An apta-aggregation based machine learning assay for rapid quantification of lysozyme through texture parameters.

A novel assay technique that involves quantification of lysozyme (Lys) through machine learning is put forward here. This article reports the tendency of the well- documented Ellington group anti-Lys aptamer, to produce aggregates when exposed to Lys. This property of apta-aggregation has been exploited here to develop an assay that quantifies the Lys using texture and area parameters from a photograph of the elliptical aggregate mass through machine learning. Two assay sets were made for the experimental procedure: one with high Lys concentration between 25-100 mM and another with low concentration between 1-20 mM. The high concentration set had a sample volume of 10 µl while the low concentration set had a higher sample volume of 100 µl, in order to obtain the statistical texture values reliably from the aggregate mass. The platform exhibited an experimental limit of detection of 1 mM and a response time of less than 10 seconds. Further, two potential operating modes for the aptamer were hypothesized for this aggregation property and the more accurate mode among the two was ascertained through bioinformatics studies.

Application of cyclic biamperometry to viability and cytotoxicity assessment in human corneal epithelial cells.

The application of cyclic biamperometry to viability and cytotoxicity assessments of human corneal epithelial cells has been investigated. Electrochemical measurements have been compared in PBS containing 5.0 mM glucose and minimal essential growth medium. Three different lipophilic mediators including dichlorophenol indophenol, 2-methyl-1,4-naphthoquinone (also called menadione or vitamin K3) and N,N,N',N'-tetramethyl-p-phenylenediamine have been evaluated for shuttling electrons across the cell membrane to the external medium. Transfer of these electrons to ferricyanide in the extra cellular medium results in the accumulation of ferrocyanide. The amount of ferrocyanide is then determined using cyclic biamperometry and is related to the extent of cell metabolic activity and therefore cell viability. To illustrate cytotoxicity assessment of chemicals, hydrogen peroxide, benzalkonium chloride and sodium dodecyl sulfate have been chosen as sample toxins, the cytotoxicities of which have been evaluated and compared to values reported in the literature. Similar values have been reported using colorimetric assays; however, the simplicity of this electrochemical assay can, in principle, open the way to miniaturization onto lab-on-chip devices and its incorporation into tiered-testing approaches for cytotoxicity assessment.

Cyclic biamperometry at micro-interdigitated electrodes.

Cyclic biamperometry was studied as an analytical method for use with commercially available, comb-type, coplanar microinterdigitated electrodes (µIDEs), using the ferri-/ferrocyanide redox couple as a model analyte. The µIDEs studied in this work were made of gold that had been deposited onto a Ti/W adhesion layer on borosilicate glass chips and had 5 and 10 µm bands with equal gap sizes. Close proximity of the two working electrodes, and their interdigitation, resulted in signal amplification by redox cycling. Results were compared with those obtained by cyclic voltammetry, where one of the two IDE electrodes was used as the working electrode and external reference and auxiliary electrodes were used. Amplification factors of almost 20 were achieved due to redox cycling. Attempts to apply cyclic voltammetry to the µIDEs, with one of the combs as the working and the other as the auxiliary electrode, were unsuccessful due to corrosion of the auxiliary electrode comb. Results of this study, and the electrochemically unique feature of biamperometry to probe but not change the net contents of the medium under examination, suggest the applicability of scanning biamperometry at µIDEs to the very small volumes and electrochemical cell dimensions that are now of great interest.

Cyclic biamperometry.

Cyclic biamperometry has been investigated as a method for the quantitation of one form of a reversibly electroactive redox couple in the presence of the other form, using the ferri-ferrocyanide couple in aqueous KCl. A triangular voltage waveform applied across two equal-area, planar gold electrodes yields peak currents that depend on the square root of the applied voltage scan rate, when one form of the redox couple is present in excess. Independent measurement of electrode-to-solution potential during biamperometric scans allowed estimation of the fractional impedance at each electrode-solution interface, and these values allow calculation of the effective potential scan rates at each electrode. Results show that when one form is present in a 5-fold excess or greater, the potential scan rate for the limiting reaction is nearly identical to the applied voltage scan rate. Similar values were obtained from impedance calculations, but discrepancies between the predicted and experimental values are evident when the two forms are present at near equivalent concentrations. When one form of the redox couple is present in excess, cyclic biamperometric peak currents depend linearly on the concentration of the limiting form, and these currents can be amplified by using cells with one electrode much larger than the other. Because this method does not require a reference electrode, it can, in principle, be readily incorporated into new electrochemical array or lab-on-a-chip devices.

