Curtis Shannon

Quantitation of Femtomolar Protein Levels via Direct Readout with the Electrochemical Proximity Assay

We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.

Thermal chemical vapor deposition growth of zinc oxide nanostructures for dye-sensitized solar cell fabrication

Dye-sensitized solar cells (DSSCs) were fabricated using a quasialigned one-dimensional zinc oxide (ZnO) nanostructure. The ZnO nanostructures were grown on indium tin oxide (ITO) coated glass substrate via a thermal chemical vapor deposition (CVD). It has been considered that thermal CVD is not suitable for the growth of ZnO nanostructure on ITO/glass due to the high processing temperature. However, we have demonstrated that a densely populated ZnO nanostructure can be prepared on ITO/glass substrate by a double-source double-tube CVD process. The power conversion efficiency of our device is 0.6%.

A Reusable Electrochemical Proximity Assay for Highly Selective, Real-Time Protein Quantitation in Biological Matrices

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)—as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Display of Solid-State Materials Using Bipolar Electrochemistry

We report the formation and characterization of one-dimensional chemical composition gradients of CdS on Au surfaces using bipolar electrodeposition. When an external electric field is applied across an electrically floating Au electrode immersed in a bipolar electrochemical cell, a position-dependent interfacial potential difference is generated along the length of the Au. This potential gradient can be used to induce variations of chemical composition within thin films electrodeposited onto the Au bipolar electrode (BPE). Thin films formed by bipolar electrodeposition represent continuous one-dimensional solid-state material libraries and were screened using resonance Raman microscopy and Auger electron spectroscopy. As predicted from simple thermodynamic considerations, we observed three distinct deposition zones scanning from the cathodic pole to the midpoint of the BPE: (i) CdS+Cd, (ii) stoichiometric CdS, and (iii) elemental S. Bipolar electrodeposition can be used to generate material libraries rapidly and without direct electrical contact to the substrate using extremely simple instrumentation.

Raman analysis of longitudinal optical phonon-plasmon coupled modes of aligned ZnO nanorods

The electronic properties of vertically aligned ZnO nanorods have been investigated using micro-Raman spectroscopy. The concentration and mobility of the charge carriers were determined via Raman line shape analysis using longitudinal-optical-phonon-plasmon coupled mode. The local laser heating and the stress effects have been considered when analyzing the Raman spectra. The mobility and carrier concentration of the aligned ZnO nanorods are 84.8cm2∕Vs and 3.8×1017cm−3, respectively. As a comparison, the mobility and carrier concentration of the undoped bulk ZnO were also obtained from the Raman line shape analysis. The mobility of the aligned ZnO nanorods is about 20% lower than that of the undoped bulk ZnO, which can be attributed to enhanced surface scattering due to the reduction in dimension.

Screening the optical properties of Ag-Au alloy gradients formed by bipolar electrodeposition using surface enhanced Raman spectroscopy.

We report the synthesis of Ag-Au alloy gradients on stainless steel substrates using bipolar electrodeposition (BP-ED), a technique based on the existence of a potential gradient at the interface of a bipolar electrode (BPE) and an electrolytic solution. The interfacial potential gradient causes the rates of electrodeposition of Ag and Au to vary along the length of the BPE, leading to the electrodeposition of a chemical concentration gradient. The surface morphology of the electrodeposits was characterized using scanning electron microscopy (SEM), and their chemical composition was determined using energy dispersive X-ray spectroscopy (EDX). Self-assembled monolayers of a Raman-active probe molecule (benzene thiol) were allowed to form on the surface of the alloy gradients, and confocal Raman microscopy was employed to determine the alloy composition that resulted in the maximum surface enhanced Raman scattering (SERS) intensity. An alloy composition of ca. 70% Ag/30% Au was found to be optimum for SERS excited using 514.5 nm radiation, and it is explained on the basis of composition-dependent changes in the local surface plasmon resonance (LSPR) of the electrodeposited Ag-Au alloy.

Synthesis of metal-semiconductor core-shell nanoparticles using electrochemical surface-limited reactions.

