Favorably orienting recombinant proteins to develop amperometric biosensors to diagnose Chagas’ disease

Clinical immunoassays often display suitable sensitivity but some lack of specificity or vice versa. As a trade-off between specificity improvement and sensitivity loss, biosensors were designed to perform indirect immunoassays with amperometric detection using tailor-made chimeric receptors to react with the analyte, specific anti-Trypanosoma cruzi immunoglobulin G (IgG). Recombinant chimeras were designed to favor their oriented covalent attachment. This allows the chimeras to properly expose their epitopes, to efficiently capture the analyte, and to withstand severe chemical treatment to reuse the biosensors. By further binding the secondary antibody, horseradish peroxidase-labeled anti-human IgG, in the presence of the soluble mediator and the enzyme substrate, a current that increased with the analyte concentration was measured. Biosensors using the chimeric constructions showed 100% specificity with samples that had revealed false-positive results when using other bioreceptors. A protein bearing a poly-Lys chain and thioredoxin as directing elements displayed the highest signal-to-noise ratio (P < 0.05). The limit of detection was 62 ng ml⁻¹, which is eight times lower than that obtained with a currently used commercial Chagas enzyme-linked immunosorbent assay (ELISA) kit. Reusability of the biosensor was assessed. The signal was approximately 80% of the original one after performing 10 consecutive determinations.     

Sequential measurement of aminotransferase activities by amperometric biosensors

An l-glutamate biosensor modified by cation exchanger membrane on a palladium (Pd) electrode was designed for the purpose of preventing interferences and electrode fouling during the measurement of serum AST and ALT activities. The rate of signal increase obtained by our sensor for the determination of AST and ALT activity was 0.259 and 0.596 nA/min U(-1)l and the response of the sensor to AST and ALT activity were linear over the range of 8-200 and 8-250 Ul(-1), respectively. Both AST and ALT activities could be measured sequentially by injecting the serum into a solution containing l-aspartate and alpha-ketoglutarate. The rate of current increase was relative to AST activity. The activity of ALT was sequentially determined after addition of l-alanine into the solution. The change in the current increase rate after the addition of l-alanine was proportional to the ALT activity. By using the proposed biosensor, the interference of 1mM ascorbic acid was negligible on a dynamical aminotransferase determination when the dynamic data are taken after the steady state of an elevated baseline has been reached. The proposed l-glutamate biosensor provides adequate sensitivity for the measurement of AST and ALT and is expectable to be applied for rapid blood screening of AST and ALT activity in clinical sample.     

Electrochemically platinized carbon paste enzyme electrodes: A new design of amperometric glucose biosensors

A platinized carbon paste prepared via electrodeposition had a preferential electrocatalytic action toward H₂O₂. Therefore, we have developed a new amperometric glucose biosensor based on the immobilization of glucose oxidase on to the electrochemically platinized carbon paste. The proposed biosensor is free of potential interferences due to its cathodic detection of glucose at the potential of 0.0 V (vs. Ag/AgCl). It also shows acceptable analytical performance in terms of linearity (6 × 10⁻⁵ to 1.2 × 10⁻² M, r = 0.998), detection limit (2 × 10⁻⁵ M), response time (20–30 s), reproducibility (RSD = 4.4%), and storage life (t _0.80 = 45 days). All these advantages of the biosensor raise potential possibilities for its medical or other biotechnical applications.     

Amperometric glucose biosensors based on layer-by-layer assembly of chitosan and glucose oxidase on the Prussian blue-modified gold electrode

A glucose biosensor based on layer-by-layer (LBL) self-assembling of chitosan and glucose oxidase (GOD) on a Prussian blue film was developed. First, Prussian blue was deposited on a cleaned gold electrode then chitosan and GOD were assembled alternately to construct a multilayer film. The resulting amperometric glucose biosensor exhibited a fast response time (within 10 s) and a linear calibration range from 6 μM to 1.6 mM with a detection limit of 3.1 μM glucose (s/n = 3). With the low operating potential, the biosensor showed little interference to the possible interferents, including ascorbic acid, acetaminophen and uric acid, indicating an excellent selectivity.     

