Biological Recognition Element Research Articles

Short-term BOD (BOD st) as a parameter for on-line monitoring of biological treatment process : Part I. A novel design of BOD biosensor for easy renewal of bio-receptor

A novel design of a biochemical oxygen demand (BOD) biosensor has been developed for on-line monitoring of easily biodegradable organic compounds in aqueous samples. The biological recognition element of the sensor could be easily renewed by injecting new bacterial paste without disassembling the sensor system. The sensor measurements were carried out in the initial-rate mode using a flow injection (FI) system, resulting in 60 s for one sample analysis followed by a recovery time less than 10 min. The sensor performance achieved showed a wide detection linearity over the range of 5–700 mg BOD 5·l −1 and a generally good agreement between the BOD values estimated by the biosensor and the conventional 5-day test. Furthermore, the precision test was in the control range (i.e. repeatability ≤ |±7.5%|, reproducibility ≤ |±7.3%|). The sensor could be used over 1 week in continuous test, however, the best performance was found within the first 24 h where standard deviation of the sensor response was ±2.4%. The design of the sensor allows easy and fast renewal of the cells used as sensing elements. Replacement of biological recognition element and calibration of the sensor responses can be performed in a rather simple procedure on a daily regular basis. By using a mixed culture as the bio-receptor, one gets a sensor that reacts to a wide range of substrates. The new sensor construction will thus allow fast and convenient replacement of the bio-receptor and on-line assay of a broad range of substrates. This makes the sensor being an interesting and promising candidate for on-line monitoring of biological treatment process.

Development of a cell-based sensing device to evaluate toxicity of inhaled materials

Health risks associated with the inhalation of materials have been a topic of great concern due to highly publicized cases of inhalation anthrax, of new regulations on the release of PM 2.5, and to increased concerns on the hazards of indoor air pollution. At this time, there are no automatable methods to evaluate the potential health impact of inhaled materials. Here, we describe the design and evaluation of a cell-based device that can determine the potential health impact of inhaled materials. The sensor platform utilizes cultured lung cells as a biological recognition element. The health and activity of these cells is characterized through a colorimetric metabolic assay that is monitored using light-emitting diodes and photodetectors. Cells are responsive to a variety of materials including low levels of Zn, of combustion derived particulate matter, and to volatizable pesticides. Substantial responses are obtained with as little as 10 −4% by volume of pesticides, to μg/ml particulate matter, and μM levels of metals. The sensitivity of this device compares well to that obtained through more traditional cell culture measurements. The device is small, portable, and has the potential to be employed outside of a laboratory setting.

Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring.

1. SUMMARYA bioluminescent bioreporter integrated circuit (BBIC) is anovel whole-cell biosensor that combines the environmentalmonitoring capabilities of genetically engineered biolu-minescent micro-organisms (bioreporters) with optical appli-cation-specific integrated circuits. A BBIC device consistsof bioreporters sustained within a micro-environment, theintegrated circuit microluminometer, and a light-tightenclosure. The bioreporter typically contains the luxCDABEreporter genes encoding the enzymes required for biolumin-escence. In the presence of a targeted analyte, a gene(transcriptional) regulatory system induces the expressionof the luxCDABE genes. Analytical benchmark data forexposure of the bioreporter Pseudomonas fluorescens 5RLto salicylate was determined using a flow-through testsystem. The detection limit (after a 45 min exposure) wasca 50 lgl

Acrylic Acid-Based Copolymers as Immobilization Matrix for Amperometric Biosensors†

Libraries consisting of acrylic acid-based polymer resins were synthesized by copolymerizing acrylic acid with different alkyl acrylates and 1-vinyl imidazole concomitantly varying the relative concentrations and the number of monomers present in the reaction mixture. The obtained polymer resins are suitable for electrochemically-induced precipitation on electrode surfaces caused by the protonation of acidic side groups at the polymer chains induced by a local electrochemical formation of protons in the diffusion zone in front of the electrode. Addition of suitable biological recognition elements such as glucose oxidase to suspensions of these electrodeposition paints (EDP) led to the entrapment of the enzyme within the slowly precipitating polymer film, and hence to the nonmanual fabrication of amperometric biosensors. The suitability of the obtained copolymers was screened with respect to the performance of the resulting glucose sensors. It could be demonstrated that the chemical composition of the used EDP significantly influences the properties of the sensors in terms of sensitivity and linear range of their response to glucose. †This article is dedicated to Prof. Dr. Pierre Coulet on the occasion of his retirement.

Design of acetylcholinesterases for biosensor applications

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure–function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.

