Robert S. Marks

Colorimetric Detection of Mercury Ions Based on Plasmonic Nanoparticles

The development of rapid, specific, cost-effective, and robust tools in monitoring Hg(2+) levels in both environmental and biological samples is of utmost importance due to the severe mercury toxicity to humans. A number of techniques exist, but the colorimetric assay, which is reviewed herein, is shown to be a possible tool in monitoring the level of mercury. These assays allow transforming target sensing events into color changes, which have applicable potential for in-the-field application through naked-eye detection. Specifically, plasmonic nanoparticle-based colorimetric assay exhibits a much better propensity for identifying various targets in terms of sensitivity, solubility, and stability compared to commonly used organic chromophores. In this review, recent progress in the development of gold nanoparticle-based colorimetric assays for Hg(2+) is summarized, with a particular emphasis on examples of functionalized gold nanoparticle systems with oligonucleotides, oligopeptides, and functional molecules. Besides highlighting the current design principle for plasmonic nanoparticle-based colorimetric probes, the discussions on challenges and the prospect of next-generation probes for in-the-field applications are also presented.

Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein.

An Electrochemical Lateral Flow Immunosensor (ELFI) is developed combining screen-printed gold electrodes (SPGE) enabling quantification together with the convenience of a lateral flow test strip. A cellulose glassy fiber paper conjugate pad retains the marker immunoelectroactive nanobeads which will bind to the target analyte of interest. The specific immunorecognition event continues to occur along the lateral flow bed until reaching the SPGE-capture antibodies at the end of the cellulosic lateral flow strip. The rationale of the immunoassay consists in the analyte antigen NS1 protein being captured selectively and specifically by the dengue NS1 antibody conjugated onto the immunonanobeads thus forming an immunocomplex. With the aid of a running buffer, the immunocomplexes flow and reach the immuno-conjugated electrode surface and form specific sandwich-type detection due to specific, molecular recognition, while unbound beads move along past the electrodes. The successful sandwich immunocomplex formation is then recorded electrochemically. Specific detection of NS1 is translated into an electrochemical signal contributed by a redox label present on the bead-immobilized detection dengue NS1 antibody while a proportional increase of faradic current is observed with increase in analyte NS1 protein concentration. The first generation ELFI prototype is simply assembled in a cassette and successfully demonstrates wide linear range over a concentration range of 1-25 ng/mL with an ultrasensitive detection limit of 0.5 ng/mL for the qualitative and quantitative detection of analyte dengue NS1 protein.

Freestanding HRP–GOx redox buckypaper as an oxygen-reducing biocathode for biofuel cell applications

Horseradish peroxidase (HRP) was immobilized on redox buckypapers followed by electropolymerization of pyrrole-modified concanavalin A enabling the subsequent additional immobilization of the glycoprotein glucose oxidase (GOx). Biocatalytic buckypapers were formed using pyrene-modified 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or bis-Pyr-ABTS, a redox mediator, as a cross-linker. ABTS-functionalized buckypaper enhances the electron transfer of the bioelectrocatalytic reduction of H2O2 by HRP. Since H2O2 is produced during glucose oxidation by GOx in the presence of oxygen, the bienzymatic GOx–HRP biocathode achieves the complete reduction of oxygen into water. A clearly improved performance of the biocathode was obtained by using an improved biocompatible immobilization strategy, enabling the prevention of enzyme loss while ensuring both diffusion of glucose and O2 and the local production of H2O2. These freestanding flexible oxygen-reducing biocathodes can operate under physiological conditions and show a high onset potential at 0.60 (±0.01) V. In the presence of glucose (5 mM), such biocathodes exhibit a stable current density output of 1.1 (±0.1) mA cm−2 at 0.1 V under continuous one-hour discharge. Furthermore, a marked increase in lifetime was observed, the biocathode displaying 64% of its initial electrocatalytic activity after 15 days.

Cloud-Enabled Microscopy and Droplet Microfluidic Platform for Specific Detection of Escherichia coli in Water

We report an all-in-one platform – ScanDrop – for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a “cloud” network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2–4 days for other currently available standard detection methods.

Biofunctionalization of Multiwalled Carbon Nanotubes by Irradiation of Electropolymerized Poly(pyrrole–diazirine) Films

A photoactivatable poly(pyrrole-diazirine) film was synthesized and electropolymerized as a versatile tool for covalent binding of laccase and glucose oxidase on multiwalled carbon nanotube coatings and Pt, respectively. Irradiation of the functionalized nanotubes allowed photochemical grafting of laccase and its subsequent direct electrical wiring, as illustrated by the electrocatalytic reduction of oxygen. Moreover, covalent binding of glucose oxidase as model enzyme, achieved by UV activation of electropolymerized pyrrole-diazirine, allowed a glucose biosensor to be realized. This original method to graft biomolecules combines electrochemical and photochemical techniques. The simplicity of this new method allows it to be extended easily to other biological systems.

