molecules. We demonstrated that carboxylic acid groups could be generated by electrochemical oxidation selectively at the apex of an AFM tip that is coated with a monolayer of oligo(ethylene glycol) derivatives for resisting nonspecific interactions. These carboxylic acid groups were used as handles to tether only one ligand molecule, such as biotin, to the tip apex for measurement of specific interaction ...[more]

PMID: 16199511

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Project description:The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH(2) groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH(2) groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker ("acetal-PEG-NHS") which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1-10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker ("aldehyde-PEG-NHS") to adjacent NH(2) groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer.

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Attaching single biomolecules selectively to the apex of AFM tips for measuring specific interactions.

Publications

Attaching single biomolecules selectively to the apex of AFM tips for measuring specific interactions.

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