Silica sol-gel composite film as an encapsulation matrix for the construction of an amperometric tyrosinase-based biosensor

An amperometric tyrosinase enzyme electrode for the determination of phenols was developed by a simple and effective immobilization method using sol-gel techniques. A grafting copolymer was introduced into sol-gel solution and the composition of the resultant organic-inorganic composite material was optimized, the tyrosinase retained its activity in the sol-gel thin film and its response to several phenol compounds was determined at 0 mV vs. Ag/AgCl (sat. KCI). The dependences of the current response on pH, oxygen level and temperature were studied, and the stability of the biosensor was also evaluated. The sensitivity of the biosensor for catechol, phenol and p-cresol was 59.6, 23.1 and 39.4 muA/mM, respectively. The enzyme electrode maintained 73% of its original activity after intermittent use for three weeks when storing in a dry state at 4 degreesC. (C) 2000 Elsevier Science S.A. All rights reserved.

Cyro-hydrogel for the construction of a tyrosinase-based biosensor

A new immobilization method for construction of a tyrosinase based biosensor is described. A simple physical freezing technique was adopted for preparation. The immobilized enzyme yields specific activities that are more than 22% of the soluble enzyme. The enzyme electrode can be stored in dry state for more than three months without any loss of activity. The biosensor was applied to the determination of several phenols and o-diphenols. The lowest detect limit is 0.02 mu mol/1 and the linear range was 1.0 X 10(-7)-1.0 X 10(-4) mol/1 for catechol. The kinetic parameters have also been calculated.

CONSTRUCTION OF A TYROSINASE-BASED BIOSENSOR IN PURE ORGANIC-PHASE

A novel immobilization method for construction of a tyrosinase-based biosensor applied in pure organic phase is described. This method gives the enzyme a hydrated shell which allows the enzyme to maintain its biocatalytic activity in a pure organic solvent The enzyme electrode was used to determine several phenols and o-diphenols in pure chloroform and chlorobenzene. The biosensor can be stored in dry state for more than 3 months without any loss of the activity. The kinetic parameters have also been calculated and are presented herein.

Self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine as an immobilization matrix for the construction of a tyrosinase-based amperometric biosensor

A tyrosinase-based amperometric biosensor using a self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine (PVA-g-PVP) as an immobilization matrix was constructed. The 4-vinylpyridine component of PVA-g-PVP enhances the adherence to a glassy carbon electrode surface. The content of 4-vinylpyridine in this immobilization matrix plays a key role in retaining the activity of tyrosinase. A simple, milder method was adopted by simply syringing the copolymer-tyrosinase aqueous solution on to the electrode surface and allowing water to evaporate at 4 degrees C in a refrigerator. Several parameters, including copolymer composition; pH, applied potential and enzyme membrane composition, ware optimized. The enzyme membrane composition can be varied to obtain higher sensitivity or a wider linear detection range. The biosensor was used for the determination of phenol, p-cresol and catechol. The biosensor exhibited excellent reproducibility, stability and sensitive response and can be used in flow injection analysis. The biosensor showed an extended linear range in hydrophilic organic solvents and it can be used in monitoring organic reaction processes. The analytical performance demonstrated this immobilization matrix is suitable for the immobilization of tyrosinase.

Electrochemical current rectification – a novel signal amplification strategy for highly sensitive and selective aptamer-based biosensor

Electrochemical aptamer-based (E-AB) sensors represent an emerging class of recently developed sensors. However, numerous of these sensors are limited by a low surface density of electrode-bound redox-oligonucleotides which are used as probe. Here we propose to use the concept of electrochemical current rectification (ECR) for the enhancement of the redox signal of E-AB sensors. Commonly, the probe-DNA performs a change in conformation during target binding and enables a nonrecurring charge transfer between redox-tag and electrode. In our system, the redox-tag of the probe-DNA is continuously replenished by solution-phase redox molecules. A unidirectional electron transfer from electrode via surface-linked redox-tag to the solution-phase redox molecules arises that efficiently amplifies the current response. Using this robust and straight-forward strategy, the developed sensor showed a substantial signal amplification and consequently improved sensitivity with a calculated detection limit of 114 nM for ATP, which was improved by one order of magnitude compared with the amplification-free detection and superior to other previous detection results using enzymes or nanomaterials-based signal amplification. To the best of our knowledge, this is the first demonstration of an aptamer-based electrochemical biosensor involving electrochemical rectification, which can be presumably transferred to other biomedical sensor systems.

Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor

Up-converting phosphor technology (UPT)-based lateral-flow immunoassay has been developed for quantitative detection of Yersinia pestis rapidly and specifically. In this assay, 400 nm up-converting phosphor particles were used as the reporter. A sandwich immumoassay was employed by using a polyclonal antibody against F1 antigen of Y. pestis immobilized on the nitrocellulose membrane and the same antibody conjugated to the UPT particles. The signal detection of the strips was performed by the UPT-based biosensor that could provide a 980 nm IR laser to excite the phosphor particles, then collect the visible luminescence emitted by the UPT particles and finally convert it to the voltage as a signal. V-T and V-c stand for the multiplied voltage units for the test and the control line, respectively, and the ratio V-T/V-C is directly proportional to the number of Y pestis in a sample. We observed a good linearity between the ratio and log CFU/ml of Y pestis above the detection limit, which was approximately 10(4) CFU/mI. The precision of the intra- and inter-assay was below 15% (coefficient of variation, CV). Cross-reactivity with related Gram-negative enteric bacteria was not found. The UPT-LF immunoassay system presented here takes less than 30 min to perform from the sample treatment to the data analysis. The current paper includes only preliminary data concerning the biomedical aspects of the assay, but is more concentrated on the technical details of establishing a rapid manual assay using a state-of-the-art label chemistry. (c) 2006 Elsevier B.V. All rights reserved.

