Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

Biotinylated anti-C-reactive protein (CRP) species were attached to the surface of streptavidin-coated magnetic beads (MB) and avidin-coated polystyrene microspheres/beads (PSB) entrapping a large number of electrogenerated chemiluminescence (ECL) labels (∼109 Ru(bpy)3[B(C6F5)4]2/bead) to form anti-CRP↔MB and Ru(II)⊂PSB/avidin↔anti-CRP conjugates, respectively. Sandwich-type Ru(II)⊂PSB/avidin↔anti-CRP 〈CRP〉 anti-CRP↔MB aggregates were formed when Ru(II)⊂PSB/avidin↔anti-CRP was mixed with anti-CRP↔MB conjugates in the presence of analyte CRP. The newly formed aggregates were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine as an ECL coreactant. ECL was carried out with a potential scan from 0 to 2.8 V vs Ag/Ag+, and the ECL intensity was found to be proportional to the analyte CRP concentration over the range of 0.010−10 μg/mL. The CRP concentration of an unknown human plasma specimen was measured by the standard addition method based on this technique. Elimination of the nonspecific adsorption of the CRP system with several different blocking agents was also studied, and 2.0% bovine serum albumin was found to be best

Electrodeposition of Single Nanometer-Size Pt Nanoparticles at a Tunneling Ultramicroelectrode and Determination of Fast Heterogeneous Kinetics for Ru(NH₃)₆³⁺ Reduction

We studied extremely fast kinetics of an outer-sphere heterogeneous electron transfer (ET) reaction at a single Pt nanoparticle (NP) using the newly adapted Kotecký–Levich (K-L) method. In this work, an electrode was prepared by nucleating and growing a single Pt NP on a tunneling ultramicroelectrode (TUME) that produces 1–40 nm or greater dimensions. Such a small-size electrode greatly enhances the mass transfer rate, thus enabling us to reliably determine ET kinetic parameters for fast ET reactions. Based on the recently demonstrated K-L model for a general UME, ET kinetic information could be measured by constructing a plot of 1/current density vs 1/mass transfer rate from the series of steady-state voltammograms obtained using Pt NP-deposited TUMEs. For this K-L plot, we altered the mass transfer rates by varying the electrode size, i.e., the Pt NP size in this work. The determined standard rate constant, <i>k</i>⁰, of heterogeneous reduction reaction for Ru­(NH₃)₆³⁺ was unprecedentedly high, at 36 ± 4 cm/s, confirmed by theoretical simulation. Extended applications to various electrocatalytic reactions with different types of electrodeposited metal NPs will show the versatility of our approach. Particularly, this novel fabrication of a nanometer-sized electrode and its application to fast ET kinetic study with simple instrumentation should be useful in studies of particle size and structure effects on given catalytic reactions

Electrogenerated Chemiluminescence. 66. The Role of Direct Coreactant Oxidation in the Ruthenium Tris(2,2‘)bipyridyl/Tripropylamine System and the Effect of Halide Ions on the Emission Intensity

We describe the electrogenerated chemiluminescence (ECL) processes of the Ru(bpy)32+ (bpy = 2,2‘-bipyridyl)/tripropylamine (TPrA) system at glassy carbon, platinum, and gold electrodes. The electrochemical behavior of TPrA on different electrode materials and its influence on the ECL process are demonstrated. At glassy carbon electrodes, the direct oxidation of TPrA began at ∼0.6 V vs SCE and exhibited a broad irreversible anodic peak. Two ECL waves were observed, one in the potential region more negative than 1.0 V vs SCE and one at more positive potentials. The first ECL process apparently occurs without the electrogeneration of Ru(bpy)33+, in contrast to that of the second ECL wave. At Pt and Au electrodes, however, the formation of surface oxides significantly blocked the direct oxidation of TPrA. An ECL wave below 1.0 V did not appear at Pt and was very weak at gold. The ECL peaks at potentials of 1.1−1.2 V were also much weaker than those observed at the glassy carbon electrode. These results showed that the direct oxidation of TPrA played an important role in the ECL processes. Therefore, the enhancement of the TPrA oxidation current might lead to an increase in the ECL intensity. Small amounts of halide species were found to inhibit the growth of surface oxides on Pt and gold electrodes and led to an obvious increase of TPrA oxidation current. The anodic dissolution of gold in halide-containing solution was also important in activating the gold electrode surface. The electrochemical catalytic effect of bromide further promoted the oxidation of TPrA. A halide effect on ECL at Pt and Au electrodes was also evident. The most effective enhancement of ECL was observed at Au electrode in a bromide-containing solution. This effect was also found in an commercial flow-through instrument (IGEN) and provided a simple way to improve the detection sensitivity at low concentrations of Ru(bpy)32+

Fabrication and Characterization of Probes for Combined Scanning Electrochemical/Optical Microscopy Experiments