Glucose fuel cell based on carbon nanotube-supported pyrene–metalloporphyrin catalysts

It is important to design a functionalization scheme for carbon nanotubes that preserves their outstanding proprieties, while adding new proprieties, thereby enabling their integration in fuel cell applications. In the present work, we describe the production of a non-covalently attached network of porphyrins to multi-walled carbon nanotubes (MWCNT) sidewalls. The approach is based on π–π stacking interactions of pyrene-modified metalloporphyrins onto MWCNT sidewalls. Two configurations of MWCNT–porphyrin hybrid electrodes were both electrochemically characterized and tested under alkaline conditions. Pyrene-functionalized rhodium deuteroporphyrin (Rh(DP)pyr2), was used as an anode in the electrocatalytic oxidation of glucose and pyrene-functionalized tetracarboxyphenyl cobalt porphyrin (Co(TCPP)pyr4) was itself used as a cathode in the electrocatalytic reduction of oxygen. Both electrodes were integrated into a glucose fuel cell system leading to a maximum power output of 0.9(±0.10) mW cm−2. Compared to alternative system approaches, pyrene-modified porphyrin hybrid electrodes and their corresponding fuel cell devices exhibited higher activity, power output, and long term stability.

Highly sensitive detection of paclitaxel by surface-enhanced Raman scattering

The surface-enhanced Raman scattering (SERS) technique was shown to be an effective molecular analytical tool due to its high sensitivity. Here, we propose to exploit soft UV assisted nanoimprint lithography (UV-NIL) for the development of a reproducible and highly-sensitive SERS biosensor. Soft lithography is known to be advantageous for biological applications since it is compatible with insulating supports and large-area samples. In the present investigations, soft UV-NIL is used for the fabrication of large-sized arrays of gold nanocylinders on glass which were shown to be highly sensitive and highly specific sensing surfaces, with a limit of detection measured down to 1 nM. Employing the UV-NIL SERS substrate enable working ranges of nanomolar to micromolar concentrations in regards to our model paclitaxel analyte.

Novel on-demand bioadhesion to soft tissue in wet environments.

Current methods of tissue fixation rely on mechanical-related technologies developed from the clothing and carpentry industries. Herein, a novel bioadhesive method that allows tuneable adhesion and is also applicable to biodegradable polyester substrates is described. Diazirine is the key functional group that allows strong soft tissue crosslinking and on-demand adhesion based on a free radical mechanism. Plasma post-irradiation grafting makes it possible to graft diazirine onto PLGA substrates. When the diazirine-PLGA films, placed on wetted ex vivo swine aortas, are activated with low intensity UV light, lap shear strength of up to 450u2009±u200950 mN cm(-2) is observed, which is one order of magnitude higher than hydrogel bioadhesives placed on similar soft tissues. The diazirine-modified PLGA thin films could be added on top of previously developed technologies for minimally invasive surgeries. The present work is focused on the chemistry, grafting, and lap shear strength of the alkyl diazirine-modified PLGA bioadhesive films.

Biofunctionalization of Multiwalled Carbon Nanotubes by Electropolymerized Poly(pyrrole-concanavalin A) Films

The synthesis and electropolymerization of a pyrrolic concanavalinu2005A derivative (pyrrole-Con A) onto a multiwalled carbon nanotube (MWCNT) deposit is reported. Glucose oxidase was then immobilized onto the MWCNT-poly(pyrrole-Con A) coating by affinity carbohydrate interactions with the polymerized Con A protein. The resulting enzyme electrode was applied to the amperometric detection of glucose exhibiting a high sensitivity of 36 mA cm(-2) mol(-1) L and a maximum current density of 350 μA cm(-2) .

Organic additives stabilize RNA aptamer binding of malachite green

Aptamer-ligand binding has been utilized for biological applications due to its specific binding and synthetic nature. However, the applications will be limited if the binding or the ligand is unstable. Malachite green aptamer (MGA) and its labile ligand malachite green (MG) were found to have increasing apparent dissociation constants (Kd) as determined through the first order rate loss of emission intensity of the MGA-MG fluorescent complex. The fluorescent intensity loss was hypothesized to be from the hydrolysis of MG into malachite green carbinol base (MGOH). Random screening organic additives were found to reduce or retain the fluorescence emission and the calculated apparent Kd of MGA-MG binding. The protective effect became more apparent as the percentage of organic additives increased up to 10% v/v. The mechanism behind the organic additive protective effects was primarily from a ~5X increase in first order rate kinetics of MGOH→MG (kMGOH→MG), which significantly changed the equilibrium constant (Keq), favoring the generation of MG, versus MGOH without organic additives. A simple way has been developed to stabilize the apparent Kd of MGA-MG binding over 24h, which may be beneficial in stabilizing other triphenylmethane or carbocation ligand-aptamer interactions that are susceptible to SN1 hydrolysis.