Weston L. Daniel

Gold nanoparticles for biology and medicine.

Gold colloids have fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications.

Colorimetric Nitrite and Nitrate detection with gold nanoparticle probes and kinetic end points

We report the development of a novel colorimetric nitrite and nitrate ion assay based upon gold nanoparticle probes functionalized with Griess reaction reagents. This assay takes advantage of the distance-dependent plasmonic properties of the gold nanoparticles and the ability of nitrite ion to facilitate the cross coupling of novel nanoparticle probes modified with aniline and naphthalene moieties. The assay works on the concept of a kinetic end point and can be triggered at the EPA limit for this ion in drinking water (highlighted in red, microM). This rapid and simple assay could be useful for on-site water quality monitoring.

Colorimetric Cu2+ detection using DNA-modified gold-nanoparticle aggregates as probes and click chemistry

Copper is a transition metal essential for life. At elevated concentrations, however, it is highly toxic to organisms such as algae, fungi and many bacteria, and in humans, may adversely affect the gastrointestinal, hepatic and renal systems.[1, 2] As such, the detection and measurement of copper ions in water has become increasingly important, especially in point-of-use formats. Several methods exist for the detection of Cu2+ ions, including those based on organic dyes,[3–6] semiconductor nanocrystals,[7] and spectroscopy.[8–10] The read out mechanisms for these methods, however, often require sophisticated instrumentation. In contrast, colorimetric methods are extremely attractive for point-of-use applications, since they can be easily interpreted with the naked eye or low-cost portable instruments.

Scavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nanoparticles

Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.

Multiplexed nanoflares: mRNA detection in live cells.

We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

Microarray-based multiplexed scanometric immunoassay for protein cancer markers using gold nanoparticle probes.

We report the use of electroless gold deposition as a light scattering signal enhancer in a multiplexed, microarray-based scanometric immunoassay using gold nanoparticle probes. The use of gold development results in greater signal enhancement than the typical silver development, and multiple rounds of metal development were found to increase the resulting signal compared to one development. Using these conditions, the assay was capable of detecting 300 aM (approximately 9000 copies) of prostate specific antigen in buffer and 3 fM in 10% serum. Additionally, the highly selective detection of three protein cancer markers at low picomolar concentrations in buffer and 10% serum was demonstrated. The use of gold deposition may have significant utility in scanometric detection schemes and broader clinical and research applications.

Scanometric microRNA array profiling of prostate cancer markers using spherical nucleic acid-gold nanoparticle conjugates.

We report the development of a novel Scanometric MicroRNA (Scano-miR) platform for the detection of relatively low abundance miRNAs with high specificity and reproducibility. The Scano-miR system was able to detect 1 fM concentrations of miRNA in serum with single nucleotide mismatch specificity. Indeed, it provides increased sensitivity for miRNA targets compared to molecular fluorophore-based detection systems, where 88% of the low abundance miRNA targets could not be detected under identical conditions. The application of the Scano-miR platform to high density array formats demonstrates its utility for high throughput and multiplexed miRNA profiling from various biological samples. To assess the accuracy of the Scano-miR system, we analyzed the miRNA profiles of samples from men with prostate cancer (CaP), the most common noncutaneous malignancy and the second leading cause of cancer death among American men. The platform exhibits 98.8% accuracy when detecting deregulated miRNAs involved in CaP, which demonstrates its potential utility in profiling and identifying clinical and research biomarkers.

Multiplexed Protein Arrays Enabled by Polymer Pen Lithography: Addressing the Inking Challenge

The ability to fabricate protein micro and nano arrays in a low-cost and high throughput manner is important for a wide variety of applications, including drug screening, materials assembly, medical diagnostics, biosensors, and fundamental biological studies.[1-3] Traditional approaches to making protein microarrays include photolithography and inkjet printing. Recently, studies also have focused on the miniaturization of protein patterns into the nanometer regime because high density protein arrays can provide increased detection sensitivity and, in principle, allow one to screen millions of biomarkers with one chip.[4] Protein nanopatterns also can provide insight into important fundamental biological processes,[5] such as cell adhesion and differentiation.[6-9] Indeed, the ability to place an array of proteins or even multiple protein structures underneath a single cell opens up the opportunity to study multivalent interactions between a cell and a surface, and points to a major capability of nanoarray technology not afforded by analogous microscale structures. Herein, we report a novel and rapid strategy for inking nanoscale probes with different proteins, which can be transferred to a surface via the technique known as Polymer Pen Lithography (PPL).[10] Using this approach, we have generated sub-100 nm structures at a rate of 150,000 features per second.

A new approach to amplified telomerase detection with polyvalent oligonucleotide nanoparticle conjugates.

We report a new assay for human telomerase activity that relies on polyvalent oligonucleotide nanoparticle conjugates as diagnostic probes and amplification units. Gold nanoparticles functionalized with specific oligonucleotide sequences can efficiently capture telomerase enzymes and subsequently be elongated. Both the elongated and unmodified oligonucleotide sequences are simultaneously measured. The two strands not only serve as internal positive controls for each other but also provide a way of amplifying signal. At high concentrations, both elongated and unmodified strands exhibit measurable responses. At low telomerase concentrations (e.g., from 10 HeLa cells), elongated strands cannot be detected, but the unmodified sequences, which come from the same probe particles, can be detected because their concentration is higher, providing a novel form of amplification. This new assay rivals the sensitivity of the conventional PCR-based method of telomerase detection.

Templated Spherical High Density Lipoprotein Nanoparticles

We report the synthesis of high density lipoprotein (HDL) biomimetic nanoparticles capable of binding cholesterol. These structures use a gold nanoparticle core to template the assembly of a mixed phospholipid layer and the adsorption of apolipoprotein A-I. These synthesized structures have the general size and surface composition of natural HDL and, importantly, bind free cholesterol (K(d) = 4 nM). The determination of the K(d) for these particles, with respect to cholesterol complexation, provides a key starting and comparison point for measuring and evaluating the properties of subsequently developed synthetic versions of HDL.