Biosensors as molecular tools for use in bioremediation

Biosensors are analytical tools, which use the biological specificity in sensing the target molecule. In this paper, the author reviews the design and application of molecular biosensors for use in bioremediation. The promoter selected from a genetic operon has been described as an interactive biological component for the target molecule to generate the signal. Different types of reporter systems have been described and as well as their application in tracking of levels of pollutants, nitrogen, phosphorus, dissolved oxygen in different habitats and toxic compounds. The paper emphasizes that in order to extend the applications of this scientific area, specialized research is needed in the aspects pertaining to bringing the biological recognition element into close proximity to the target molecules so that it can be integrated with the signal analysis system.

Amperometric enzyme biosensors based on optimised electron-transfer pathways and non-manual immobilisation procedures

Development of reagentless biosensors implies the tight and functional immobilisation of biological recognition elements on transducer surfaces. Specifically, in the case of amperometric enzyme electrodes, electron-transfer pathways between the immobilised redox protein and the electrode surface have to be established allowing a fast electron transfer concomitantly avoiding free-diffusing redox species. Based on the specific nature of different redox proteins and non-manual immobilisation procedures possible biosensor designs are discussed, namely biosensors based on (i) direct electron transfer between redox proteins and electrodes modified with self-assembled monolayers; (ii) anisotropic orientation of redox proteins at monolayer-modified electrodes; (iii) electron-transfer cascades via redox hydrogels; and (iv) electron-transfer via conducting polymers.

Electrochemical biosensors for environmental monitoring.

Highly sensitive electrochemical biosensors offer precision, sensitivity, rapidity, and ease of operation for on-site environmental analysis. An electrochemical biosensor is an analytical device in which a specific biological recognition element (bioreceptor) is integrated within or intimately associated with an electrode (transducer) that converts the recognition event to a measurable electrical signal for the purpose of detecting a target compound (analyte) in solution. The signal generated allows both qualitative and quantitative measurements of an analyte in real time. In most cases, a miniaturized electrochemical cell contains a low volume of analyte, which is vital when dealing with hazardous materials and makes such devices ideal for environmental monitoring. This approach not only provides the means for on-site analysis but also removes the time delay and sample alteration that can occur during transport to a centralized laboratory. We first address the basic principles of electroanalytical measurement and the merger of electrochemistry and biology into a biosensing system, and then we discuss various environmental monitoring strategies involving this technology.

Immobilization method for the preparation of biosensors based on pH shift-induced deposition of biomolecule-containing polymer films.

Miniaturization of amperometric biosensors is crucially dependent on the availability of methods for the nonmanual immobilization of biological recognition elements on the transducer surface. From an aqueous polymer suspension, the precipitation of a polymer film with entrapped biological recognition elements is initiated by electrochemically induced oxidation of H20 at the electrode surface. Using the locally generated H+ gradient, acidic side chains of the polymer are titrated, leading to a change in the polymer solubility and hence to the controlled deposition of a polymer film. To investigate the properties and limitations of this immobilization technology, the specific features of a glucose biosensor based on polymer-entrapped glucose oxidase and amperometric detection of enzymatically generated H202 were investigated. Besides the reproducibility of the immobilization procedure, the sensitivity (14.59 mA cm(-2) M(-1) at pH 7), long-term stability (up to 5000 measurements in a sequential-injection analyzer), dependence on enzyme concentration, polymer thickness, and possibilities to fabricate multilayer sensor architectures were exploited. In addition, the miniaturization potential of this nonmanual immobilization technology was evaluated by investigating the modification of microband electrode arrays and cross talk between the neighboring microsensors.

Electrochemical impedance spectroscopy as a platform for reagentless bioaffinity sensing

Simple reagentless immunosensor formats have been difficult to achieve, particularly for electrochemical devices, since antigen/hapten recognition by an antibody does not directly lead to a reaction cascade. A direct reading electrochemical immunosensor would have major advantages with respect to speed, de-skilled analysis and the development of multi-analyte sensors. Electrically conducting polymers, such as poly(pyrrole), allow for the intimate association between a biological recognition element and a potential reporter polymeric chain. We have polymerised poly(pyrrole), loaded with avidin or antibody to luteinising hormone (LH), on gold interdigitated electrodes (IDE) and employed two-electrode electrochemical impedance spectroscopy (EIS) to “visualise” charge transfer through the polymer as the basis for a reagentless protocol. We investigated bulk redox processes in the poly(pyrrole) films by cyclic voltammetry in order to ascertain the redox state of the film prior to EIS. The redox process of the immobilised protein molecule was identified and allowed the focusing of the EIS studies on polymer associated with the immobilised bioaffinity molecule. The polymer displayed both polaronic and electronic charge transfer during EIS studies. A possible binding-dependent response, observed as a decrease in peak polaronic phase angle, occurred when a redox cycle was performed on the film following exposure to the appropriate analyte. The response of poly(pyrrole) films loaded with avidin to d-biotin and two derivatives was assessed, which was shown to be sensitive to electrode pre-treatment. Poly(pyrrole) films loaded with antibody to LH allowed a calibration for LH to be constructed between 1 and 800 IU/l. Importantly, the films were responsive to LH within the clinically relevant range of 1–10 IU/l.

A fiber-optic lactate sensor based on bacterial cytoplasmic membranes

A new type of fiber-optic biosensor based on bacterial cytoplasmic membranes (CPM) as the biological recognition element and an oxygen sensitive dye layer as the transducer is described for the detection of lactate. CPMs from bacteria with an induced lactate oxidase system are adsorbed onto a cellulose disk. The disk is fixed mechanically over an oxygen sensitive siloxane layer on the distal end of an optical fiber. This system detects lactate with no interference from glucose, fructose or glutamic acid.

Electrooxidation mechanism of biogenic amines at amine oxidase modified graphite electrode.

Amine oxidase (AO, EC. 1.4.3.6) was previously shown to be a very efficient biological recognition element of amperometric biosensors for monitoring biogenic amines. The enzyme was effectively working in both mono- and bienzyme electrode designs, based on either a direct or a mediated electron-transfer pathway. This work focuses on the elucidation of the electron-transfer mechanism of the monoenzymatic unmediated AO-modified biosensor. The observed unmediated catalytic currents were assumed to be caused by (i) a direct electron-transfer process, (ii) the electrooxidation of the formed product, or (iii) their combination. Experiments supporting these assumptions are discussed in detail.

A Pyruvate Oxidase Electrode Based on an Electrochemically Deposited Redox Polymer

A redox polymer-modified, multilayer biosensor for the determination of pyruvate and phosphate in oxygen-free samples has been developed. The new, highly conductive redox polymer was produced by potentiostatic copolymerization (+1.4 V vs. Ag/AgCl) of Os(bipy)2pyCl-modified pyrrole monomer (6×10–3 mol L–1) and thiophene (1×10–3 mol L–1) on top of a platinized glassy-carbon electrode. The redox polymer-coated platinum black layer with increased active surface area permitted the adsorption of pyruvate oxidase as the biological recognition element and efficient electron transfer from enzyme-bound FAD-groups to the electrode. Pyruvate was detected at anodic potentials (350–500 mV) in oxygen-free solution in the presence of phosphate as the cosubstrate with a linear range from 0.02×10–3 to 0.3×10–3 mol L–1. A sensitivity as high as 0.2 A cm–2 mol–1 L was obtained. Phosphate was measured similarly between 0.02×10–3 and 0.5×10–3 mol L–1 in the presence of pyruvate as co-substrate. The sensitivity of the sensor dropped to about 12 % after 10 days. Since interference by ascorbate, due to the high formal potential of the used Os(bipy)2pyCl-group, could be a problem in real samples, coverage of the adsorbed enzyme by a polycationic size exclusion layer of polypyrrole was investigated. Compared to former enzyme electrodes utilizing pyruvate oxidase, the new approach offered an unprecedentedly high sensitivity, O2- and thiamindiphosphate-independent operation and presented a large step towards electrochemical pyruvate determination in vivo.

Recent developments towards disposable screen-printed biosensors incorporating a carbon ink modified with the redox mediotor. Meldola's Blue

Screen-printed carbon electrodes (SPCEs) can be mass-produced at low cost and are readily adaptable as base transducers for incorporation into disposable biosensors. As biological recognition elements, the NAD + -dependent dehydrogenase enzymes have wide ranging application for the determination of many analytes in biological samples. In order to achieve the desired selectivity, it has proved necessary to reduce the overpotential required to electrochemically regenerate the cofactor, NAD + , by incorporating redox mediators into the carbon ink prior to screen-printing. One such electrocatalyst, Meldola's Blue (MB), has been used successfully and has been combined with dehydrogenase enzymes for the determination of some important biological analytes, including lactate, alcohol and ammonia. These examples from our laboratory using MB-modified SPCEs will be reviewed in this article, together with a recent application to determine aspartate aminotransferase (AST) in serum.

An enzyme based biosensor for the direct determination of diisopropyl fluorophosphate

The ability of organophosphate hydrolase (OPH, EC 3.1.8.1) from Pseudomonas diminuta to hydrolyze diisopropyl fluorophosphate (DFP) was used to develop a biosensor for the direct determination of DFP. A fluoride sensitive ion-selective electrode was exploited as the physical transducer for a batch-mode biosensor, and OPH enzyme immobilized on silica gel was used as a biological recognition element. The correlation between DFP concentration and hydrolyzed fluoride extended over a concentration range of 2.5×10 −5–5×10 −3 M. The influence of MeOH on DFP hydrolysis by immobilized OPH was greatly reduced from that of the soluble enzyme, and the effect of paraoxon on DFP detection in mixed samples was investigated.

Electrochemical Biosensors: Recommended Definitions and Classification

Abstract Two Divisions of the International Union of Pure and Applied Chemistry (IUPAC), namely Physical Chemistry (Commission I.7 on Biophysical Chemistry formerly Steering Committee on Biophysical Chemistry) and Analytical Chemistry (Commission V.5 on Electroanalytical Chemistry) have prepared recommendations on the definition, classification and nomenclature related to electrochemical biosensors; these recommendations could, in the future, be extended to other types of biosensors. An electrochemical biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, we recommend that a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device which is both disposable after one measurement, i.e., single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration should be designated a single use biosensor. Biosensors may be classified according to the biological specificity-conferring mechanism or, alternatively, to the mode of physico-chemical signal transduction. The biological recognition element may be based on a chemical reaction catalysed by, or on an equilibrium reaction with macromolecules that have been isolated, engineered or present in their original biological environment. In the latter cases, equilibrium is generally reached and there is no further, if any, net consumption of analyte(s) by the immobilized biocomplexing agent incorporated into the sensor. Biosensors may be further classified according to the analytes or reactions that they monitor: direct monitoring of analyte concentration or of reactions producing or consuming such analytes; alternatively, an indirect monitoring of inhibitor or activator of the biological recognition element (biochemical receptor) may be achieved. A rapid proliferation of biosensors and their diversity has led to a lack of rigour in defining their performance criteria. Although each biosensor can only truly be evaluated for a particular application, it is still useful to examine how standard protocols for performance criteria may be defined in accordance with standard IUPAC protocols or definitions. These criteria are recommended for authors, referees and educators and include calibration characteristics (sensitivity, operational and linear concentration range, detection and quantitative determination limits), selectivity, steady-state and transient response times, sample throughput, reproducibility, stability and lifetime.

A Low-Volume Electrochemical Cell for the Deposition of Conducting Polymers and Entrapment of Enzymes

A new small-volume electrochemical cell for the entrapment of biological recognition elements in electrochemically deposited conducting polymer films has been developed. It allows one to save significant amounts of the monomer and especially of the used biological recognition element, and hence permits one to choose very expensive enzymes for the development of respected biosensors. Since the conductivity, morphology and film thickness of the obtained conducting polymer films are dependent on the presence of molecular oxygen, the electrochemical cell allows a facilitated handling of the electrode and electrolyte solution under strict exclusion of any oxygen. To prove the applicability of the new electrochemical cell, the entrapment of glucose oxidase into polypyrrole films has been performed as an example.

Micromachined multichannel systems for the measurement of cellular metabolism

Using living cells as the biological recognition element in a biosensor has two important advantages: cell receptors provide functional rather than structural specificity, and the cells provide second messenger pathways that amplify the receptor/ligand binding events. Cellular responses can be detected by measuring extracellular acidification with a silicon sensor. By using silicon micromachining technology, we can integrate the fluidic pathways and chambers for immobilizing cells with the silicon sensor. We describe a multichannel chip in which each channel is etched into the surface, and has its own flow of tissue-culture medium past the cells. Measurements have been made on adherent cells in which a muscarinic acetylcholine receptor is transfected. Responses to the agonist carbachol are shown.

Enhanced performance of fibre-optic immunoprobes using refunctionalized fluoropolymers as the substratum

Surface refunctionalized fluoropolymers are compared to quartz substrata as means of stabilizing covalently-immobilized biological recognition elements. Results show that antibodies immobilized on the refunctionalized fluoropolymers have greater stability (by at least a factor of 20). Fluoropolymer-immobilized antibodies retain up to 85% of their initial affinity after one year of refrigerated (4°C) storage. A new fibre-optic immunoprobe, based on these refunctionalized fluorpolymers, is described. Comparison studies between quartz and the refunctionalized fluoropolymer substrata show that the detection limits and dynamic range are similar and probe stability is improved significantly using the new materials.

Biosensor development.

This article reviews the recent biosensor developments for medical applications, focusing on the various biological recognition elements used in biosensors and the systems transduction mechanisms. Available instruments utilizing biosensor technology are also examined from a commercial perspective.