We report the synthesis of Au/CuI and Au/CdS core-shell nanoparticle (NP) thin films using codeposition and electrochemical atomic layer deposition (EC-ALD). Au nanoparticle films were prepared on glassy carbon supports by depositing alternating layers of poly(diallyl dimethylammonium)-stabilized Au nanoparticles and CoP(2)W(17)O(61)(8-) polyoxometallate interlayers. From there, CuI was deposited onto the surface of Au nanoparticles using electrochemical atomic layer deposition, while CdS films were grown by an atom-by-atom codeposition method. The semiconductor-Au core-shell nanoparticles were characterized by electrochemistry, photoluminescence spectroscopy, and Raman spectroscopy. Our results indicate that the semiconductors deposit onto the AuNP surface by surface limited electrochemical reactions.

Preparation and Characterization of Polyoxometalate/Protein Ultrathin Films Grown on Electrode Surfaces Using Layer-by-Layer Assembly

We report a new electrostatic layer-by-layer assembly method for the controlled deposition of electrocatalytically active enzymes onto electrode surfaces using polyoxometalate as the counteranion. Cytochrome c (cyt c), a redox active protein, and P(2)W(18)O(62)(6-), a Dawson-type polyoxometalate, were deposited onto glassy carbon electrodes by two procedures: static dipping and electrochemical cycling. Cyclic voltammetry and UV-vis spectroscopy reveal that approximately 1.5 x 10(-10) mol/cm(2) of P(2)W(18)O(62)(6-) and 2.2 x 10(-11) mol/cm(2) of cytochrome c are deposited per cycle, which correspond to approximately one monolayer of each molecule. The thicknesses of the resulting films measured by atomic force microscopy also indicate that the films are formed in a layer-by-layer fashion. Experimental factors that affect electron-transfer rate in these films, such as scan rate and film thickness, were systematically analyzed. The use of {P(2)W(18)O(62)(6-)/cyt c}n films to catalyze hydrogen peroxide reduction was demonstrated.

Detection of ferrocenemethanol and molecular oxygen based on electrogenerated chemiluminescence quenching at a bipolar electrode.

Small molecules, such as ferrocenemethanol (FcMeOH) and O2, that are capable of quenching the Ru(bpy)3(2+) excited state via energy or electron transfer can be quantitatively detected in a bipolar electrochemical cell based on the attenuation of steady-state electrogenerated chemiluminescence (ECL). FcMeOH quenches ECL generated by the Ru(bpy)3(2+) oxalate coreactant system, exhibiting a linear dependence on [FcMeOH] with a Stern-Volmer slope of 921 M(-1), corresponding to a quenching rate constant of 2 × 10(9) M(-1) s(-1). We used the bipolar ECL quenching platform to measure dissolved O2 and validated the results using a standard Clark electrode. The detection limit for local [O2] measured using ECL quenching was found to be 300 ppb. This work opens up the possibility of utilizing ECL quenching at bipolar electrodes for a wide range of applications.

Resonance Raman scattering and scanning tunneling spectroscopy of CdS thin films grown by electrochemical atomic layer epitaxy-thickness dependent phonon and electronic properties

Abstract The thickness dependence of electron–phonon coupling and the band gap of ultrathin CdS films deposited on Au substrates using electrochemical atomic layer epitaxy (EC-ALE) have been measured by resonance Raman scattering and scanning tunneling spectroscopy (STS). Polarization dependent resonance Raman experiments indicate that electron–phonon coupling is strongly dependent on film thickness when measured using p-polarized radiation, but is essentially thickness independent when measured using s-polarized light. We find that the electron–phonon coupling reaches its limiting value at a film thickness of seven monolayers, yielding an estimated apparent exciton diameter of 2.3 nm. STS measurements of the electronic band gap of the same samples confirm this behavior. The band gap is observed to shift from its bulk value of 2.4 to >2.7 eV for a three monolayer film. The size dependence of the band gap can be described qualitatively using the strong confinement model.