A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays.

Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.

Multicolored silver nanoparticles for multiplexed disease diagnostics: distinguishing dengue, yellow fever, and Ebola viruses.

Rapid point-of-care (POC) diagnostic devices are needed for field-forward screening of severe acute systemic febrile illnesses. Multiplexed rapid lateral flow diagnostics have the potential to distinguish among multiple pathogens, thereby facilitating diagnosis and improving patient care. Here, we present a platform for multiplexed pathogen detection using multi-colored silver nanoplates. This design requires no external excitation source and permits multiplexed analysis in a single channel, facilitating integration and manufacturing.

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 (SPR), which range from 5.7 × 108 to 26.8 × 108 M-1cm-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.

Selective targeting of antibody conjugated multifunctional nanoclusters (nanoroses) to epidermal growth factor receptors in cancer cells.

The ability of smaller than 100 nm antibody (Ab) nanoparticle conjugates to target and modulate the biology of specific cell types may enable major advancements in cellular imaging and therapy in cancer. A key challenge is to load a high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A versatile method called thin autocatalytic growth on substrate (TAGs) has been developed in our previous study to form ultrathin and asymmetric gold coatings on iron oxide nanocluster cores producing exceptional near-infrared (NIR) absorbance. AlexaFluor 488 labeled Abs were used to correlate the number of Abs conjugated to iron oxide/gold nanoclusters (nanoroses) with the hydrodynamic size. A transition from submonolayer to multilayer aggregates of Abs on the nanorose surface was observed for 54 Abs and an overall particle diameter of ∼60-65 nm. The hydrodynamic diameter indicated coverage of a monolayer of 54 Abs, in agreement with the prediction of a geometric model, by assuming a circular footprint of 16.9 nm diameter per Ab molecule. The targeting efficacy of nanoclusters conjugated with monoclonal Abs specific for epidermal growth factor receptor (EGFR) was evaluated in A431 cancer cells using dark field microscopy and atomic absorbance spectrometry (AAS) analysis. Intense NIR scattering was achieved from both high uptake of nanoclusters in cells and high intrinsic NIR absorbance of individual nanoclusters. Dual mode imaging with dark field reflectance microscopy and fluorescence microscopy indicates the Abs remained attached to the Au surfaces upon the uptake by the cancer cells. The ability to load intense multifunctionality, specifically strong NIR absorbance, conjugation of an Ab monolayer in addition to a strong r2 MRI contrast that was previously demonstrated in a total particle size of only 63 nm, is an important step forward in development of theranostic agents for combined molecular specific imaging and therapy.

Utility of biodegradable plasmonic nanoclusters in photoacoustic imaging.

Plasmonic metal nanoparticles are used in photoacoustic imaging as contrast agents because of their resonant optical absorption properties in the visible and near-IR regions. However, the nanoparticles could accumulate and result in long-term toxicity in vivo, because they are generally not biodegradable. Recently, biodegradable plasmonic gold nanoclusters, consisting of sub-5 nm primary gold nanoparticles and biodegradable polymer stabilizer, were introduced. In this Letter, we demonstrate the feasibility of biodegradable nanoclusters as a photoacoustic contrast agent. We performed photoacoustic and ultrasound imaging of a tissue-mimicking phantom with inclusions containing nanoclusters at various concentrations. The results indicate that the biodegradable gold nanoclusters can be used as effective contrast agents in photoacoustic imaging.

Controlled assembly of biodegradable plasmonic nanoclusters for near-infrared imaging and therapeutic applications.

Metal nanoparticles with surface plasmon resonance (SPR) in the near-infrared region (NIR) are of great interest for imaging and therapy. Presently, gold nanoparticles with NIR absorbance are typically larger than 50 nm, above the threshold size of approximately 5 nm required for efficient renal clearance. As these nanoparticles are not biodegradable, concerns about long-term toxicity have restricted their translation into the clinic. Here, we address this problem by developing a flexible platform for the kinetically controlled assembly of sub-5 nm ligand-coated gold particles to produce metal/polymer biodegradable nanoclusters smaller than 100 nm with strong NIR absorbance for multimodal application. A key novel feature of the proposed synthesis is the use of weakly adsorbing biodegradable polymers that allows tight control of nanocluster size and, in addition, results in nanoclusters with unprecedented metal loadings and thus optical functionality. Over time, the biodegradable polymer stabilizer degrades under physiological conditions that leads to disassembly of the nanoclusters into sub-5 nm primary gold particles which are favorable for efficient body clearance. This synthesis of polymer/inorganic nanoclusters combines the imaging contrast and therapeutic capabilities afforded by the NIR-active nanoparticle assembly with the biodegradability of a polymer stabilizer.

Small multifunctional nanoclusters (nanoroses) for targeted cellular imaging and therapy.

The ability of 20-50 nm nanoparticles to target and modulate the biology of specific types of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. Herein we report approximately 30 nm stable uniformly sized near-infrared (NIR) active, superparamagnetic nanoclusters formed by kinetically controlled self-assembly of gold-coated iron oxide nanoparticles. The controlled assembly of nanocomposite particles into clusters with small primary particle spacings produces collective responses of the electrons that shift the absorbance into the NIR region. The nanoclusters of approximately 70 iron oxide primary particles with thin gold coatings display intense NIR (700-850 nm) absorbance with a cross section of approximately 10(-14) m(2). Because of the thin gold shells with an average thickness of only 2 nm, the r(2) spin-spin magnetic relaxivity is 219 mM(-1) s(-1), an order of magnitude larger than observed for typical iron oxide particles with thicker gold shells. Despite only 12% by weight polymeric stabilizer, the particle size and NIR absorbance change very little in deionized water over 8 months. High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast in dark field and hyperspectral microscopy, both in cell culture and an in vivo rabbit model of atherosclerosis. Small nanoclusters with optical, magnetic, and therapeutic functionality, designed by assembly of nanoparticle building blocks, offer broad opportunities for targeted cellular imaging, therapy, and combined imaging and therapy.