H+ISFET-based biosensor for determination of penicillin G

A biosensor based on an H+ ion sensitive field effect transistor (H+-ISFET) and penicillin G acylase has been developed. The response time of the sensor to different concentrations of penicillin G was 30 s. In a 20 mM phosphate buffer at pH 7.0, the linear range of the calibration curve was from 0.5 to 8 mM. The coefficients of variation for three samples with 20 repeated measurements were below 5%. Stability of the sensor could reach about 6 months and more than 1000 runs were performed without a significant decrease of the output value. The sensor was tested for measurement of the penicillin G content in penicillin fermentation broth. Forty samples with low and high concentrations of penicillin G were chosen for the correlation test. The values assayed by the sensor method were compared with the values assayed by HPLC method, the correlation coefficient (r) was 0.9944 and the regression equation was y = 1.034X - 2083.7 respectively. The different measuring methods are discussed in the text. (C) 1998 Published by Elsevier Science S.A. All rights reserved.

[Ru(bpy)(2)(dcbpy)NHS] Labeling/Aptamer-Based Biosensor for the Detection of Lysozyme by Increasing Sensitivity with Gold Nanoparticle Amplification

A novel [Ru(bpy)(2) (dcbpy)NHS] labeling/aptamer-based biosensor combined with gold nanoparticle amplification for the determination of lysozyme with an electrochemiluminescence (ECL) method is presented. In this work, an aptamer, an ECL probe, gold nanoparticle amplification, and competition assay are the main protocols employed in ECL detection. With all the protocols used, an original biosensor coupled with an aptamer and [Ru(bpy)(2)(dcbpy)NHS] has been prepared. Its high selectivity and sensitivity are the main advantages over other traditional [Ru(bpy)(3)](2+) biosensors. The electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) characterization illustrate that this biosensor is fabricated successfully. Finally, the biosensor was applied to a displacement assay in different concentrations of lysozyme solution, and an ultrasensitive ECL signal was obtained. The ECL intensity decreased proportionally to the lysozyme concentration over the range 1.0 x 10-(13)-1.0 x 10(-8) mol L-1 with a detection limit of 1.0 x 10(-13) mol L-1.

i-Motif Quadruplex DNA-Based Biosensor for Distinguishing Single- and Multiwalled Carbon Nanotubes

The increasing worldwide demand for carbon nanotubes (CNTs) and increasing concern regarding how to safely develop and use CNTs are requiring a low-cost, simple, and highly sensitive CNT detection assay for toxicological evaluation and environmental monitoring. However, this goal is still far from being achieved. All the current CNT detection techniques are not,applicable for automation and field analysis because they are dependent on highly expensive special instruments and complicated sample preparation. On the basis of the capability of single-walled carbon nanotubes (SWNTs) to specifically induce human telomeric i-motif formation, we design an electrochemical DNA (E-DNA) sensor that can distinguish single- and multiwalled carbon nanotubes both in buffer and in cell extracts. The E-DNA sensor can selectively detect SWNTs; with a direct detection limit of 0.2 ppm and has been demonstrated in cancer cell extracts. To the best of our knowledge, this is the first demonstration of a biosensing technique that can distinguish different types of nanotubes. Our work will provide new insights into how to design a biosensor for detection of carbon nanotubes.

Electrochemical biosensors based on advanced bioimmobilization matrices

Biosensors have experienced rapid, extensive development. To maintain the bioactivity of biomolecules and to give the electrochemical output signal required, appropriate bioimmobilization matrices for biomolecules are critical.In this review, we describe some advanced membrane materials (including hydrogels, sol-gel-derived organic-inorganic composites and lipid membranes), introduce electrochemical biosensors based on bioimmobilization materials and describe their performance.Biosensors operating in extreme conditions and displaying direct electron transfer with electrodes based on these advanced membrane materials are attractive. Recent developments in nanomaterials include biosensors, so we emphasize the intersection of nanomaterials with advanced membrane materials in biosensors.

Organic-phase enzyme electrode for phenolic determination based on a functionalized sol-gel composite

An amperometric biosensor for monitoring phenols in the organic phase was constructed by the silica sol-gel immobilization of tyrosinase on a glassy carbon electrode. The organic-inorganic hybrid materials with different sol-gel precursors and polymers were optimized, and the experimental conditions, such as the effect of the solvent, operational potential and enzyme loading were explored for the optimum analytical performance of the enzyme electrode. The biosensor can reach 95% of steady-state current in about 18 s, and the trend in the sensitivity of different phenols is as follows: catechol > phenol >p-cresol. In addition, the apparent Michaelis-Menten constants (K-m(app)) and the stability of the enzyme electrode were discussed. (C) 2000 Elsevier Science S.A. All rights reserved.

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH• radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADHmeasured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50nMof AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

beta-Lactamase-based biosensor for the electrochemical determination of benzylpenicillin in milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

New PON1-based biosensor for the detection of homocysteine-thiolactone in human plasma

Poster presented at the XXIII International Symposium on Bioelectrochemistry and Bioenergetics, 14-18 June 2015, Malmo, Sweden.