Application of biotin-4-fluorescein in homogeneous fluorescence assays for avidin, streptavidin, and biotin or biotin derivatives.

Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 microM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 microM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 microg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution.

Protein-resistant self-assembled monolayers on gold with latent aldehyde functions.

In the present study, oligo(ethylene glycol) (OEG)-linked alkanethiols were synthesized which carry a vicinal diol on one end of the OEG chain. After self-assembled monolayer (SAM) formation on gold, the vicinal diols were converted into aldehyde functions by exposure to aqueous NaIO4, as previously used for SAMs with OEG chains buried in the center of the SAM [Jang et al. Nano Lett. 2003, 3, 691-694]. Mixed SAMs with latent aldehydes on 5% of the OEG termini showed high protein resistance, which greatly slowed the kinetics of protein coupling on the time scale of minutes. Small bioligands (such as biocytin hydrazide) or small heterobifunctional crosslinkers (maleimidopropionyl hydrazide, pyridyldithiopropionyl hydrazide) with hydrazide functions were efficiently bound to the aldehyde functions on the SAM, providing for specific capture of streptavidin or for fast covalent binding of proteins with free thiols or maleimide functions, respectively. In conclusion, OEG-terminated SAMs with latent aldehydes serve as protein-resistant sensor surfaces which are easily functionalized with small ligands or with heterobifunctional crosslinkers to which the bait molecule is attached in a subsequent step.

A new, simple method for linking of antibodies to atomic force microscopy tips.

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.

Self-assembled monolayers with latent aldehydes for protein immobilization.

Aldehyde functions are widely used for immobilization of biomolecules on glass surfaces but have found little attention for biofunctionalization of self-assembled monolayers (SAMs) on gold, due to interference between thiol and aldehyde functions. This problem was recently solved by synthesis of an alkanethiol that carried a vicinal diol group [Jang et al. (2003) Nano Lett. 3, 691-694]. The latter served as a latent aldehyde function that was unmasked by short exposure of the vicinal diol-terminated SAM to aqueous periodate. However, the synthesis of the new vicinal diol-terminated alkane thiol was time-consuming and had an overall yield of approximately 3.5%. In the present study, a general modular strategy was introduced by which SAM components with vicinal diol functions were rapidly synthesized with high yield: this was accomplished by amide bond formation between a SAM-forming carboxylic acid (exemplified by lipoic acid and 16-mercaptohexadecanoic acid) with 3-aminopropane-1,2-diol, using suitable protecting groups. The disulfide or free thiol group afforded SAM formation on gold and, after periodate oxidation of the vicinal diol functions, proteins were covalently bound via their lysine residues. At 1 mg/mL protein concentration, complete surface coverage was reached within minutes. No further protein was bound by nonspecific adsorption, but cognate proteins were specifically bound with high capacity. Pyrogallol-O-hexadecanoic acid and 10-undecenoic acid were also coupled with 3-aminopropane-1,2-diol by amide bond formation, thereby producing latent aldehyde-containing SAM components for metal oxides and hydrogen-terminated silicon, respectively, to show the general usefulness of the new synthetic design.

Antibody linking to atomic force microscope tips via disulfide bond formation.

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.