Quantifying the nanomachinery of the nanoparticle-biomolecule interface

A study is presented of the nanomechanical phenomena experienced by nanoparticle-conjugated biomolecules. A thermodynamic framework is developed to describe the binding of thrombin-binding aptamer (TBA) to thrombin when the TBA is conjugated to nanorods. Binding results in nanorod aggregation (viz. directed self-assembly), which is detectable by absorption spectroscopy. The analysis introduces the energy of aggregation, separating it into TBA–thrombin recognition and surface-work contributions. Consequently, it is demonstrated that self-assembly is driven by the interplay of surface work and thrombin-TBA recognition. It is shown that the work at the surface is about −10 kJ mol−1 [mol superscript -1] and results from the accumulation of in-plane molecular forces of pN magnitude and with a lifetime of <1 s, which arises from TBA nanoscale rearrangements fuelled by thrombin-directed nanorod aggregation. The obtained surface work can map aggregation regimes as a function of different nanoparticle surface conditions. Also, the thermodynamic treatment can be used to obtain quantitative information on surface effects impacting biomolecules on nanoparticle surfaces.MIT-IQS Exchange Progra

Selective Light-Triggered Release of DNA from Gold Nanorods Switches Blood Clotting On and Off

Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs) to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.National Science Foundation (U.S.) (Grant DMR #0906838

Designing polymer surfaces via vapor deposition

Chemical Vapor Deposition (CVD) methods significantly augment the capabilities of traditional surface modification techniques for designing polymeric surfaces. In CVD polymerization, the monomer(s) are delivered to the surface through the vapor phase and then undergo simultaneous polymerization and thin film formation. By eliminating the need to dissolve macromolecules, CVD enables insoluble polymers to be coated and prevents solvent damage to the substrate. Since de-wetting and surface tension effects are absent, CVD coatings conform to the geometry of the underlying substrate. Hence, CVD polymers can be readily applied to virtually any substrate: organic, inorganic, rigid, flexible, planar, three-dimensional, dense, or porous. CVD methods integrate readily with other vacuum processes used to fabricate patterned surfaces and devices. CVD film growth proceeds from the substrate up, allowing for interfacial engineering, real-time monitoring, thickness control, and the synthesis of films with graded composition. This article focuses on two CVD polymerization methods that closely translate solution chemistry to vapor deposition; initiated CVD and oxidative CVD. The basic concepts underlying these methods and the resultant advantages over other thin film coating techniques are described, along with selected applications where CVD polymers are an enabling technology.Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract DAAD-19-02-D-0002

Patterning Nanodomains with Orthogonal Functionalities: Solventless Synthesis of Self-Sorting Surfaces

A simple method to fabricate a multifunctional patterned platform on the nanometer scale is demonstrated. The platform contains two reactive functional groups on the surface: one is an acetylene group which can be functionalized via click chemistry, and the other is an amine group which can also be functionalized by classic carbodiimide chemistry with N-hydroxysuccinimide (NHS). The click-active and amine surface could be obtained from polymer coating of poly(propargyl methacrylate) (PPMA) via initiated chemical vapor deposition (iCVD) and poly(allylamine) (PAAm) via a plasma polymerization process, respectively, utilizing commercially available monomers. A capillary force lithography (CFL) process was applied on a stacked film of a PPMA layer on PAAm, and CFL could selectively pattern PPMA maintaining the bottom PAAm layer intact, which completes the multifunctional nanopatterns. The minimum feature size of this nanopattern was 110 nm. The entire fabrication process is solventless and low temperature, which can minimize the loss of functionalities. The click and NHS reactions are highly orthogonal to each other so that nonspecific immobilization can be minimized. These advantageous characteristics enable the covalent functionalization of two independent components in a one-pot functionalization process in self-recognized way. The one-pot orthogonal functionalization was performed in an aqueous solution at room temperature, which is biocompatible. Considering the versatility and generality of the reactions used here, we believe this platform can be easily extended to various biodevice applications.close631

TBA and antidote affect coagulation in whole human blood.