An amperometric cholesterol biosensor based on multiwalled carbon nanotubes and organically modified sol-gel/chitosan hybrid composite film

A new type of amperometric cholesterol biosensor based on sol-gel chitosan/silica and multiwalled carbon nanotubes (MWCNTs) organic-inorganic hybrid composite material was developed. The hybrid composite film was used to immobilize cholesterol oxidase on the surface of Prussian blue-modified glass carbon electrode. Effects of some experimental variables such as enzyme loading, concentration of Triton X-100, pH, temperature, and applied potential on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results show that analytical performance of the biosensor can be improved greatly after introduction of the MWCNTs. Response time, sensitivity, linear range, limit of detection (S/N=3), and apparent Michaelis-Menten constant Km are 25s, 0.54 microA mM(-1), 8.0 x 10(-6) to 4.5 x 10(-4) M, 4.0 x 10(-6) M, and 0.41 mM for the biosensor without MWCNTs and 13 s, 1.55 microA mM(-1), 4.0 x 10(-6) to 7.0 x 10(-4) M, 1.0 x 10(-6) M, and 0.24 mM for the biosensor with MWCNTs, respectively. The activation energy of the enzyme-catalyzed reaction was measured to be 42.6 kJ mol(-1). This method has been used to determine the free cholesterol concentration in real human blood samples.     

Enzyme logic gates for the digital analysis of physiological level upon injury

A biocomputing system composed of a combination of AND/IDENTITY logic gates based on the concerted operation of three enzymes: lactate oxidase, horseradish peroxidase and glucose dehydrogenase was designed to process biochemical information related to pathophysiological conditions originating from various injuries. Three biochemical markers: lactate, norepinephrine and glucose were applied as input signals to activate the enzyme logic system. Physiologically normal concentrations of the markers were selected as logic 0 values of the input signals, while their abnormally increased concentrations, indicative of various injury conditions were defined as logic 1 input. Biochemical processing of different patterns of the biomarkers resulted in the formation of norepi-quinone and NADH defined as the output signals. Optical and electrochemical means were used to follow the formation of the output signals for eight different combinations of three input signals. The enzymatically processed biochemical information presented in the form of a logic truth table allowed distinguishing the difference between normal physiological conditions, pathophysiological conditions corresponding to traumatic brain injury and hemorrhagic shock, and abnormal situations (not corresponding to injury). The developed system represents a biocomputing logic system applied for the analysis of biomedical conditions related to various injuries. We anticipate that such biochemical logic gates will facilitate decision-making in connection to an integrated therapeutic feedback-loop system and hence will revolutionize the monitoring and treatment of injured civilians and soldiers.     

Stable mediated amperometric biosensors using a graphite electrode embedded with tetrathiafulvalene in silicone oil

A new method has been developed to incorporate the mediator, tetrathiafulvalene (TTF), to the electrode/solution interface of an amperometric biosensor. TTF was dissolved in methylphenyl polysiloxane (silicone oil) and embedded in a graphite disc electrode. The mediator was able to diffuse to the electrode surface at an electrocatalytically significant speed. The storage of TTF in the inert polysiloxane provided a long-lasting and stable mediator supply.

TTF-silicone oil electrodes with immobilized glucose oxidase, xanthine oxidase, or amino acid oxidase exhibited sensitive, fast and reproducible responses. The glucose oxidase electrode was very stable for at least 2 months when stored at 4°C. Together with flow injection analysis (FIA), the enzyme electrodes were reused for at least 500 repeated analyses during a 25 h operation without losing their initial activity.     

An amperometric glucose biosensor based on poly(o-aminophenol) and Prussian blue films at platinum electrode

Prussian blue (PB), as a good catalyst for the reduction of hydrogen peroxide, has been combined with nonconducting poly(o-aminophenol) (POAP) film to assemble glucose biosensor. Compared with PB-modified enzymatic biosensor, the biosensor based on glucose oxidase immobilized in POAP film at PB-modified electrode shows much improved stability (78% remains after 30 days) in neutral medium. Additionally, the biosensor, at an applied potential of 0.0 V, exhibits other good characteristics, such as relative low detection limit (0.01 mM), short response time (within 5s), large current density (0.28 mA/cm2), high sensitivity (24 mAM(-1)cm(-2)), and good antiinterferent ability. The apparent activation energy of enzyme-catalyzed reaction and apparent Michaelis-Menten constant are 34.2 KJmol(-1) and 10.5 mM, respectively. In addition, effects of temperature, applied potential used in the determination, pH value of the detection solution, and electroactive interferents on the amperometric response of the sensor were investigated and are discussed.     

