Interfacing Nanoparticles to Biological Systems

Nature has created biomolecular machines that function with remarkable efficiency and precision. In recent years, science has attained an unprecedented understanding of the mechanisms of biological “machines.” This has inspired utilization of Nature’s engineering for applications in computation, self-assembly, and mechanics. Because biomolecules are inherently on the nanoscale, nanotechnology has emerged as an appropriate means for controlling biology. This requires both understanding the inorganic properties of the nanoparticle as well as creating an interface that is compatible with the complex and highly disordered environments of real biological systems. We will discuss the use of nanoparticles composed of Au, Fe3O4, Fe2O3, CoFe2O4, and similar materials in biological applications by engineering both the inorganic properties of the nanoparticles along with creating optimal biological interfaces. We study the interface between the nanoparticle and covalently linked proteins and DNA. Labeling proteins with nanoparticles has been utilized for many applications but often the structure of the protein in the conjugate is not characterized. In addition, site-specific labeling of the protein with a nanoparticle has been achieved for only a limited set of proteins and nanoparticles. We present work in which we study the interface between nanoparticles and the protein cytochrome c. We vary nanoparticle ligand and composition, as well as labeling site on the protein. Biophysical techniques such as quantitative gel electrophoresis, circular dichroism, and optical spectroscopy are used to characterize the structure of the protein in the conjugate. These experiments allow us to understand some of the chemical interactions involved in non-specific adsorption, and come up with general design rules for optimal conjugation

Release mechanism of octadecyl rhodamine B chloride from Au nanorods by ultrafast laser pulses

We investigated the release of octadecyl rhodamine B chloride (R[subscript 18]) loaded onto cetyltrimethylammonium bromide (CTAB) coated gold nanorods (NR) by pulsed ultrafast laser excitation. R[subscript 18] intercalates into the hydrophobic CTAB bilayer on the NR surface and can exchange on and off the NR with free CTAB micelles in solution. We find that laser excitation accelerates the rate of both R[subscript 18] release from the NR and R[subscript 18] binding to the NR with increasing fluence. However, at laser fluences >220 μJ/cm[superscript 2] thermal degradation of the R[subscript 18] dominates. We also find that the concentration of CTAB, particularly around the critical micelle concentration, strongly influences the release and binding rates

Protein Thin Film Machines

We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fuelled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model

Extinction Coefficient of Gold Nanostars

Gold nanostars (NStars) are highly attractive for biological applications due to their surface chemistry, facile synthesis, and optical properties. Here, we synthesize NStars in HEPES buffer at different HEPES/Au ratios, producing NStars of different sizes and shapes and therefore varying optical properties. We measure the extinction coefficient of the synthesized NStars at their maximum surface plasmon resonances (SPRs), which range from 5.7 × 10⁸ to 26.8 × 10⁸ M⁻¹ cm⁻¹. Measured values correlate with those obtained from theoretical models of the NStars using the discrete dipole approximation (DDA), which we use to simulate the extinction spectra of the nanostars. Finally, because NStars are typically used in biological applications, we conjugate DNA and antibodies to the NStars and calculate the footprint of the bound biomolecules.United States. National Institutes of Health (AI100190

Designing Paper-Based Immunoassays for Biomedical Applications

Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture

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

Site-directed nanoparticle labeling of cytochrome c

Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein–nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol–Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N- and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle–protein conjugates

Rapid Diagnostics for Infectious Disease using Noble Metal Nanoparticles

Rapid point-of-care (POC) diagnostic devices are needed for field-forward screening of severe acute systemic febrile illnesses such as dengue, Ebola, chikungunya, and others. Multiplexed rapid lateral flow diagnostics have the potential to distinguish among multiple pathogens, thereby facilitating diagnosis and improving patient care. We present a platform for multiplexed pathogen detection which uses gold or silver nanoparticles conjugated to antibodies to sense the presence of biomarkers for different infectious diseases. We exploit the size-dependent optical properties of Ag NPs to construct a multiplexed paperfluidic lateral flow POC sensor. AgNPs of different sizes were conjugated to antibodies that bind to specific biomarkers. Red AgNPs were conjugated to antibodies that could recognize the glycoprotein for Ebola virus, green AgNPs to those that could recognize nonstructural protein 1 for dengue virus, and orange AgNPs for non structural protein 1 for yellow fever virus. Presence of each of the biomarkers resulted in a different colored band on the test line in the lateral flow test. Thus, we were able to use NP color to distinguish among three pathogens that cause a febrile illness. Because positive test lines can be imaged by eye or a mobile phone camera, the approach is adaptable to low-resource, widely deployable settings. This design requires no external excitation source and permits multiplexed analysis in a single channel, facilitating integration and manufacturing. We will also discuss engineering the nanoparticle physical properties and surface chemistry for improving detection and also optimizing device properties, and expansion of the device to detect other diseases

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

Death from mantle cell lymphoma limits sequential therapy, particularly after first relapse: Patterns of care and outcomes in a series from Australia and the United Kingdom

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma characterised by a heterogeneous clinical course. Patients can often receive sequential treatments, yet these typically yield diminishing periods of disease control, raising questions about optimal therapy sequencing. Novel agents, such as chimeric antigen receptor T-cell therapies and bispecific antibodies, show promise in relapsed MCL, but are often reserved for later treatment lines, which may underserve patients with aggressive disease phenotypes who die early in the treatment journey. To assess the problem of patient attrition from lymphoma-related death limiting sequential treatment, we performed a multicentre retrospective cohort analysis of 389 patients treated at Australian and UK centres over a 10-year period. Deaths from MCL increased after each treatment line, with 7%, 23% and 26% of patients dying from uncontrolled MCL after first, second and third lines respectively. Patients with older age at diagnosis and early relapse after induction therapy were at particular risk of death after second-line treatment. This limitation of sequential treatment by lymphoma-related death provides support for the trial of novel therapies in earlier treatment lines, particularly in high-risk patient populations