A technique that combines scanning electrochemical microscopy (SECM) and optical microscopy (OM) was implemented with a new probe tip. The tip for scanning electrochemical/optical microscopy (SECM/OM) was constructed by insulating a typical gold-coated near-field scanning optical microscopy tip using electrophoretic anodic paint. Once fabricated, the tip was characterized by steady-state cyclic voltammetry, as well as optical and electrochemical approach experiments. This tip generated a stable steady-state current and well-defined SECM approach curves for both conductive and insulating substrates. Durable tips whose geometry was a ring with <1 μm as outer ring radius could be consistently fabricated. Simultaneous electrochemical and optical images of an interdigitated array electrode were obtained with a resolution on the micrometer scale, demonstrating good performance of the tip as both an optical and an electrochemical probe for imaging microstructures. The SECM feedback current measurements were successfully employed to determine tip−substrate distances for imaging

Electrogenerated Chemiluminescence. 72. Determination of Immobilized DNA and C-Reactive Protein on Au(111) Electrodes Using Tris(2,2‘-bipyridyl)ruthenium(II) Labels

Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2‘-bipyridyl)ruthenium(II) (Ru(bpy)32+) labels. A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5‘ end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)32+ ECL labels. Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide. CRP and anti-CRP tagged with Ru(bpy)32+ labels were then conjugated to the surface layer. ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution. A series of electrode treatments, including blocking free −COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species. The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1−24 μg/mL. CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique

Observing Single Nanoparticle Collisions at an Ultramicroelectrode by Electrocatalytic Amplification

The collisions of single platinum nanoparticles at an ultramicroelectrode were observed electrochemically by their characteristic current−time transients for a particle-catalyzed reaction. A single event is characterized by the current generated by an electrocatalyzed reaction of an indicator species (proton, hydrogen peroxide) present in solution. Since the indicator reaction does not occur at the selected ultramicroelectrode and can involve a high concentration of indicator species with a much larger diffusion coefficient than the nanoparticle, large amplification (10 orders of magnitude or more) in the current occurs. Every collision produces a unique current−time profile that can be correlated with the particle size, the particle residence time, and the nature of the particle interaction with the electrode surface. Applications to studying heterogeneous kinetics at single nanoparticles, determining particle size distributions, and as a very sensitive electroanalytical technique are suggested

On the Absence of a Diffuse Double Layer at Electronically Conductive Polymer Film Electrodes. Direct Evidence by Atomic Force Microscopy of Complete Charge Compensation

On the Absence of a Diffuse Double Layer at Electronically Conductive Polymer Film Electrodes. Direct Evidence by Atomic Force Microscopy of Complete Charge Compensatio

Electrogenerated Chemiluminescence. 77. DNA Hybridization Detection at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2‘-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)3[B(C6F5)4]2), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)3[B(C6F5)4]2 species (∼7.5 × 109 molecules/bead). With this approach a large amplification factor of Ru(bpy)3[B(C6F5)4]2 molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA−MB ↔ t-ssDNA−PSB/Ru(bpy)32+ conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag+, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-μm diameter MB required to bind and magnetically separate a single 10-μm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays

Use of Atomic Force Microscopy for the Study of Surface Acid−Base Properties of Carboxylic Acid-Terminated Self-Assembled Monolayers

Atomic force microscopy was used to measure the forces between a silica probe and a carboxylic acid-terminated self-assembled monolayer (SAM) on a gold substrate in the presence of KCl electrolyte solutions of different pH. Silica−silica interaction force measurements were conducted at different pH values to determine the silica probe surface electrostatic potentials under these conditions. The interaction between two silica surfaces is repulsive and can be accurately predicted (except at short distances) by the Derjaguin−Landau−Verwey−Overbeek theory. The interaction between silica and clean gold surfaces exhibits an attractive interaction at neutral pH. The interaction between a silica probe and carboxylic acid-terminated SAM-covered gold substrate was a strong function of the pH value of the electrolyte. The surface electrostatic potentials of the surface-confined monolayers of carboxylic acid were obtained by theoretical fits of the force data to solutions of the complete nonlinear Poisson−Boltzmann equation, with the knowledge of silica probe surface potentials, at different solution pH values. The surface titration curve was obtained by correlating the surface potentials to the different electrolyte pH values. A theoretical fit to the titration curve provides the surface pKa and an explanation for the broadening of the titration curve

Electrophoretic Migration and Particle Collisions in Scanning Electrochemical Microscopy

We report for the first time how electrophoretic migration of ions and charged nanoparticles (NPs) in low electrolyte concentration solutions affects positive feedback in scanning electrochemical microscopy (SECM). The strength of the electric field in the gap between either the tip and the substrate, or the tip and counter electrodes, is shown to increase proportionally to the decrease in gap size. This field affects the flux of the charged redox species as expected for dilute electrolyte solutions. However, the shape of the normalized approach curve is unaffected by the electrophoretic migration. We also report that the rate of collisions of charged insulating NPs with the tip electrode decreases as the tip is brought closer to the substrate electrode. This rather unexpected result (negative feedback) can be explained by the blocking of the particle flux with the glass insulating layer around the metal microwires. Observation of simultaneous changes in the faradaic current at the tip and substrate electrodes due to particle collisions with the tip confirms a high rate of mass transport between the two electrodes under the conditions of positive feedback SECM