Interference elimination of an amperometric glucose biosensor using poly(hydroxyethyl methacrylate) membrane

A permselective membrane fabricated from photo‐cross‐linked poly(hydroxyethyl methacrylate) (pHEMA) was studied as a potential selective membrane that can eliminate electrochemical interferences commonly faced by a hydrogen peroxide‐based biosensor. The quantitative selection of the permselective membrane was based on the permeabilities of hydrogen peroxide and acetaminophen (AC). AC was used as a model of the interfering substance due to its neutral nature. pHEMA membrane with the cross‐linking ratio of 0.043 was found to achieve a selectivity of hydrogen peroxide over AC of 10, while maintaining an acceptable degree of hydrogen peroxide response. A two‐layer glucose biosensor model consisting of glucose oxidase entrapped within a freeze‐thawed poly(vinyl alcohol) matrix and the cross‐linked pHEMA membrane was challenged with AC, ascorbic acid and uric acid. 0.2 mM AC and 0.2 mM ascorbic acid were completely eliminated. However, 0.2 mM uric acid could not be completely eliminated and still gave a bias of approximately 6.6% relative to 5 mM glucose. The results showed that cross‐linked pHEMA was quite promising as an interference eliminating inner membrane.     

Site-specific immobilization of a (His)6-tagged acetylcholinesterase on nickel nanoparticles for highly sensitive toxicity biosensors

This paper reports site-specific affinity immobilization of (His)6-tagged acetylcholinesterase (AChE) onto Ni/NiO nanoparticles for the development of an electrochemical screen-printed biosensor for the detection of organophosphate pesticides. The method is based on the specific affinity binding of the His-tagged enzyme to oxidized nickel nanoparticle surfaces in the absence of metal chelators. This approach allows stable and oriented attachment of the enzyme onto the oxidized nickel through the external His residue in one-step procedure, allowing for fast and sensitive detection of paraoxon in the concentration range from 10⁻⁸ to 10⁻¹³ M. A detection limit of 10⁻¹² M for paraoxon was obtained after 20 min incubation. This method can be used as a generic approach for the immobilization of other His-tagged enzymes for the development of biosensors.     

Amperometric ethanol biosensor based on poly(vinyl alcohol)-multiwalled carbon nanotube-alcohol dehydrogenase biocomposite

A novel amperometric ethanol biosensor was constructed using alcohol dehydrogenase (ADH) physically immobilized within poly(vinyl alcohol)–multiwalled carbon nanotube (PVA–MWCNT) composite obtained by a freezing–thawing process. It comprises a MWCNT conduit, a PVA binder, and an ADH function. The measurement of ethanol is based on the signal produced by β-nicotinamide adenine dinucleotide (NADH), the product of the enzymatic reaction. The homogeneity of the resulting biocomposite film was characterized by atomic force microscopy (AFM). The performance of the PVA–MWCNT–ADH biocomposite modified glassy carbon electrode was evaluated using cyclic voltammetry and amperometry in the presence of NADH and in the presence of ethanol. The ethanol content in standard solutions was determined and a sensitivity of 196 nA mM⁻¹, a linear range up to 1.5 mM, and a response time of about 8 s were obtained. These characteristics allowed its application for direct detection of ethanol in alcoholic beverages: beer, red wine, and spirit.     

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine)

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.     

Determination of catalase-like activity in plants based on the amperometric monitoring of hydrogen peroxide consumption using a carbon paste electrode modified with ruthenium(IV) Oxide

A carbon paste electrode containing ruthenium(IV) oxide as a modifier was tested as an effective hydrogen peroxide amperometric sensor in bulk measurements (hydrodynamic amperometry). Factors that influence its overall analytical perform ance, such as pH and the applied potential, were examined. The RuO2-modified electrode displayed high sensitivity towards hydrogen peroxide, with detection limits as low as 0.02 mm at pH 7.4 and 0.007 mM at pH 9.0. The method was applied for monitoring the decomposition of hydrogen peroxide (by catalase) in phosphate buffer of pH 7.4. The relative response of the electrode towards ascorbic acid was assessed and it was found that the selectivity of the RuO2-modified electrode towards hydrogen peroxide over ascorbic acid could be significantly improved by electro-polymerizing m-phenylenediamine on its surface prior to measurements. The RuO2-modified electrode was used for the kinetic (fixed time) determination of catalase activity in the range of 4-40 U/mL (detection limit 1.2 U/mL). The method was applied to the determination of catalase-like activity in various plant materials (recov-ery ranged from 93 to 101%, detection limit 480 U/100 g).     

