Susan R. Mikkelsen

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.

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.

Chemometric analysis of square wave voltammograms for classification and quantitation of untreated beverage samples

Abstract Square wave voltammetry at platinum electrodes of 55 untreated liquid samples, including wines, beers, coffees, milks and fruit juices was performed, and the resulting voltammograms were analyzed using principal components analysis (PCA). Pattern recognition, using plots of the first two principal components, as well as cluster analysis, was used to determine subpopulation groupings. Variable selection, using subsets of the complete voltammograms, allowed finer classification of nonalcoholic beverages based on type and brand. Principal components regression (PCR) and partial least squares (PLS) were applied to voltammograms of diluted pure orange juice samples to determine whether adulteration can be detected by these methods. RMS errors in volume fractions in the calibration set were 2.0% and 1.8%, respectively, for PCR and PLS, and 4.0% (PCR) and 5.4% (PLS) in the validation set.

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-beta-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2 micrograms/ml. The calibration curves are linear from 0.4 to 16 micrograms/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.

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.

Ionic‐complementary peptide‐modified highly ordered pyrolytic graphite electrode for biosensor application

Ionic‐complementary peptides are promising new biomaterials with potential applications in bionanotechnology. In the present investigation, a typical ionic‐complementary peptide, EFK16‐II, was used to modify a highly ordered pyrolytic graphite (HOPG) electrode. Upon modification, peptide nanofibers, parallel or oriented 60° or 120° to each other, were formed on the surface of HOPG electrode. Surface wettability of the electrode was improved as indicated by a significant decrease in the water contact angle. The electrochemical response of the EFK16‐II nanofiber‐modified HOPG electrode for the ferricyanide/ferrocyanide redox couple was characterized. Cyclic voltammograms indicated that the presence of peptide nanofibers on the HOPG electrode did not block electron transfer at slow scan rates (∼2 mV/s), but did so at high scan rates (∼ 100 mV/s). A model enzyme glucose oxidase (GOx) was covalently immobilized onto this nanofiber‐modified electrode, and its potential as an enzyme‐based biosensor for glucose was examined. At an applied potential of +0.45 V (vs. Ag/AgCl), the current increased linearly with glucose concentration up to 7.5 mM and a relative high sensitivity was obtained at 11.3 ± 1.0 nA/(mM mm2). The immobilized GOx showed high affinity for glucose, with a Michaelis–Menten constant Km of 6.8 ± 0.9 mM. It also exhibited relatively good storage and operational stabilities, and reflected in only a small decrease (13%) in the current response after 1 month storage and negligible changes upon 50 cyclic voltammetric scans. The results presented here demonstrate an excellent potential of the use of ionic‐complementary peptides to modify electrode surfaces for biomolecular sensing and diagnostics.

Rotating disc electrode characterization of immobilized glucose oxidase

The kinetic properties of glucose oxidase (EC 1.1.3.4) which has been covalently immobilized to a rotating glassy carbon electrode surface have been investigated. Analysis of the rotation rate dependence of the hydrogen peroxide-derived current suggests that oxygen mass transport to the enzyme-electrode surface is rate controlling at low rotation rates. Only as the diffusion layer approaches zero thickness (i.e., infinitely fast rotation rate) does mass transport become unimportant. A diffusion-free glucose Km for air-saturated buffer is found to be 66 mM using this methodology. The importance of mass transport restrictions in two-substrate enzymes such as glucose oxidase is discussed in the context of biosensor design.

Determination of ferrocene iron in protein matrices

Abstract Trichloroacetic acid (TCA; 5%) was found to release iron quantitatively from a number of ferrocene derivatives. Free iron was then determined spectrophotometrically, using 564 nm absorbance maximum of the Fe(ferrozine) 2+ 3 complex [ferrozine = 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine]. As TCA also deproteinizes solutions, the ferrocene concentration of solutions initially containing up to 7 mg ml −1 protein was readily found by the spectrophotometric determination of iron in protein-free filtrates. Use of ferrozine as a chromogenic reagent led to a lower detection limit (ca. 0.2 μM ferrocene, ca. 0.01 mg l −1 Fe) than flame atomic absorption spectrometry for the detection of ferrocene iron in protein matrices. Total protein was determined spectrophotometrically using Coomassie Brillant Blue R250, a ferrocene did not interfere with this protein assay. The procedure was used to determine routinely the extent of covalent modification of glucose oxidase and lysozyme with a number of ferrocene derivatives.

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.

Single-Pulse Coulostatic Analysis

Abstract An instrument which performs oxidative and reductive single- pulse coulostatic analysis has been designed and constructed. An IBM Personal Computer with a DT2801 Single Board Analog and Digital I/O System coupled to an external potentiostat/coulostat circuit performs data acquisition, processing, and storage. Details of the hardware and software are presented along with results for the analysis of ferrocene in a methanol/ tetrabutyl ammonium tetrafluoroborate medium.