Microplate-compatible biamperometry array for parallel 48-channel amperometric or coulometric measurements.

We report a new reusable electrochemical array for parallel biamperometric measurements that has been designed for use with standard microplates. The 48-channel array uses half of the available 96 wells and has 48 pairs of Pt wire electrodes. Applications to the quantitation of a variety of oxidizable species, including acetaminophen, ascorbic acid, hydroquinone, trolox, and uric acid, are demonstrated in assays that use potassium ferricyanide as an oxidant to produce a mixture of ferri- and ferrocyanide. Hydrogen peroxide quantitation is also demonstrated, based on an assay in which ferrocyanide is oxidized, again to produce a mixture of ferri- and ferrocyanide. Detection limits (signal-to-noise ratio (S/N) = 3) in these assays range from 1 (acetaminophen, R2 = 0.994) to 8 microM (ascorbic acid, R2 = 0.967), and linearity was observed to analyte concentrations of at least 100 microM. We also demonstrate the application of the biamperometric array to enzymatic assays, using the glucose oxidase reaction as an example; following a 20 min enzyme reaction time, a detection limit of 0.1 mM glucose was obtained. These results indicate that applications to other oxidase-based assays are feasible in this high-throughput format. The new electrochemical array employs standard, inexpensive microplates, and the biamperometric measurements are simple, precise, and rapid, requiring only 2 min for 48 parallel measurements.

Antibiotic susceptibility testing at a screen-printed carbon electrode array.

Screen-printed carbon electrode arrays were treated to allow respiratory activity-based measurement of antibiotic susceptibility with Escherichia coli JM105. Carbon working electrodes were examined for antibiotic adsorption and were pretreated with various electrochemical and chemical protocols to minimize antibiotic adsorption. Treatment by voltammetry in basic solution or by chemical modification with poly-L-lysine or chitosan were found to be effective methods for the elimination of adsorption of the studied group of 17 antibiotics, which comprised several classes and modes of action. Measurements consisted of two-electrode amperometry of the bacterial suspension after 10 min of incubation with antibiotic followed by addition of an oxidative cocktail of ferricyanide and dichlorophenolindophenol for a further 10 min; response currents, which indicate the extent of reduction of ferricyanide to ferrocyanide by cellular respiratory activity, decrease with increasing concentration of antibiotic present in the initial 10 min incubation. IC50 values obtained for chloramphenicol with these electrode modification methods are consistent at 2.0 +/- 0.2 mM, in approximate agreement with previously reported respiration-based results for this organism but significantly higher than values reported for growth-based antibiotic susceptibility testing methods.

Rapid identification of viable Escherichia coli subspecies with an electrochemical screen-printed biosensor array.

Rapid identification of Escherichia coli strains is an important diagnostic goal in applied medicine as well as the environmental and food sciences. This paper reports an electrochemical, screen-printed biosensor array, where selective recognition is accomplished using lectins that recognize and bind to cell-surface lipopolysaccharides and coulometric transduction exploits non-native external oxidants to monitor respiratory cycle activity in lectin-bound cells. Ten different lectins were separately immobilized onto porous membranes that feature activated surfaces (ImmunodyneABC). Modified membranes were exposed to untreated E. coli cultures for 30 min, rinsed, and layered over the individual screen-printed carbon electrodes of the sensor array. The membranes were were incubated 5 min in a reagent solution that contained the oxidants menadione and ferricyanide as well as the respiratory substrates succinate and formate. Electrochemical oxidation of ferrocyanide for 2 min provided chronocoulometric data related to the quantities of bound cells. These screen-printed sensor arrays were used in conjunction with factor analysis for the rapid identification of four E. coli subspecies (E. coli B, E. coli Neotype, E. coli JM105 and E. coli HB101). Systematic examination of lectin-binding patterns showed that these four E. coli subspecies are readily distinguished using only five essential lectins.