Immobilization of catalase via adsorption on poly(styrene-d-glycidylmethacrylate) grafted and tetraethyldiethylenetriamine ligand attached microbeads

Fibrous poly(styrene-d-glycidylmethacrylate) (P(S-GMA)) brushes were grafted on poly(styrene-divinylbenzene) (P(S-DVB)) beads using surface initiated-atom transfer radical polymerization (SI-ATRP). Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The multi-modal ligand attached beads were used for reversible immobilization of catalase. The influences of pH, ionic strength and initial catalase concentration on the immobilization capacities of the P(S-DVB)-g-P(S-GMA)-TEDETA beads have been investigated. Catalase adsorption capacity of P(S-DVB-g-P(S-GMA)-TEDETA beads was found to be 40.8 ± 1.7 mg/g beads at pH 6.5 (with an initial catalase concentration 1.0 mg/mL). The K_m value for immobilized catalase on the P(S-DVB-g-P(S-GMA)-TEDETA beads (0.43 ± 0.02 mM) was found about 1.7-fold higher than that of free enzyme (0.25 ± 0.03 mM). Optimum operational temperature and pH was increased upon immobilization. The same support was repeatedly used five times for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity.     

Monitoring the enzymatic conversion of urea to ammonium by conventional or microchip capillary electrophoresis with contactless conductivity detection

Interleukin-6 (IL-6), an inflammatory cytokine, is one of the most important mediators of fever, the acute phase response, and inflammatory conditions. Described here is an integrated microfluidic immunosensor capable of detecting the concentration of IL-6 in human serum samples by use of an electrochemical method in a microfluidic biochip format. The detection of IL-6 was carried out using a sandwich immunoassay method based on the use of anti-IL-6 monoclonal antibodies, immobilized on a 3-aminopropyl-modified controlled-pore glass (APCPG) packet in a central channel (CC) of the microfluidic system. The IL-6 in the serum sample is allowed to react immunologically with the immobilized anti-IL-6 and biotin-labeled second antibodies specific to IL-6. After washing, the streptavidin–alkaline phosphatase conjugate is added. p-Aminophenyl phosphate is converted to p-aminophenol by alkaline phosphatase, and the electroactive product is quantified on a gold electrode at 0.10 V. For electrochemical detection and enzyme immunoassay, the LOD was 0.41 and 1.56 pg mL⁻¹, respectively. Reproducibility assays employed repetitive standards of IL-6, and the intra- and inter-assay coefficients of variation were below 6.5%. Compared with the traditional IL-6 sensing method, the integrated microfluidic immunosensor required smaller amounts of sample to perform faster detection.     

Immobilization of Candida antarctica lipase B (CALB) on surface-modified rice husk ashes (RHA) via physical adsorption and cross-linking methods

In the present study, the recovery of activity of Candida antarctica lipase B (CALB) immobilized onto surface-modified rice husk ash (RHA) was 90% for both cross-linking and adsorption methods. Both cross-linked and adsorbed immobilized preparations were very stable, retaining more than 48% of their activity over the range of temperatures studied. The optimum temperature and optimum pH values were 37?°C and 7.0, respectively for both immobilized preparations, while the relative activities after storage at 4.0?°C for 60 days were 55% and 65% using cross-linking and adsorption methods, respectively. Also, the activity of the immobilized lipase began to decrease after 10 cycles, more than 58% of the initial activities were still retained after 10 cycles for both immobilization methods. These results indicated that lipase immobilized by cross-linking and adsorption not only effected activity recovery, but also remarkably effected stability, reusability and application adaptability. It can be concluded that, surface-modified RHA can be used as alternative supports for immobilization of CALB for polymerization reactions.     

Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod (G. morhua) using a 96-well microplate technique

Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0-40 degrees C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60 degrees C with high stability and accuracy in any data acquisition mode. At 4 degrees C the accuracy of the temperature control was +/-0.1 degrees C and temperature homogeneity across the microplate was +/-0.3 degrees C. As examples, analyses of the temperature dependence of Michaelis-Menten (K'(PYR)(m) and substrate inhibition (K'(PYR)(si) constants for pyruvate, of the maximal rate of reaction (V'(max), of the apparent Arrhenius activation energy (E(A), and of the Gibbs free-energy change (deltaG) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4 degrees C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change.     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.