Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

American Chemical Society full text link American Chemical Society Free PMC article
Full text links

Actions

Share

.2012 Jun 5;84(11):4907-14.
doi: 10.1021/ac3001622. Epub 2012 May 15.

High density single-molecule-bead arrays for parallel single molecule force spectroscopy

Affiliations

High density single-molecule-bead arrays for parallel single molecule force spectroscopy

Michael J Barrett et al. Anal Chem..

Abstract

The assembly of a highly parallel force spectroscopy tool requires careful placement of single-molecule targets on the substrate and the deliberate manipulation of a multitude of force probes. Since the probe must approach the target biomolecule for covalent attachment, while avoiding irreversible adhesion to the substrate, the use of polymer microspheres as force probes to create the tethered bead array poses a problem. Therefore, the interactions between the force probe and the surface must be repulsive at very short distances (<5 nm) and attractive at long distances. To achieve this balance, the chemistry of the substrate, force probe, and solution must be tailored to control the probe-surface interactions. In addition to an appropriately designed chemistry, it is necessary to control the surface density of the target molecule in order to ensure that only one molecule is interrogated by a single force probe. We used gold-thiol chemistry to control both the substrate's surface chemistry and the spacing of the studied molecules, through binding of the thiol-terminated DNA and an inert thiol forming a blocking layer. For our single molecule array, we modeled the forces between the probe and the substrate using DLVO theory and measured their magnitude and direction with colloidal probe microscopy. The practicality of each system was tested using a probe binding assay to evaluate the proportion of the beads remaining adhered to the surface after application of force. We have translated the results specific for our system to general guiding principles for preparation of tethered bead arrays and demonstrated the ability of this system to produce a high yield of active force spectroscopy probes in a microwell substrate. This study outlines the characteristics of the chemistry needed to create such a force spectroscopy array.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Representation of a force profile for a probe (microsphere) approaching a surface. To bind to the DNA molecule, the probe must interact with the amine terminal group by passing into the area represented by the blue-gray shaded box. If the probe comes in contact with the surface, it will most likely adhere strongly, so it must not pass completely through the region indicated by a shaded box. The graph shows three representative force-distance profiles as the probe approaches the surface. The repulsive electrostatic force can be too strong and prevent the probe from approaching the target molecule. Conversely, the van der Waals attractive forces can be too strong and cause the probe to stick irreversibly to the surface. If the forces become balanced, the probe will be attracted to the point where it may bind to the DNA, but is repelled at a very close proximity to the surface.
Figure 2
Figure 2
The effects of tuning the parameters of the system based on DLVO interactions between a polymer bead (4.5 μm diameter) and an organic layer (thiol monolayer) on gold in water. The zero distance is set at the organic layer/water interface. The black curves in each graph represent a standard set of conditions, where ionic strength = 100 mM, zeta potential = 35 mV, blocking layer thickness = 2 nm, the blocking layer-water-probe Hamaker constant = 2 zJ, and the substrate-water-probe Hamaker constant of 15 zJ. The effects of zeta potential (a), ionic strength (b), substrate-water-probe Hamaker constant (c), and the blocking layer thickness (d) were evaluated.
Figure 3
Figure 3
The effects of changing the pH with a constant ionic strength of 1 mM on an MHA surface, (a, b, c), the solutions ionic strength with a constant pH of 7 on an MHA surface, (d, e, f), and the surface chemistry with constant pH of 7 and ionic strength of 1 mM (g, h, i) on the electrostatic forces. The measured force profiles are shown as an AFM cantilever lowers a probe towards a surface under various conditions (a, d, g). The pH (a), ionic strength (d), and the surface chemistry (g) are labeled on each plot. From these force profiles, electrostatic properties, such as Debye length and the surface potential of the probe and substrate, are measured by fitting to DLVO theory (b, e, h). In b, the data is plotted with the measured zeta potential of the probes only (blue crosses) and shown with the fit of the zeta potential (blue dotted line) to expected pH titration curve. Finally the fraction of remaining probes on the surface after a magnet is applied is shown for all the conditions measured (c, f, i).
Figure 4
Figure 4
AFM measurements of van der Waals interactions. (a) Representative force curves for the bead approaching substrates with blocking layers of varying thicknesses. The Hamaker constants for the probe attraction with the gold for each thickness (b) are calculated from the data in (a). The results of the probe binding assays (c) show the levels of adhesion for the different thicknesses in a series of carboxyl-terminated SAMs (formed by HS-(CH2)n-COOH, n=2, 5, 10, 15) at pH 3.
Figure 5
Figure 5
Force spectroscopy of ssDNA conducted using DEP tweezers on a system optimized using design principles based on results from this paper. Force probes tethered to DNA in wells before (a) and after (b) they were exposed to a DEP force. Free force probes settled in wells before (c) and after (d) the DEP force was applied. The brightness of the probes (a–d) varies due to Gaussian profile of the illumination laser beam and variations in the bead sizes. (e) A single frame from a movie of the probes, showing the indexing of the force probes for data analysis. (f) A representative intensity vs. voltage plot for one probe. (g) Fits of the data from the plot in part f.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Mukhopadhyay R. Analytical Chemistry. 2009;81:1736. - PubMed
    1. Fuller CW, Middendorf LR, Benner SA, Church GM, Harris T, Huang X, Jovanovich SB, Nelson JR, Schloss JA, Schwartz DC, Vezenov DV. Nat Biotechnol. 2009;27:1013. - PubMed
    1. Oliver PM, Park JS, Vezenov D. Nanoscale. 2010;3:581. - PMC - PubMed
    1. Song L, Ahn S, Walt DR. Analytical Chemistry. 2006;78:1023. - PubMed
    1. Nelson PC, Zurla C, Brogioli D, Beausang JF, Finzi L, Dunlap D. The Journal of Physical Chemistry B. 2006;110:17260. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
American Chemical Society full text link American Chemical Society Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp