Reagentless Bioluminescent Sensor for NADH

Abstract

A reagentless fiber optic biosensor specific for NADH, associated with flow injection analysis and based on bacterial bioluminescence is described. Only a buffer solution as flowing stream is required for the probe which works without supply of its coreactants (FMN and N-decyl-aldehyde). These are coentrapped in a poly(vinyl)alcohol (PVA) matrix which allows their internal release in the vicinity of the immobilized enzymes. Two PVA matrices differing by the reticulation process have been tested : first, by polymerization with glutaraldehyde and secondly, by a cyclic freezing-thawing process. The self-containment working time was estimated at 1 and 1.5 h of continuous measurements, respectively. NADH was determined using flow injection analysis. The sensor gave excellent reproducibility (RSD ≤ 3%) in the linear dynamic range 5 — 500 pmol with an average cycle-time of 2.5 min.     

Highly Stable Bioluminescence-Based Fiber-Optic Sensor Using Immobilized Enzymes from Vibrio Harveyi

Abstract

The properties of the bioluminescent enzymatic systems from V. fischeri and V.harveyi immobilized on preactivated polyamide membranes, as well as the characteristics of a fiber-optic biosensor equipped with such membranes, have been studied. Particular attention has been paid to the stability of the bioactive matrices under working conditions, since this remains a key-point for designing operational biosensors of practical use. Whatever the origin of the bioluminescent system, the microdetermination of NADH could be performed at the nanomolar level with detection limits of 2 nM and 0.3 nM with the systems from V. harveyi and V. fischeri, respectively. the use of polyethyleneglycol (PEG 600) improves the operational stability of the bi-enzymatic system from V. fischeri, but a recalibration must be carried out every ten assays. Although the immobilized system from V. harveyi exhibited a lower activity than the enzymes from V. fischeri, its excellent operational stability (100 assays performed within a day without loss in activity) makes it more suitable in designing a truly operational bioluminescence-based NADH sensor.     

Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors

Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.     

High‐sensitive Electrochemical Determination of Ethyl Carbamate Using Urethanase and Glutamate Dehydrogenase Modified Electrode

An amperometric biosensor for ethyl carbamate (EC) was developed for the first time through the cascade reactions of urethanase and glutamate dehydrogenase (GLDH). Urethanase decomposes ethyl carbamate to produce ammonia, which converts to L‐glutamate under the catalysis of GLDH in the presence of α‐ketoglutarate and NADH. Then the change of NADH can be detected chronoamperometrically. The two enzymes were entrapped into chitosan/gelatine/γ‐glycidoxy propyl trimethoxy silane sol‐gel and immobilized on the surface of pyrolytic graphite electrode (PGE). The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimized conditions, the amperometric EC biosensor exhibits a linear detection range from 0.5 to 40 μM with a low detection limit of 5.30 nM. The biosensor was successfully used to detect EC in mimic Chinese rice wine samples, and satisfactory recovery and relative standard deviation were achieved.     

Fabrication of Redox‐Mediator Supported Potentiometric Nitrate Biosensor with Nitrate Reductase

Fabrication of a more superior nitrate potentiometric biosensor than previously achieved with NaR and NADH has been accomplished by co‐entrapment of redox mediators and NaR into polypyrrole (PPy) film during galvanostatic polymerization of pyrrole. The replacement of NADH with redox mediators such as thionin acetate (ThAc), safranin (Saf), and azure A (AzA) gave more sensitive potentiometric responses, better minimum detectable concentration, linear concentration range and response time for nitrate than possible with NADH. The co‐entrapment of ThAc, Saf, AzA and methyl viologen (MV) with NaR into PPy films also improved the Nernstian behavior of the electrode process beyond the capability of the PPy‐NaR‐NADH biosensor. Substantial reduction in volume and quantity of cofactor/mediator and, hence cost, was achieved by the replacement of NADH with a redox mediator. Only 50 μM of AzA was required to form a PPy‐NaR‐AzA biosensor which gave the most sensitive potentiometric response for nitrate, achieving a minimum detectable concentration of 10 μM, a linear concentration range of 50–5000 μM and a response time of 2–4 s.     

Luciferin incorporation in the structure of acrylic microspheres with subsequent confinement in a polymeric film: A new method to develop a controlled release-based biosensor for ATP, ADP and AMP

The controlled release of coreactants at the sensing tip of a biosensor is a possible approach to develop self-contained devices. For luciferin which is a firefly luciferase cosubstrate, a new method of retention is evaluated. The two-step procedure consists of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a PVA SbQ film. When associated with a compartmentalised trienzymatic sequence (adenylate kinase, creatine kinase and luciferase), such a complex matrix ensures the internal delivery of the cosubstrate in the enzymatic microenvironment at a controlled rate. For the three adenylic nucleotides (ATP, ADP and AMP), the self-containment working time is 3 h of continuous and reproducible assays. The sensitivity of the fibre optic biosensor represents, for ATP, 30% of that obtained when luciferin is supplied in solution whereas for ADP and AMP, the values are about 80% of the reference ones.     

Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose

NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10^-5 M and 5.8% at 2 × 10^-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10^-4 M and 3.2% at 1 × 10^-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.     

Porous and electrically conductive polypyrrole-poly(vinyl alcohol) composite and its applications as a biomaterial

Bulk modification of polypyrrole (PPY) with poly(vinyl alcohol) (PVA) was carried out by the electropolymerization of pyrrole in the presence of PVA in the reaction solution, with tetraethylammonium perchlorate (TEAP) as the electrolyte. The surface morphology of the as-synthesized PPY-TEAP-PVA film was investigated using scanning electron microscopy, and the film was further characterized using X-ray photoelectron spectroscopy, electrical conductivity, the water contact angle, and BET surface area measurements. The PPY-TEAP-PVA composite is electrically conductive, hydrophilic, and microporous with a high surface area. Its potential as a biomaterial was investigated with respect to its blood compatibility and function as a substrate for biosensor fabrication and cell culture. The presence of PVA in the film attenuates blood protein adsorption, and the porous nature of the PPY-TEAP-PVA film results in a 10-fold increase in the amount of glucose oxidase covalently immobilized on the film over that on a nonporous PPY film. PC12 cell attachment and growth on the PPY-TEAP-PVA film was also shown to be enhanced compared with that on tissue culture polystyrene. The attached cells proliferated and formed a monolayer on the film surface after 48 h of seeding.     

Detection of fragmented genomic DNA by PCR-free piezoelectric sensing using a denaturation approach

Label-free and real-time DNA sequence detection in PCR-amplified DNA samples can now be achieved by different approaches. On the contrary, only few works have been reported dealing with direct sequence detection in nonamplified genomic DNA. Here, a piezoelectric biosensor for direct detection of sequences in nonamplified genomic DNA is described. The system relies on real-time and label-free detection of the hybridization reaction between an immobilized probe and the complementary sequence in solution. The DNA probe is immobilized on the sensing surface (10 MHz quartz crystals), while the complementary sequence is present in the genomic DNA, previously fragmented with restriction enzymes.     

A sensitive continuous-flow bioluminescent system for determining ethanol in serum and saliva

A continuous-flow system, based on NAD(P)H:FMN oxidoreductase and bacterial luciferase co-immobilized on a nylon coil placed in front of a photomultiplier, and alcohol dehydrogenase separately immobilized on a second nylon coil, is described for the assay of ethanol in serum and saliva. The flow is air-segmented and 5–50-μl samples are required. The sample throughput is 25–30 h^?1 with no carryover. The detection limit is 1 μmol l^?1 ethanol (50 pmol injected) and the response is linear between 50 and 2500 pmol of ethanol. The relative standard deviation is 3–10% for both within-day and between-day assays, and serum and saliva can be analyzed directly. The immobilized enzymes are satisfactorily stable and up to 900 samples can be analyzed with one enzyme reactor.     

Development and application of a biosensor for hypoxanthine in fish extract

A hypoxanthine biosensor was constructed using immobilized xanthine oxidase and a polarographic electrode. The enzyme was covalently immobilized on a commercially available preactivated nylon membrane. The polarographic electrode detected hydrogen peroxide and uric acid released during the enzymatic reaction. The electrode responded linearly to hypoxanthine concentration in the range 3.6–107 μM. When applied to the determination of hypoxanthine in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method. More than 40 assays could be performed with the same membrane and each sample could be assayed in ca. 2–3 min. The biosensor provides a reliable, simple, rapid and economical method for the measurement of hypoxanthine, a useful indicator of fish freshness.     

Highly Sensitive Impedimetric Biosensor Based on Thermolysin Immobilized on a GCE Modified with AuNP-decorated Graphene for the Detection of Ochratoxin A

The fabrication of a thermolysin-based biosensor capable of detecting ochratoxin A (OTA) from food samples is described. The electrochemical deposition of calcium cross-linked cellulose film (CCLC) and gold nanoparticles (AuNPs) on graphene (GR) for modification of a glassy carbon electrode (GCE) is the first step. Then the thermolysin (TLN) enzyme in a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix is immobilized. The impedimetric biosensor response is linear from 0.2 nM to 100 nM with a detection limit of 0.2 nM. The obtained stable and reproducible biosensor is then applied for the detection of OTA in spiked extracts from coffee beans.     

