Antibody linking to atomic force microscope tips via disulfide bond formation.
ABSTRACT 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.
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ABSTRACT: A new strategy for AFM (atomic force microscopy) tip functionalization based on the use of aldehyde-phosphorus dendrimers for the immobilization of biomolecules such as proteins (e.g. antibodies) is presented. Firstly functionalized with amino groups, the tips are reacted with dendrimers leading to dendrimer-activated tips (so-called dendritips). Free aldehyde functions on the dendrimer are therefore available to react with amino-functions present on every protein and many biomolecules. Using biofunctionalized-dendritip, single molecule force interactions between glutathione-S-transferase (GST) and its cognate antibody immobilized on dendritips (67 ± 11 pN for single interaction) were probed by AFM spectroscopy. The specificity of our measurements was demonstrated by performing blocking tests resulting in the loss of interactions.Sensors and Actuators B Chemical 06/2012; 168:436–441. · 3.84 Impact Factor
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ABSTRACT: Atomic Force Microscopy (AFM) has been extensively used in studies of biological interactions. Particularly, AFM based force spectroscopy and recognition imaging can sense biomolecules on a single molecule level, having great potential to become a tool for molecular diagnostics in clinics. These techniques, however, require affinity molecules to be attached to AFM tips in order to specifically detect their targets. The attachment chemistry currently used on silicon tips involves multiple steps of reactions and moisture sensitive chemicals, such as (3-aminopropyl)triethoxysilane (APTES) and N-hydroxysuccinimide (NHS) ester, making the process difficult to operate in aqueous solutions. In the present study, we have developed a user-friendly protocol to functionalize the AFM tips with affinity molecules. A key feature of it is that all reactions are carried out in aqueous solutions. In summary, we first synthesized a molecular anchor composed of cyclooctyne and silatrane for introduction of a chemically reactive function to AFM tips and a bi-functional polyethylene glycol linker that harnesses two orthogonal click reactions, copper free alkyne-azide cycloaddition and thiol-vinylsulfone Michael addition, for attaching affinity molecules to AFM tips. The attachment chemistry was then validated by attaching anti-thrombin DNA aptamers and cyclo-RGD peptides to silicon nitride (SiN) tips respectively, and measuring forces of unbinding these affinity molecules from their protein cognates human α-thrombin and human α5β1-integrin immobilized on mica surfaces. In turn, we used the same attachment chemistry to functionalize silicon tips with the same affinity molecules for AFM based recognition imaging, showing that the disease-relevant biomarkers such as α-thrombin and α5β1-integrin can be detected with high sensitivity and specificity by the single molecule technique. These studies demonstrate the feasibility of our attachment chemistry for the use in functionalization of AFM tips with affinity molecules.Langmuir 11/2013; · 4.38 Impact Factor
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ABSTRACT: Introduction: The therapeutic effects of medicinal drugs not only depend on their properties, but also on effective transport to the target receptor. Here we highlight recent developments in this discipline and show applications of atomic force microscopy (AFM) that enable us to track the effects of drugs and the effectiveness of nanoparticle delivery at the single molecule level. Areas covered: Physiological AFM imaging enables visualization of topographical changes to cells as a result of drug exposure and allows observation of cellular responses that yield morphological changes. When we upgrade the regular measuring tip to a molecular biosensor, it enables investigation of functional changes at the molecular level via single molecule force spectroscopy. Expert opinion: Biosensing AFM techniques have generated powerful tools to monitor drug delivery in (living) cells. While technical developments in actual AFM methods have simplified measurements at relevant physiological conditions, understanding both the biological and technical background is still a crucial factor. However, due to its potential impact, we expect the number of application-based biosensing AFM techniques to further increase in the near future.Expert Opinion on Drug Delivery 05/2014; · 4.87 Impact Factor