A force-based protein biochip

Abstract

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for proteinprotein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Facile Preparation of Complex Protein Architectures with Sub‐100‐nm Resolution on Surfaces

“…This has spurred the development of novel bioinspired materials and of nanofabrication routes. [7][8][9] The ability to control the patterning of proteins is, therefore, not only important for gaining insight into biological phenomena, [10] but is also a prerequisite for highperformance biosensors [11,12] and novel fabrication paradigms. [13] Many approaches have been pursued for patterning proteins on surfaces with high resolution, including dip-pen lithography, [14] microcontact printing, [15][16][17][18][19] self-assembly, [20] ablation of patterns into monolayers of proteins or organic molecules using various techniques, [21] and nanografting based on scanning-probe methods.…”

Mechanically Induced Trapping of Molecular Interactions and Its Applications

“…7 Single-molecule force spectroscopy is a technique used to measure the binding strength between two molecules, which are usually in the order of piconewton forces. 49a Although several techniques exist, such as atomic force microscopy, optical tweezers, and magnetic tweezers, they are time-consuming, extremely low-throughput, costly, and difficult to set up.…”

“…4b Molecularly, the basis for the interaction between two biological molecules is a combination of noncovalent bonds (i.e., electrostatic, van der Waals, and hydrogen) or hydrophobic interactions, 2,5 yet much of the molecular behavior between two biomolecules can be accounted for by two biochemical parameters: the strength of binding (affinity) and the kinetics of reaction (association and dissociation rates). Binding force is a less frequently used parameter, given the technical difficulties to obtain it, 6 but it can provide complementary information such as the specificity of the molecular interactions, 7 and resolve low-and high-affinity interactions. 8 Nevertheless, affinity and binding rates are the principal thermodynamic properties that characterize molecular interactions.…”

“…15 High-throughput sequencingfluorescent ligand interaction profiling (HTS-FLIP) is a new technique that has been developed to study protein-DNA interactions, and it can determine disassociation constants of one transcription factor to millions of DNA targets. 16 RNA on a massive parallel array (RNA-MaP) is another recent highthroughput technique used to measure the binding affinities and disassociation rates of a single, fluorescently labeled protein to >10 7 RNA targets. 17 However, both HTS-FLIP and RNA-Map require a second-generation DNA-sequencing instrument.…”

A high throughput molecular force assay for protein–DNA interactions

“…To solve these issues a new technique, the molecular force assay (MFA), has been introduced by our group. 19,20 The MFA measures unbinding forces with a high sensitivity like single mismatches in DNA 21 and with low-budget and simple instrumentation compared to AFM, optical or magnetic tweezers. Since with the state-of-the-art instrumentation the force resolution is limited only by thermal fluctuations of the force sensor, shrinking the sensor size improves the signal-to-noise ratio.…”