Preparation of PVA membrane for immobilization of GOD for glucose biosensor

A membrane was prepared using polyvinyl alcohol (PVA) with low and high degree of polymerization (DOP), acetone, benzoic acid (BA) and was cross-linked by UV treatment. Membrane composition was optimized on the basis of swelling index. Membrane prepared with 12% low DOP and 8% high DOP of PVA, 2% BA, dissolved in buffer containing 20% acetone and cross-linked with UV treatment exhibited lower swelling index. Fourier transform infrared (FTIR) study of the membranes showed appearance of a strong band at approximately 2337 cm(-1) when UV was used for cross-linking in the presence of benzoic acid. Scanning electron microscope (SEM) study revealed that membrane cross-linked with UV treatment was smoother. Glucose oxidase (GOD)-PVA membrane was associated with the dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose to gluconolactone. A wide detection range, 0.9-225 mg/dl was estimated from the linear range of calibration plot of biosensor reading. Membranes were reused for 32 reactions without significant loss of activity and stored for 30 days (approximately 90% activity) at 4 degrees C. Membranes were also used with real blood samples.     

Protein-immobilized electrode for rapid and convenient sensing of thyroid hormone receptor-ligand interaction.

The recombinant human thyroid hormone receptor (TR) was expressed as an in-frame fusion with ten consecutive histidine residues using a bacterial system; then the receptor was immobilized on an Au-electrode with Ni(II)-mediated chemisorption using the histidine tag and thiol-modified nitrilotriacetic acid. The receptor-modified electrode could rapidly detect ligand binding to hTR without any separation procedures, and showed a good response in a concentration-dependent manner. The sensitivity of this biosensor based on ligand-receptor interactions was comparable to those of conventional competitive ligand binding assays using radio-labeled ligands. Our results strongly suggest that our new biosensor can be applied to the identification of new ligands for hTR.     

Potentiometric Biosensor System Based on Recombinant Urease and Creatinine Deiminase for Urea and Creatinine Determination in Blood Dialysate and Serum

A biosensor system for simultaneous determination of creatinine and urea in blood serum and dialysate samples was developed. It consisted of creatinine and urea biosensors based on a potentiometric transducers with two identical pH‐sensitive field‐effect transistors. In creatinine biosensor, creatinine deiminase immobilized via photopolymerization in PVA/SbQ polymer on one transistor served as a biorecognition element, while bovine serum albumin in PVA/SbQ polymer placed on the second transistor was used for reference. The urea biosensor was created in the same way but recombinant urease was used instead of creatinine deiminase. The linear ranges of creatinine and urea measurement were 0.02–2 mM and 0.5–15 mM, correspondingly, which allowed simultaneous determination of the metabolites. Response time of the biosensor system was 2–3 min; RSD of responses did not exceeded 5 %. The biosensors demonstrated absence of non‐selective response towards components of blood dialysate and serum. Urea and creatinine concentrations were determined in 20 samples of blood dialysate and serum. The results correlated well with traditional methods of analysis. Creatinine and urea biosensors were stable during five months of storage (during this time the responses decreased by about 10 %). The proposed biosensor system can be effectively used for analysis of serum samples and for hemodialysis control.     

An optical fiber biosensor for chlorpyrifos using a single sol-gel film containing acetylcholinesterase and bromothymol blue

An optical fiber biosensor consisting of acetylcholinesterase (AChE) and bromothymol blue (BTB) doped sol-gel film was employed to detect organophosphate pesticide chlorpyrifos. The main advantage of this optical biosensor is the use of a single sol-gel film with immobilized AChE and BTB. The compatibility of this mixture (AChE and BTB) with the sol-gel matrix has prevented leaching of the film. The immobilization of the enzyme and indicator was simple without chemical modification. The biosensing element on single sol-gel film has been placed inside the flow-cell for flow system. In the presence of a constant AChE, a color change of the BTB and the measured reflected signal at wavelength 622nm could be related to the pesticide concentration in the sample solutions. The performance of optical biosensor in the flow system has been optimized, including chemical and physical parameters. The response time of the biosensor is 8min. A linear calibration curve of chlorpyrifos against the percentage inhibition of AChE was obtained from 0.05 to 2.0mg/L of chlorpyrifos (18-80% inhibition, R(2)=0.9869, n=6). The detection limit for chlorpyrifos was 0.04mg/L. The results of the analysis of 0.5-1.5mg/L of chlorpyrifos using this optical biosensor agreed well with chromatographic method.     