<p>a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (<i>t_plasma</i>) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti]  =  0 to 1000 nM (anti/TBA  =  0 to 2.0).</p

Release from NR- and NB-coronas and their comparison to covalently loaded NRs.

<p>a) Absorption spectrum of NB-HS-TBA before (black) and after (red) 1100 nm irradiation, where [NB-HS-anti]  = 0.3 nM, and released [anti]  = 129±5 nM (430±17 anti released/NB). Inset: fluorescence spectrum of released TBA before (black) and after (red) 1100 nm irradiation. b) Absorption spectrum of NR-HS-TBA before (black) and after (red) 800nm irradiation where [NR-HS-TBA]  = 2.9 nM, and released [TBA]  = 663±23 nM (223±8 DNA released/NR). Inset: fluorescence spectrum of released TBA before (black) and after (red) 800 nm irradiation, c) Effect of the released TBA in blood. Comparing normalized <i>t_plasma</i> from released TBA from the coronas [NR-HS-TBA] = 2.9 nM (red), where released [TBA] = 663±23 nM in a clotting test. Supernatant of NR-HS-TBA with exposed to no irradiation and added to blood is defined as <i>t_plasma</i>  = 1.0 (gray dotted line). A significant difference (p≤0.05) from baseline <i>t_plasma</i> is indicated with a * (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068511#pone.0068511.s005" target="_blank">Table S1</a>), d) <i>t_plasma</i> (normalized) calibration curve of free TBA in a thrombin test (stars). Released TBA from NR-HS-TBA (red circle) and extrapolated equivalent concentration (red dashed line). e) <i>t_plasma</i> (normalized) calibration curve of free thiolated TBA (blue X’s). Released thiolated TBA from NR-thiol-TBA (1460±108 nM, blue square) and extrapolated equivalent concentration (blue dashed line).</p

Gold nanoparticles synthesized and loaded for triggered release.

<p>Absorption spectra of a) NRs, NR-HSA-TBA coronas (LSPR max  = 777 nm), b) NB, NB-HSA-antidote (LSPR max  = 1093 nm). c) TEM image of NRs, scale bar  = 20 nm, d) TEM image of NBs, scale bar  = 100 nm, e) <i>D_H</i> (DLS) of NRs, NR-HS-TBA, NBs, NB-HS-antidote, indicating that a corona contains multiple not a single NR or NB, but multiple ones. f) Zeta potential of NRs, NR-HS-TBA  = −9.8 mV, NBs, NB-HS-antidote  = −10.1 mV g) Quantified DNA payloads of NR-HS-TBA (674±74 TBA/NR), NB-HS-antidote (1307±255 antidote/NB). h) mixture of NR-CTAB + NB-CTAB before (black) and after (red) 800 nm irradiation. i) NR-CTAB + NB-CTAB before (black) and after (red) 1100 nm irradiation.</p

Selective melting of NRs and NBs to switch blood clotting off and on.

<p>NR-HS-TBA and NB-HS-antidote were mixed at a ratio so that the TBA:antidote was 1:1. a) NR-HS-TBA + NB-HS-anti mixture before (black) and after 800 nm irradiation (red), after 800 nm and then 1100 nm irradiation (blue) b) fluorescence of released supernatant before (black) and after 800 nm irradiation (red), and after 800 nm+1100 nm irradiation (blue). c) Normalized <i>t_plasma</i> for samples before irradiation (defined as 1.0, and used to compare the statistical parameters of all samples) of mixture of NR-HS-TBA + NB-HS-antidote with excitation at 800 nm (<i>t_plasma</i> increases to 1.73), and 800 nm+1100 nm (<i>t_plasma</i> restored to 0.88), demonstrating restoration of clotting time. 1100 nm irradiation alone of the mixture NR-HS-TBA + NB-HS-anti, showing no significant increase in clotting time. Irradiation at 800 nm of NR-HS (without TBA) + NB-HS-anti showing no increase in clotting time. To test the effect of the presence of the nanoparticles, HS-NR-TBA+NB-HS-anti were not exposed to any irradiation in blood. Significant differences (p≤0.05) from baseline <i>t_plasma</i> are indicated with an * (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068511#pone.0068511.s005" target="_blank">Table S1</a>).</p