Bifunctional nanoarrays for probing the immune response at the single-molecule level

Cai, Haogang; Depoil, David; Palma, Matteo; Sheetz, Michael P.; Dustin, Michael L.; Wind, Shalom J.

doi:10.1116/1.4823764

Cited by 11 publications

(30 citation statements)

References 18 publications

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“…The surrounding surface was functionalized with a his-tag ICAM-1 linked to a PEG-silane via a nickel-NTA bridge to facilitate binding of LFA-1, which enhances cell adhesion. 37 Figure 5a schematically shows a live T-cell on this bifunctional surface. Bleaching analysis of 1 μ m spaced heptamer arrays revealed that nanodots ( D = 7.5 nm, x = 50%) had a Fab′ occupancy of ∼1 ( N streptavidin = 0.92 ± 0.28; N Fab′ = 0.98 ± 0.58, Supporting Information Figure S4), albeit with a higher coefficient of variation (CV) than streptavidin.…”

Section: Resultsmentioning

confidence: 99%

“…In the case of T-cell assays, 37 1 mg silane-PEG-NHS (MW 5000, Nanocs) solution in 25 mL anhydrous methanol with 1 mM NTA-L-lysine was incubated overnight to synthesize silane-PEG-NTA, which was used in place of mPEG-silane. After PEGylation, samples were first incubated in 40 mM aqueous NiSO 4 for 1 h to adsorb Ni(II) to the NTA groups, followed by 1 μ g/mL His-tag ICAM-1 solution in PBS for 2 h. Then the samples were incubated in streptavidin solution in PBS for 30 min, followed by 2 μ g/mL biotinylated UCHT1 Fab-568 (F/P 0.45) for another 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…5,28,37,40–42,55 The so-called top-down approaches enable heterogeneous arrays on the same surface, with arbitrary geometry and various dot sizes. This is important for both the molecular occupancy analysis and cell assays, because the integration of various patterns on the same sample enables direct comparison between them, avoiding the variations from sample to sample.…”

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confidence: 99%

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Molecular Occupancy of Nanodot Arrays

Cai¹,

Wolfenson²,

Depoil

et al. 2016

ACS Nano

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Single-molecule nanodot arrays, in which a biomolecule of choice (protein, nucleic acid, etc.) is bound to a metallic nanoparticle on a solid substrate, are becoming an increasingly important tool in the study of biomolecular and cellular interactions. We have developed an on-chip measurement protocol to monitor and control the molecular occupancy of nanodots. Arrays of widely spaced nanodots and nanodot clusters were fabricated on glass surfaces by nanolithography and functionalized with fluorescently labeled proteins. The molecular occupancy was determined by monitoring individual fluorophore bleaching events, while accounting for fluorescence quenching effects. We found that the occupancy can be interpreted as a packing problem, and depends on nanodot size and binding ligand concentration, where the latter is easily adjusted to compensate the flexibility of dimension control in nanofabrication. The results are scalable with nanodot cluster size, extending to large area close packed arrays. As an example, the nanoarray platform was used to probe the geometric requirement of T-cell activation at the single-molecule level.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Molecular Occupancy of Nanodot Arrays

Cai¹,

Wolfenson²,

Depoil

et al. 2016

ACS Nano

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“…22, 23, 49 A 12 nm-thick Ti hard mask was deposited at an angle of 30° by e-beam evaporation (Semicore SC2000); the angled deposition reduces the opening size to sub-15 nm, and creates a negative resist profile suitable for lift-off. The hard mask also protects the unexposed resist in the descumming by oxygen plasma (Oxford PlasmaLab 80).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Thin films of PEG can be covalently bound to glass through silane chemistry 27 using either a two-step process using silane reagents with reactive organic moieties ( e.g. , amines 4,5 , epoxides 28 ), or a one-step formation of a self-assembled monolayer (SAM) of PEG-silane 22–25, 28–31 . Unfortunately, regardless of the recipe used, the PEG film is usually not a uniform, defect-free monolayer.…”

Section: Introductionmentioning

confidence: 99%

Improved Glass Surface Passivation for Single-Molecule Nanoarrays

Cai

Wind

2016

Langmuir

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Single-molecule fluorescence techniques provide a critical tool for probing biomolecular and cellular interactions with unprecedented resolution and precision. Unfortunately, many of these techniques are hindered by a common problem, namely the nonspecific adsorption of target biomolecules. This issue is mostly addressed by passivating the glass surfaces with a poly(ethylene glycol) (PEG) brush. This is effective only at low concentrations of the probe molecule, because there are defects inherent to polymer brushes formed on glass coverslips, due to the presence of surface impurities. Tween-20, a detergent, is a promising alternative that can improve surface passivation, but it is incompatible with living cells, and it also possesses limited selectivity for glass background over metallic nanoparticles, which are frequently used as anchors for the probe molecules. To address these issues, we have developed a more versatile method to improve the PEG passivation. A thin film of hydrogen silsesquioxane (HSQ) is spin-coated and thermally cured on glass coverslips in order to cover the surface impurities. This minimizes the formation of PEG defects and reduces nonspecific adsorption, resulting in an improvement comparable to Tween-20 treatment. This approach was applied to single-molecule nanoarrays of streptavidin bound to AuPd nanodots patterned by e-beam lithography (EBL). The fluorescence signal to background ratio (SBR) on HSQ coated glass was improved by ~ 4-fold as compared to PEG directly on glass. This improvement enables direct imaging of ordered arrays of single molecules anchored to lithographically patterned arrays of metallic nanodots.

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The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

Chang

Kim

Thompson

et al. 2016

Small

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Methods for patterning biomolecules on a substrate at the single molecule level have been studied as a route to sensors with single-molecular sensitivity or as a way to probe biological phenomena at the single-molecule level. However, the arrangement and orientation of single biomolecules on substrates has been less investigated. Here, the arrangement and orientation of two rod-like coiled-coil proteins, cortexillin and tropomyosin, around patterned gold nanostructures is examined. The high aspect ratio of the coiled coils makes it possible to study their orientations and to pursue a strategy of protein orientation via two-point attachment. The proteins are anchored to the surfaces using thiol groups, and the number of cysteine residues in tropomyosin is varied to test how this variation affects the structure and arrangement of the surface-attached proteins. Molecular dynamics studies are used to interpret the observed positional distributions. Based on initial studies of protein attachment to gold post structures, two 31-nm-long tropomyosin molecules are aligned between the two sidewalls of a trench with a width of 68 nm. Because the approach presented in this study uses one of twenty natural amino acids, this method provides a convenient way to pattern biomolecules on substrates using standard chemistry.

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Bifunctional nanoarrays for probing the immune response at the single-molecule level

Cited by 11 publications

References 18 publications

Molecular Occupancy of Nanodot Arrays

Molecular Occupancy of Nanodot Arrays

Improved Glass Surface Passivation for Single-Molecule Nanoarrays

The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

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