The Use of Poly(Vinyl Alcohol) to Cross‐link Penicillinase for the Fabrication of a Penicillin Potentiometric Biosensor

Chemically cross‐linked PVA films are permeable matrices for the fabrication of biosensors. PVA provides an attractive immobilisation method as it preserves the enzymatic activity. Penicillinase (P’nase) was cross‐linked with poly(vinyl alcohol) (PVA) and bovine serum albumin (BSA). The optimum conditions for the of BSA‐PVA‐P’nase film were: 2.5 % w/v PVA, 0.006 % w/v BSA, 2.4 mM penicillin (Pen) and 16 U/mL P’nase. The minimum detectable concentration was 1.7 µM. The linear concentration range obtained for the BSA‐PVA film was 7.5–283 µM. The BSA‐PVA P’nase biosensor detected penicillin in amoxycillin with an average percentage recovery of 97±12 %. Higher penicillin concentrations (10–20 ppm) were detected more successfully than lower concentrations (≤5 ppm). These results indicate that further work is required to enable the successful detection of lower penicillin concentrations such as 5 ppm.     

A low-cost,fast, disposable and sensitive biosensor study: flow injection analysis of glucose at poly-methylene blue-modified pencil graphite electrode

In this work, firstly methylene blue (MB) was electropolymerized onto pencil graphite electrode (PGE) surface for the electrocatalytic oxidation of NADH. Cyclic voltammograms show that oxidation potential of NADH at Poly-MB/PGE shifted to negative direction about 300 mV compared with bare PGE. These results indicate that Poly-MB/PGE exhibits a good electrocatalytic activity toward NADH oxidation. Then, a glucose biosensor study was performed based on the determination of enzymatically generated NADH by glucose dehydrogenase (GDH) which immobilized onto Poly-MB/PGE using glutaraldehyde cross-linking procedure. The biosensing of glucose in flow injection analysis (FIA) system was performed at GDH/Poly-MB/PGE for the first time. The electrocatalytic oxidation currents of enzymatically produced NADH obtained from FI amperometric current–time curves recorded at + 200 mV and in phosphate buffer solution at pH 7.0 containing 1.0 M KCl were linearly related to the concentration of glucose. Linear calibration plots are obtained in the concentration range from 0.01 to 1.0 mM. The limit of detection (LOD) was found to be 4.0 µM. A fast, sensitive, low-cost and disposable glucose biosensor was constructed in FIA system using GDH/Poly-MB/PGE; therefore, it might provide a new perspective for the fabrication of biosensor of other compounds such as glutamate, lactate and alcohol.     

Immobilized Laccase on Activated Poly(Vinyl Alcohol) Microspheres For Enzyme Thermistor Application

Poly(vinyl alcohol) (PVA) microspheres were prepared by inverse suspension crosslinked method, with glutaraldehyde as a crosslinking agent. PVA microspheres activated with aldehyde groups were employed for Trametes versicolor laccase immobilization. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the activated PVA microspheres and PVA microspheres with immobilized laccase (Lac/PVA microspheres), which show that laccase was successfully immobilized on the PVA microspheres. The optimum pH and temperature coupling conditions for the immobilized laccase were determined to be 3.3 and 30 °C, respectively. Residual activity was also investigated by soaking the immobilized laccase in organic solvents at different concentrations, proving it chemically stable. Immobilized laccase exhibited good storage stability at 4 °C. The enzyme biosensor showed good performance in 2,2-azinobis(3-ethylthiazoline-6-sulfonate) and bisphenol A, with concentration ranges of 2 to 8 mM and 0.05 to 0.25 mM, respectively. Therefore, PVA microspheres may have high potential as support for enzyme thermistor applications.     

Aptamer-based Electrochemical Biosensors for Highly Selective and Quantitative Detection of Adenosine

A new adenosine biosensor based on aptamer probe is introduced in this article. An amino-labeled aptamer probe was immobilized on the gold electrode modified with an o-phenylenediamine electropolymerized film. When adenosine is bound specifically to the aptamer probe, the interface of the biosensor is changed, resulting in the decrement of the peak current. The response current is proportional to the amount of adenosine in sample. The used electrode can be easily regenerated in hot water. The proposed biosensor represents a linear response to adenosine over a concentration range of 1.0x 10^-7-l.0x10^-4 mol/L with a detection limit of 1.0xl0^-8 mol/L. The presented biosensor exhibits a nice specificity towards adenosine. It offers a promising approach for adenosine assay due to its excellent electrochemical properties that are believed to be very attractive for electrochemical studies and electroanalytical applications.