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.

We report the electrochemistry of surface-confined monolayers of 4-aminothiophenol (4-ATP) and mixed monolayers of 4-ATP and thiophenol (TP) on Au surfaces. Cyclic voltammograms of the 4-ATP monolayer in acidic aqueous perchlorate solutions are characterized by an irreversible oxidative wave at 0.730 V vs Ag/AgCl on the first scan and, upon scan reversal, by a persistent, reversible, surface-confined wave centered at approximately 0.500 V and a transient peak at about 0.300 V. We propose an ECE mechanism to account for this electrochemistry:  4-ATP is first oxidized to the cation radical, followed by chemical coupling to form an adsorbed dimer. The dimer is subsequently hydrolyzed in the presence of H2O to yield an adsorbed quinone species that is reversibly electroactive. Grazing angle FTIR spectroscopy was used to identify the product. The transient peak is due to the coupling of desorbed molecules and is consistent with the formation of a phenazine species. We then show that mixed monolayers of 4-ATP and TP can be used to study the coverage dependence of surface-confined reactions. The chemical composition of the mixed systems was determined using two independent Auger electron spectroscopic techniques and grazing angle FTIR spectroscopy. Using 20 min assembly times, we find that the surface concentration of 4-ATP is directly proportional to its mole fraction in solution. Interestingly, TP does not participate in the 4-ATP electrochemistry and functions only to dilute the surface concentration of 4-ATP. We find that the efficiency of the conversion of 4-ATP to product is somewhat higher at low mole fractions of 4-ATP.

Transition metal heteropolyanions have been used to catalyze a variety of organic oxidations but have not previously been used for O2 generation, despite sharing some structural similarities with dioxoruthenium water-oxidation catalysts. In this study, we report that the di-Ru-substituted polyoxometalate (POM) [Ru2Zn2(H2O)2(ZnW9O34)2]14- can be used to catalyze the electrochemical generation of O2. By comparing the behavior of this compound to that observed using a mono-Ru-substituted POM catalyst, we show that adjacent Ru sites are necessary to observe O2 generation. These observations suggest a reaction pathway involving two Ru-bound oxygen species combining to form O2 and are consistent with the accepted mechanism of electrochemical oxygen evolution. Finally, analysis of the observed electrode kinetics yields a Tafel slope of roughly 120 mV, which is similar to values reported previously for perovskite anodes.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for bio-sensing 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 5 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.

We report a study of the physical and electrochemical properties of two-component self-assembled monolayers (SAMs) composed of both electroactive (4-aminothiophenol, 4-ATP) and electroinactive (n-octadecanethiol, ODT) species. In all of the experiments reported here, relatively short (3 h) assembly times were used to prepare the mixed SAMs. We have characterized the macroscopic composition and the microscopic structure of these SAMs using Auger electron spectroscopy (AES), coulometry, grazing angle Fourier transform infrared spectroscopy, and lateral force microscopy (LFM). The adsorption isotherms determined by AES and coulometry show significant deviation from Langmuirian behavior and are suggestive of phase separation. LFM images obtained at three points near the critical region of the isotherm ([4-ATP]/[ODT] ∼ 4) indicate that these two-component SAMs display complex phase behavior:  At relatively low 4-ATP coverages, the surface consists of small islands of 4-ATP imbedded in an ordered film of ODT. At higher coverages of 4-ATP, however, we find evidence of both separation into distinct phases and mixing of the two components. In a second series of experiments, we demonstrate that phase domains of 4-ATP are electroactive and can be used to carry out localized electrochemistry. That is, the islands of 4-ATP, which are randomly distributed in the ODT matrix, behave as an array of ultramicroelectrodes. Surface-confined 4-ATP molecules can be used to nucleate the growth of polyaniline selectively from the phase separated domains of 4-ATP. We find that if a 4-ATP/ODT mixed monolayer is oxidized in the presence of aniline, nanometer scale polyaniline features are formed. The size and distribution of these features have been characterized by AFM and can be controlled through a combination of monolayer composition and the concentration of aniline in solution.