Glenn J. Lubrano

An enzyme electrode for the amperometric determination of glucose.

Abstract An amperometric method utilizing a glucose electrode has been developed for the determination of blood glucose. The time of measurement is less than 12 s if a kinetic method is used and 1 min if a steady-state method is used. The long-term stability of the electrode is ca. 0.1% change from maximum response per day when stored at room temperature for over 10 months. The enzyme electrode determination of blood sugar compares favorably with commonly used methods with respect to accuracy, precision, and stability. The only reagent required for blood sugar determinations is a buffer solution. The electrode consists of a metallic sensing layer covered by a thin film of immobilized glucose oxidase held in place by means of cellophane. When poised at the correct potential, the current produced is proportional to the glucose concentration.

A study of interferences in glucose measurements in blood by hydrogen peroxide based glucose probes

The main blood constituents which could interfere in clinical glucose measurements using a hydrogen peroxide based glucose electrode have been investigated using several different membranes and constant and sweeping potentials. Both diluted and whole undiluted sera were investigated. With a 3500 molecular weight cut-off (MWCO) membrane, acetaminophen, cysteine, and ascorbic acid can interfere. With a 100 MWCO membrane, only acetaminophen interfered.

Non-invasive biosensors in clinical analysis

Several amperometric biosensors have been developed and applied for the non invasive determination of metabolites in body fluids. Advantages of saliva or sweat analysis are the ease of sample collection and that samples can be collected more frequently with much less stress on the patient. An alcohol biosensor has been developed with a hydrogen peroxide based electrode utilizing immobilized alcohol oxidase. Immobilization parameters have been optimized to increase the stability of the enzyme. An auter hydrophobic gas membrane was used to improve the selectivity of the probe. A hydrogen peroxide based amperometric biosensor has also been developed that utilized the enzyme glucose oxidase. The biosensor was applied to the determination of sera and saliva glucose content. Two hydrogen peroxide based amperometric biosensors that utilized lactate oxidase were also developed for determination of lactate in saliva and sweat. To discriminate against electroactive substances, the biosensor for assay of lactate in saliva utilized a dual electrode with one side active and one inactive, while the biosensor for assay of sweat lactate content utilized a hydrophobic hydrogen peroxide membrane to improve selectivity. Lactate content of saliva and sweat samples were measured after an intense physical exercise. A new procedure to measure glucose via transbuccal mucosa was developed using a dual glucose probe similar to that used for lactate. Correlation between glucose in blood and in transbuccal mucosa has been evaluated.

Amperometric enzyme electrodes

Abstract An amperometric method based on an L-amino acid oxidase electrode has been developed for the determination of several L-amino acids. The time of measurements is less than 12 s if a kinetic method is used, and 1 min if a steady-state method is used. The only reagent required is a phosphate buffer solution.

A quartz crystal microbalance displacement assay for Listeria monocytogenes

Abstract A novel, piezoelectric, quartz crystal microbalance (QCM) biosensor has been developed for the detection of Listeria monocytogenes . The Listeria was immobilized on the gold surface of the crystals using different immobilization approaches. Using a liquid flow cell, it was possible to directly monitor the antigen (Ag)-antibody (Ab) binding without the use of a label, i.e. enzymes, radioactive compounds, etc. The calibration curve was prepared using a displacement assay with a response range from 2.5 × 10 5 to 2.5 × 10 7 cells crystal . The assay was also performed in milk which was injected with the non-specific antigen Serratia or Listeria . The method is as sensitive as ELISA and results can be obtained in less than 15 min.

Amperometric enzyme electrodes: Part III. Alcohol oxidase

Abstract A rapid, simple method for the measurement of ethanol or methanol has been developed by coupling amperometric hydrogen peroxide monitoring to the alcohol oxidase enzyme system. Alcohols can be measured within several seconds at concentrations as low as 0.5 mg/100 ml. The only reagent required is alcohol oxidase in phosphate buffer.

Amperometric biosensor for determination of lactate in sweat

Abstract An amperometric hydrogen peroxide based biosensor has been developed for non-invasive determination of l -lactate. The biosensor utilizes lactate oxidase immobilized between a polycarbonate membrane and a polytetrafluoroethylene (PTFE) blocking membrane to effectively eliminate electrochemical interferences. Both the steady state current and maximum rate of current change were measured. The response times were 2 min and 10 s, respectively. Because of the addition of polycarbonate and PTFE membranes, the linear range of lactate was extended up to 140 mg dl−1 and the apparent Michaelis-Menten constant was almost two orders or magnitude higher than that of the free enzyme. The biosensor was applied to the analysis of sweat l -lactate content of healthy subjects during physical exercise.

Piezoelectric Detection of Ricin and Affinity-Purified Goat Anti-ricin Antibody

Abstract Detection of ricin, in a piezoelectric quartz crystal microbalance format, can be accomplished with the use of capture antibody techniques. These techniques allow for the specific attachment of ricin to immobilized capture antibodies on the quartz crystal transducer area. A reversed format can also be used to detect antibody in solution. In this case, the antigen is immobilized and antibody attaches specifically, thus increasing the overall mass and decreasing the resonant frequency. In this report we describe detection of both ricin and anti-ricin antibody using immunological piezoelectric quartz crystal microbalance techniques. Partial support for this research was supplied by an Army Phase II SBIR Grant #DAAA/5-92-C-0083 from the Department of Defense, Edgewood Arsenal.

Determination of lactate in human saliva with an electrochemical enzyme probe

Abstract Electrochemical biosensors for lactate were assembled and used for the determination of lactic acid in saliva. Saliva was collected from healthy subjects and immediately screened for its lactate content. The electrochemical and biological interferences from saliva were discriminated by using a dual platinum electrode and blocking membranes. The stability, reproducibility and lifetime of the probe were studied. Lactate was measured in eight subjects in fasting conditions and after eating, showing an increase in lactate for each subject after meals. Correlation with a spectrophotometric lactate measurement is reported. Subjects before, during and after physical exercise showed consistent variations of lactate in saliva.

AMPEROMETRIC ASPARTATE ELECTRODE

Abstract An amperometric enzyme electrode for L -aspartate determination was developed. The probe consisted of a platinum electrode which senses hydrogen peroxide produced from the reactions catalyzed by two enzymes co-immobilized on a preactivated polymeric membrane, α-Ketoglutarate in the presence of L -aspartate was transaminated to L -glutamate by aspartate aminotransferase and the glutamate produced was oxidized by glutamate oxidase, with concomitant production of hydrogen peroxide. Additional protective membranes eliminated interferences from glutamate and most electroactive compounds. The response curve of the probe was linear over the concentration range 1.0 × 10−6 M to 2.0 × 10−4 M aspartate and was useful for at least two months. Aspartic acid in some pharmaceutical products was determined and the results correlated well with a liquid chromatographic reference method and the manufacturers specification.