Brendan D. Smith

Regenerable DNA-Functionalized Hydrogels for Ultrasensitive, Instrument-Free Mercury(II) Detection and Removal in Water

Mercury is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, new materials are required to not only detect but also effectively remove mercury from environmental sources such as water. We herein describe a polyacrylamide hydrogel-based sensor functionalized with a thymine-rich DNA that can simultaneously detect and remove mercury from water. Detection is achieved by selective binding of Hg(2+) between two thymine bases, inducing a hairpin structure where, upon addition of SYBR Green I dye, green fluorescence is observed. In the absence of Hg(2+), however, addition of the dye results in yellow fluorescence. Using the naked eye, the detection limit in a 50 mL water sample is 10 nM Hg(2+). This sensor can be regenerated using a simple acid treatment and can remove Hg(2+) from water at a rate of approximately 1 h(-1). This sensor was also used to detect and remove Hg(2+) from samples of Lake Ontario water spiked with mercury. In addition, these hydrogel-based sensors are resistant to nuclease and can be rehydrated from dried gels for storage and DNA protection. Similar methods can be used to functionalize hydrogels with other nucleic acids, proteins, and small molecules for environmental and biomedical applications.

DNA-Functionalized Monolithic Hydrogels and Gold Nanoparticles for Colorimetric DNA Detection

Highly sensitive and selective DNA detection plays a central role in many fields of research, and various assay platforms have been developed. Compared to homogeneous DNA detection, surface-immobilized probes allow washing steps and signal amplification to give higher sensitivity. Previously research was focused on developing glass or gold-based surfaces for DNA immobilization; we herein report hydrogel-immobilized DNA. Specifically, acrydite-modified DNA was covalently functionalized to the polyacrylamide hydrogel during gel formation. There are several advantages of these DNA-functionalized monolithic hydrogels. First, they can be easily handled in a way similar to that in homogeneous assays. Second, they have a low optical background where, in combination with DNA-functionalized gold nanoparticles, even ∼0.1 nM target DNA can be visually detected. By using the attached gold nanoparticles to catalyze the reduction of Ag+, as low as 1 pM target DNA can be detected. The gels can be regenerated by a simple thermal treatment, and the regenerated gels perform similarly to freshly prepared ones. The amount of gold nanoparticles adsorbed through DNA hybridization decreases with increasing gel percentage. Other parameters including the DNA concentration, DNA sequence, ionic strength of the solution, and temperature have also been systematically characterized in this study.

Assembly of DNA-Functionalized Nanoparticles in Alcoholic Solvents Reveals Opposite Thermodynamic and Kinetic Trends for DNA Hybridization

DNA has been a key molecule in biotechnology and nanotechnology. To date, the majority of the experiments involving DNA have been performed in aqueous solutions, which may be related to the perception that DNA hybridization is slower and less stable in organic solvents. All studies on the effect of organic solvents have focused on thermodynamic properties such as DNA melting temperature and the B-to-A form transition for very long DNAs, but not on the hybridization kinetics of short synthetic DNAs. We employed DNA-functionalized gold nanoparticles (AuNPs) as a model system and found that if the alcohol content is less than approximately 30%, more alcohol leads to a faster DNA hybridization, although with a decreased melting temperature. The generality of this observation was independently verified with two molecular beacon systems (in the absence of AuNPs) using fluorophore and quencher-labeled DNAs. With 25% ethanol, the hybridization rates are three to four times faster than in the case with water. This discovery will extend the application of DNA bio- and nanotechnology to organic solvents with improved performance.

Exploring the thermal stability of DNA-linked gold nanoparticles in ionic liquids and molecular solvents

While water is the most commonly used solvent for DNA, many co-solvents have been added for various applications. Ionic liquids (ILs) are molten salts at around room temperature. ILs have been tested as a green solvent for many reactions and many biopolymers can also be dissolved in ILs. In this work, we study DNA-linked gold nanoparticles (AuNPs) in seven types of ILs. DNA-functionalized AuNPs possess a high density of negative charges and thus may generate new physical properties in ILs. We have identified the role of ILs to transit from salts to increase DNA duplex stability to solvents to decrease DNA melting temperature. The onset of this transition depends on the structure of ILs, where more hydrophobic cations destabilize DNA at lower IL concentrations. This trend is opposite to molecular solvents (e.g. ethanol, DMSO, ACN and DMF) that destabilize DNA at low solvent concentration. Specific DNA base pairing is disrupted at high DMSO concentrations, and AuNPs are held together by non-specific interactions. The other tested molecular solvents are able to maintain DNA base pairs, although strong non-specific interactions are also present. Several ILs can release proton and thus drastically change pH, which also changes the melting temperature of DNA. This study also reveals the feasibility of using ILs as solvents for DNA-functionalized nanomaterials.

Catalyst Self-Assembly for Scalable Patterning of Sub 10 nm Ultrahigh Aspect Ratio Nanopores in Silicon

Nanoporous silicon (NPSi) has received significant attention for its potential to contribute to a large number of applications, but has not yet been extensively implemented because of the inability of current state-of-the-art nanofabrication techniques to achieve sufficiently small pore size, high aspect ratio, and process scalability. In this work we describe the fabrication of NPSi via a modified metal-assisted chemical etching (MACE) process in which silica-shell gold nanoparticle (SiO2-AuNP) monolayers self-assemble from solution onto a silicon substrate. Exposure to the MACE etchant solution results in the rapid consumption of the SiO2 spacer shell, leaving well-spaced arrays of bare AuNPs on the substrate surface. Particles then begin to catalyze the etching of nanopore arrays without interruption, resulting in the formation of highly anisotropic individual pores. The excellent directionality of pore formation is thought to be promoted by the homogeneous interparticle spacing of the gold core nanocatalysts, which allow for even hole injection and subsequent etching along preferred crystallographic orientations. Electron microscopy and image analysis confirm the ability of the developed technique to produce micrometer-scale arrays of sub 10 nm nanopores with narrow size distributions and aspect ratios of over 100:1. By introducing a scalable process for obtaining high aspect ratio pores in a novel size regime, this work opens the door to implementation of NPSi in numerous devices and applications.

