Enzymatically enhanced collisions on ultramicroelectrodes for specific and rapid detection of individual viruses

Abstract

We report the specific collision of a single murine cytomegalovirus (MCMV) on a platinum ultramicroelectrode (UME, radius of 1 μm). Antibody directed against the viral surface protein glycoprotein B functionalized with glucose oxidase (GOx) allowed for specific detection of the virus in solution and a biological sample (urine). The oxidation of ferrocene methanol to ferrocenium methanol was carried out at the electrode surface, and the ferrocenium methanol acted as the cosubstrate to GOx to catalyze the oxidation of glucose to gluconolactone. In the presence of glucose, the incident collision of a GOx-covered virus onto the UME while ferrocene methanol was being oxidized produced stepwise increases in current as observed by amperometry. These current increases were observed due to the feedback loop of ferrocene methanol to the surface of the electrode after GOx reduces ferrocenium methanol back to ferrocene. Negative controls (i) without glucose, (ii) with an irrelevant virus (murine gammaherpesvirus 68), and (iii) without either virus do not display these current increases. Stepwise current decreases were observed for the prior two negative controls and no discrete events were observed for the latter. We further apply this method to the detection of MCMV in urine of infected mice. The method provides for a selective, rapid, and sensitive detection technique based on electrochemical collisions.collisions | cytomegalovirus | electrochemistry | electrovirology | ELISA T he development of methods to observe collisions on ultramicroelectrodes (UMEs) has allowed for the study of single entities, such as single nanoparticles (1), emulsion droplets (2, 3), vesicles (4, 5), biological macromolecules (6), and even single ions (7), in a digital manner (8). However, many of the techniques developed lack specificity. In the field of collisions, the study of biologically relevant analytes of interest, such as viruses and cells, has been largely unexplored. Perhaps the greatest challenges in these experiments are achieving specificity and understanding the electrochemical response. Overcoming these challenges will ultimately lead to the development of sensitive detection methodologies based on facile and inexpensive electrochemical methods, which will be of importance in the diagnosis of infectious diseases.Rapid, accurate diagnosis is a critical first step in the care of those suffering from viral infections. ELISAs, PCR-based techniques, or culture methods are fairly reliable diagnostic measures with varying degrees of sensitivity (9-11). However, these diagnostic techniques require a significant degree of sample and experimental preparation, increasing the amount of time between a positive diagnosis and the beginning of treatment. Diagnostic tools that are able to directly sample from complex media (i.e., blood, serum, urine, etc.) are necessary to expedite the diagnostic process.One such infectious agent that requires rapid and early detection is human cytomegalovirus (HCMV). HCMV is a prototypical betaherpesvirus, establ...

Stochastic Collision Nanoelectrochemistry: A Review of Recent Developments

“…In a recent work, they improved the selectivity of the detection of MCMV by using a glucose oxidase (GOx)‐functionalized specific antibody (Figure B). Instead of detecting changes in the magnitude of the current reduction and in the collision frequency, as demonstrated in the previous work, they detect the specific collision of a single MCMV on a UME.…”

“…Stochastic collision nanoelectrochemistry (SCNEC) has become a particularly important electrochemical method in recent years owing to its unique advantage in single entity electrochemical analysis . Since the establishment of SCNEC, a variety of research studies have been published, which have greatly accelerated the development of SCNEC.…”

“…Since the establishment of SCNEC, a variety of research studies have been published, which have greatly accelerated the development of SCNEC. As a convenient and rapid strategy, SCNEC has broad applications in many areas, including electrocatalysis, bioelectrochemistry, and biosensing . Moreover, a key finding has shown that SCNEC is particularly powerful for obtaining the properties of single nanoparticles (SNPs), such as the particle size, concentration, aggregation, and catalytic reactivity, by using electrochemical signals.…”

“…Metal and metal oxide NPs have been widely used by researchers in SCNEC studies over the past decade . Currently, “soft particles” (SPs) offer a promising new trend in SCNEC research, which has tremendously expanded the application of SCNEC in the area of interface‐transfer analysis, immuno‐detection, and single bio‐particle monitoring …”

Zellzählung mittels Einzelobjektelektrochemie

“…Eine jüngste Veröffentlichung von Compton und Mitarbeitern in der Angewandten Chemie beschreibt nun einen großen Fortschritt auf diesem Gebiet in Form von Aufprallexperimenten mit roten Blutkörperchen unter physiologischen Bedingungen. Was hier so einzigartig ist, ist nicht, dass biologische Objekte durch Aufprallvoltammetrie nachgewiesen werden; Viren, die mit Silber‐Nanopartikeln oder Glukoseoxidase‐Antikörper‐Konjugaten markiert wurden, wurden bereits früher in Aufprallexperimenten eingesetzt. Das vielmehr Besondere ist die Verwendung von Säugerzellen, die vollständig unmarkiert sind und in Blut erfasst werden (verdünnt auf 25 %, um die Viskosität zu reduzieren und Verklumpung zu vermeiden).…”