DNA-modified gold nanoparticles (AuNPs) are useful signal-reporters for detecting diverse molecules through various hybridization- and enzyme-based assays. However, their performance is heavily dependent on the probe DNA surface coverage, which can influence both target binding and enzymatic processing of the bound probes. Current methods used to adjust the surface coverage of DNA-modified AuNPs require the production of multiple batches of AuNPs under different conditions, which is costly and laborious. We here develop a single-step assay utilizing dithiothreitol (DTT) to fine-tune the surface coverage of DNA-modified AuNPs. DTT is superior to the commonly used surface diluent, mercaptohexanol, as it is less volatile, allowing for the rapid and reproducible controlling of surface coverage on AuNPs with only micromolar concentrations of DTT. Upon adsorption, DTT forms a dense monolayer on gold surfaces, which provides antifouling capabilities. Furthermore, surface-bound DTT adopts a cyclic conformation, which reorients DNA probes into an upright position and provides ample space to promote DNA hybridization, aptamer assembly, and nuclease digestion. We demonstrate the effects of surface coverage on AuNP-based sensors using DTT-regulated DNA-modified AuNPs. We then use these AuNPs to visually detect DNA and cocaine in colorimetric assays based on enzyme-mediated AuNP aggregation. We determine that DTT-regulated AuNPs with lower surface coverage achieve shorter reaction times and lower detection limits relative to those for assays using untreated AuNPs or DTT-regulated AuNPs with high surface coverage. Additionally, we demonstrate that our DTT-regulated AuNPs can perform cocaine detection in 50% urine without any significant matrix effects. We believe that DTT regulation of surface coverage can be broadly employed for optimizing DNA-modified AuNP performance for use in biosensors as well as drug delivery and therapeutic applications.

DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds—a universal feature of DNA-modified particles—to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.

We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout and demonstrate its value for monitoring NAD+-driven enzymatic reactions. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in a Au3+-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer sandwiched between two plastic cover layers. In the absence of NADH, the Au3+-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au3+ is rapidly reduced to Au+, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 μL sample to a surface-confined test zone to achieve a detection limit of 12.5 μM NADH. We used the enzyme glucose dehydrogenase as a model to demonstrate that our paper-based device can monitor NAD+-driven biochemical processes with and without selective dehydrogenase inhibitors by naked-eye observation within 4 min at room temperature in a small sample volume. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD+-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.

Thin gold films offer intriguing material properties for potential applications including fuel cells, supercapacitors, and electronic and photonic devices. We describe here an ambient filtration method that provides a simple and novel way to generate rapidly porous and thin gold films without the need for sophisticated instruments, clean-room environments, and any postgrowth process or sintering steps. Using this approach, we can fabricate highly conductive gold films composed of gold nanoparticles layered atop a matrix of metallic single-walled carbon nanotubes on mixed cellulose ester filter paper within 20 min. These hybrid films (thickness ∼40 nm) exhibit fast electron transfer and excellent electrocatalytic properties that are similar to purchased gold films, but with a larger electroactive surface that lends itself to more sensitive analyte detection. We used the neurotransmitters dopamine and serotonin as benchmark analytes to demonstrate that our hybrid gold films can clearly discriminate the presence of both molecules in a mixture with resolution that greatly exceeds that of either purchased gold slides or electrodeposited gold films. Importantly, we postulate that this new approach could readily be generalized for the rapid fabrication of films from various other metals under ambient conditions, and could also be used as a prelude to transferring the resulting films onto glass or other flexible substrates.

Single nucleotide polymorphism (SNP) detection is important for early diagnosis, clinical prognostics, and disease prevention, and a rapid and sensitive low-cost SNP detection assay would be valuable for resource-limited clinical settings. We present a simple platform that enables sensitive, naked-eye detection of SNPs with minimal reagent and equipment requirements at room temperature within 15 min. SNP detection is performed in a single tube with one set of DNA probe-modified gold nanoparticles (AuNPs), a single exonuclease (Exo III), and the target in question. Exo III’s apurinic endonucleolytic activity differentially processes hybrid duplexes between the AuNP-bound probe and DNA targets that are perfectly matched or contain a single-base mismatch. For perfectly matched targets, Exo III’s exonuclease activity facilitates a process of target recycling that rapidly shears DNA probes from the particles, generating an AuNP aggregation-induced color change, whereas no such change occurs for mismatched targets. This color change is easily observed with as little as 2 nM of target, 100-fold lower than the target concentration required for reliable naked eye observation with unmodified AuNPs in well-optimized reaction conditions. We further demonstrate that this system can effectively discriminate a range of different mismatches.