Nanoparticle fabrication by geometrically confined nanosphere lithography

Abstract. Arrays of metal nanoparticles, typically gold or silver, exhibit localized surface plasmon resonance, a phenomenon that has many applications, such as chemical and biological sensing. However, fabrication of metal nanoparticle arrays with high uniformity and repeatability, at a reasonable cost, is difficult. Nanosphere lithography (NSL) has been used before to produce inexpensive nanoparticle arrays through the use of monolayers of self-assembled microspheres as a deposition mask. However, control over the size and location of the arrays, as well as uniformity over large areas is poor, thus limiting its use to research purposes. In this paper, a new NSL method, called here geometrically confined NSL (GCNSL), is presented. In GCNSL, microsphere assembly is confined to geometric patterns defined in photoresist, allowing high-precision and large-scale nanoparticle patterning while still remaining low cost. Using this new method, it is demonstrated that 400 nm polystyrene microspheres can be assembled inside of large arrays of photoresist patterns. Results show that optimal microsphere assembly is achieved with long and narrow rectangular photoresist patterns. The combination of microsphere monolayers and photoresist patterns is then used as a deposition mask to produce silver nanoparticles at precise locations on the substrate with high uniformity, repeatability, and quality.

Ultra-high aspect ratio functional nanoporous silicon via nucleated catalysts

Nanoporous silicon (NPSi) has drawn recent interest because of its potential in a range of applications such as battery anodes, photocatalysis, thermoelectrics, and filtration membranes. However, the inexpensive and scalable manufacturing of high aspect ratio porous structures on the nanometer scale has been difficult due to the reliance of current methods on complex and expensive equipment used for techniques such as anodization or photolithography. Here, we report a method of producing NPSi with sub-10 nm pore sizes and aspect ratios as high as 400 : 1 by leveraging the nucleation of sputtered noble metals on the Si surface, followed by metal-assisted chemical etching (MACE). The technique is capable of producing NPSi in an intrinsically scalable manner. Samples are characterized with SEM and TEM, along with vertical and horizontal FIB cross-sectional milling to elucidate the porous structure at several μm of depth within the substrate. Following preparation of the NPSi, it is functionalized with Al2O3 and TiO2 via atomic layer deposition (ALD). TiO2-functionalized NPSi exhibits reflectivity of 6–8% for visible wavelengths, and 2–3% in the infrared – showing its promise as a robust and functional porous substrate. The developed approach of employing MACE with sputtered nucleated catalysts facilitates the scalable fabrication of functional ultra-high aspect-ratio nanopores in silicon.

Fabrication of large-area metal nanoparticle arrays by nanosphere lithography for localized surface plasmon resonance biosensors

The localized surface plasmon resonance (LSPR) phenomenon that is characteristic of gold and silver nanoparticles has applications in areas such as portable and remote chemical and biological sensing. However, fabrication of metal nanoparticle arrays with high uniformity and repeatability, at a reasonable cost, is difficult. Nanosphere lithography (NSL) has been used to produce inexpensive nanoparticle arrays, through the use of monolayers of self-assembled microspheres as a deposition mask. However, lack of control over the location and size of the arrays, as well as poor uniformity over large areas, limits its use to research purposes. Here, we present large-area fabrication of nanoparticle arrays through both convective self-assembly NSL (CSANSL) and our new method, geometrically confined NSL (GCNSL). In GCNSL, microsphere assembly is confined to geometric patterns defined in photoresist. We show that 400nm polystyrene microspheres can be assembled inside of large arrays of photoresist trenches from 4-20μm in width and 500μm in length, with high uniformity, repeatability, and quality. Compared to CSANSL, GCNSL allows precise patterning of nanoparticle arrays for use in practical LSPR sensing devices, while still remaining inexpensive.

Enhancing conjugation rate of antibodies to carboxylates: Numerical modeling of conjugation kinetics in microfluidic channels and characterization of chemical over-exposure in conventional protocols by quartz crystal microbalance

This research reports an improved conjugation process for immobilization of antibodies on carboxyl ended self-assembled monolayers (SAMs). The kinetics of antibody/SAM binding in microfluidic heterogeneous immunoassays has been studied through numerical simulation and experiments. Through numerical simulations, the mass transport of reacting species, namely, antibodies and crosslinking reagent, is related to the available surface concentration of carboxyl ended SAMs in a microchannel. In the bulk flow, the mass transport equation (diffusion and convection) is coupled to the surface reaction between the antibodies and SAM. The model developed is employed to study the effect of the flow rate, conjugating reagents concentration, and height of the microchannel. Dimensionless groups, such as the Damköhler number, are used to compare the reaction and fluidic phenomena present and justify the kinetic trends observed. Based on the model predictions, the conventional conjugation protocol is modified to increase the yield of conjugation reaction. A quartz crystal microbalance device is implemented to examine the resulting surface density of antibodies. As a result, an increase in surface density from 321 ng/cm(2), in the conventional protocol, to 617 ng/cm(2) in the modified protocol is observed, which is quite promising for (bio-) sensing applications.