Continuous Nanoflow-Scanning Electrochemical Microscopy: Voltammetric Characterization and Application for Accurate and Reproducible Imaging of Enzyme-Labeled Protein Microarrays

Abstract

The coupling of scanning electrochemical microscopy (SECM) to a continuous nanoflow (CNF) system is accomplished with the use of a microconcentric ring electrode/injector probe. The gold microring electrode encapsulated by a glass sheath is robust and can be beveled and polished. The CNF system, comprising a precision gas displacement pump and a rotary valve, is capable of delivering solution to the center of the SECM probe in the range of 1-150 nL/min. Major advantages of the CNF-SECM imaging mode over the conventional SECM generation/collection (G/C) mode include higher imaging resolution, immunity from interferences by species in the bulk solution or at other sites of the substrate, elimination of the feedback current that could interfere with the G/C data interpretation, and versatility of initiating surface reactions/processes via introducing different reactants into the flowing stream. Parameters such as flow rates, probe/substrate separations, and collection efficiencies are examined and optimized. Higher resolution, reproducibility, and accuracy are demonstrated through the application of CNF-SECM to horseradish peroxidase (HRP)-amplified imaging of protein microarrays. By flowing H2O2 and ferrocenemethanol through the injector and detecting the surface-generated ferriceniummethanol, human IgG spots covered with HPR-labeled antihuman IgG can be detected in the range of 13 nM-1.333 μM with a detection limit of 3.0 nM. In addition, consistent images of microarray spots for selective and high-density detection of analytes can be attained.

Imaging of localized enzymatic peroxidase activity over unbiased individual gold nanowires by scanning electrochemical microscopy

“…The enzyme horseradish peroxidase (HRP) was used as the model enzyme for peroxidase activity imaging, as in addition to having an extensive biosensor potential, it is a commonly used tagging enzyme (enzyme label) for antibody or ligand molecules for molecular recognition or signal amplication in immunoassays, protein microarrays and SECM imaging studies. 17,23,[32][33][34] 2. Materials and methods…”

Micro‐ and Nanoscopic Imaging of Enzymatic Electrodes: A Review

“…Moreover, the possibility to put whatever is needed in the capillary ensures the versatility of the method. [117] In tip generation/substrate collection mode, SECM enables locally triggering enzymatic activity. This is also possible thanks to combination of a scanning microelectrode and light emitting methods, like fluorescence or ECL.…”

“…This lead to higher imaging resolution because no interference from other active sites, which causes overlapping of the diffusion layer, was observed, neither interference from the bulk (Figure ). Moreover, the possibility to put whatever is needed in the capillary ensures the versatility of the method …”

“…AFM was conducted in contact mode while SECM simultaneously enabled detection of ferrocinium methylhydroxide (FCMH + ), the electroactive byproduct of the enzymatic reaction . Another method, inspired by a work by Bard and coworkers, consisted in coupling SECM with continuous nano‐flow to overcome a general problem linked to the SG/TC mode: the increasing background current resulting from steady build‐up of the reaction products in the diffusion layer. At long experience times, activity spots are not anymore distinguishable from the background.…”

Biological imaging with scanning electrochemical microscopy

“…SECM was also coupled to a continuous nanoflow system [124]. The specially designed tip consisted of a capillary injector surrounded by a microring electrode (r = 11.5 µm).…”