Flow-injection Biosensor System Research Articles

Development of a Flow Injection Biosensor System Enables Glucose and Cortisol Simultaneous Measurement for the Evaluation of Fish Stress

The continued growth of the aquaculture industry has led fish farmers to increase production capacity. Increasing production capacity requires high-density aquaculture and excess feeding, which are stressful to the fish and negatively affect fish health and immunity. Further, the accumulation of residual feed and excretions deteriorates the water quality of the aquaculture environment. Although medicine can be administered to the fish for disease prevention, there are concerns about the effects of the medicine that remain in the fish, which may be harmful to the consumer. Thus, a rapid and convenient, non-scheduled fish stress check would be an ideal solution. To evaluate fish stress, people always focus on the change of some plasma indicator, such as cortisol and glucose. Fish respond to stressors by the release of stress hormones such as cortisol. These stress hormones singly and in combination increase glucose production in fish. Because glucose is an important energy substrate for several tissues, the higher glucose production is thought to assist the animal in metabolically coping with the increased energy demand caused by stress. Currently, blood tests are performed to evaluate fish stress states. Conventional methods, such as enzyme-linked immunosorbent assay or colorimetric method, are both very time-consuming and costly, however, making it impractical to perform regular stress evaluations. On the other hand, cortisol, which is secreted rapidly after exposure to a stressor, is not easy to be detected. In addition, though glucose changes are easy to monitor, it may also arise after the feeding. Therefore, rather than measure both cortisol and glucose separately, simultaneous detection of both cortisol and glucose will enhance our understanding of fish stress conditions. In this study, we developed a flow injection biosensor system for measuring fish glucose and cortisol simultaneously. System schematic diagram was shown in the supplement image. For the glucose measurement, it was based on the decrease of the oxygen concentration detected by optical probe due to a glucose oxidation reaction. While, the measurement of cortisol based on competitive immunologic reactions, magnetic separation, and an electrochemical measurement. An anti-cortisol antibody was immobilised on magnetic beads and injected into the system. At first, the glucose concentration was detected first from the change on oxygen decrease by optical probe which immobilized glucose oxidase on its surface. After that, a specific quantity of acetylcholinesterase-labelled cortisol (cort-AChE) was injected into the flow system and the sample will go through a reaction coil as a competitive reaction unit. After reacting in the reaction coil, the sample was separated magnetically using a neodymium magnet. The cort-AChE was detached from the magnetic beads and transferred into the enzyme reaction unit with acetylthiocholine (ATCh). ATCh was hydrolysed by the cort-AChE to produce thiocholine. The thiocholine was quantified downstream by electrochemical detection using a Pt-Ir electrode. We also found that the performance of the proposed biosensor system was optimised under the following conditions: pH 7.5, temperature 25°C, flow rate 0.16 ml min− 1. After the understanding the optimal parameters, we performed the measurement on both glucose and cortisol. The fluorescence intensity was well correlated with the concentration of the cortisol standard solution (range: 0–30 mg dL− 1, R= 0.9785). While the output current was well correlated with the concentration of the cortisol standard solution (range: 0.06–40 ng mL− 1, R= 0.9812). The results obtained using the proposed flow method were compared with those obtained using conventional method (correlation coefficient was 0.9869 for glucose and 0.9848 for cortisol). These findings suggest that the proposed system can be used to analyse glucose and cortisol in fish plasma samples simultaneously and help us to understand the relationship between the stress hormone and glucose change. Figure 1

A polymeric dual-channel amperometric biosensor chip capable of symmetrically splitting sample bands for parallel micro flow injection determination of glucose and lactate

This paper presents a chip-based dual-channel micro flow injection biosensor system for parallel determination of two components that potentially interference with each other. With glucose and lactate being the model analytes, a hybrid polydimethylsiloxane (PDMS)–polycarbonate (PC) microfluidic chip with a Y-shaped main-to-branch channel network was designed. Glucose oxidase (GOD) and lactate oxidase (LOD) biosensors of amperometric type were separately prepared inside each of the two branch channels. A sample band injected into the main channel was split into two daughter bands at the bifurcated point of the Y-shaped channel. The daughter bands were delivered into branches where the two analytes in the daughter bands were independently detected by the GOD and LOD sensors, preventing the potential chemical crosstalk between the two sensors. Factors influencing the symmetry of splitting a sample band, in turn, the sensitivity and reproducibility of the system, were discussed. Symmetric splitting was realized by using back pressure regulators (BPR). Under optimized conditions, detection limits of 78 μmol L−1 for glucose and 127 μmol L−1 for lactate were observed with a sample volume of 20 nL and a sample throughput rate of 40 h−1. RSDs of 0.8–0.9% for peak heights were observed for 11 runs of a standard solution containing glucose and lactate both at 2 mmol L−1. The designed dual-channel biosensor chip was applied to the simultaneous determination of glucose and lactate in human serum samples.

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G

We report a flow-injection biosensor system with a capacitive transducer for assay and quality control of human immunoglobulin G (hIgG). The sensing platform is based on self-assembled monolayers (SAMs) of carboxylic acid terminated alkyl-thiols with covalently attached concanavalin A. The electrochemical characteristics of the sensor surface were assessed by cyclic voltammetry using a permeable redox couple (potassium ferricyanide). The developed biosensor proved capable of performing a sensitive label-free assay of hIgG with a detection limit of 1.0 microg mL(-1). The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 microg mL(-1), in a logarithmic plot. Typical measurements were performed in 15 min and up to 18 successive assays were achieved without significant loss of sensitivity using a single electrode. In addition, the biosensor can detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content (5.0 microg mL(-1)). Hence, it represents a potential post-size-exclusion chromatography-UV (post-SEC-UV) binding assay for in-process quality control of hIgG, which cannot be detected by SEC-UV singly at concentrations below 0.3% of the total hIgG content.

Simultaneous Determination of l-Glutamate, Acetylcholine and Dopamine in Rat Brain by a Flow-Injection Biosensor System with Microdialysis Sampling

A flow-injection biosensor system with an on-line microdialysis sampling system is proposed for the simultaneous detection of neurotransmitters (L-glutamate, acetylcholine and dopamine) released from rat brain cells. The dialysate collected in the sample loop from the microdialysis probe was automatically injected into the flow-injection line with a triple electrode arranged perpendicular to the flow direction. The triple electrode was constructed by hybridizing a poly(1,2-diaminobenzene) film to two enzyme sensing-parts which respond to L-glutamate and acetylcholine, and by coating a Nafion film on a remaining sensing part which responds to dopamine, respectively, without any cross-reactivity. The three sensing parts of the triple electrode responded linearly to the concentrations of L-glutamate and acetylcholine in the range of 0.002-5 mM and to that of dopamine in the range of 0.002-20 mM, respectively, without any interference from oxidizable species present in the dialysate. The proposed flow-injection analytical method could be applied to an in vivo assay of these neurotransmitters released from rat-brain cells by the continuous KCl stimulation.

On-line microdialysis assay of l-lactate and pyruvate in vitro and in vivo by a flow-injection system with a dual enzyme electrode

A flow-injection biosensor system with an on-line microdialysis sampling is proposed for the simultaneous assay of l-lactate and pyruvate in serum and rat brain. The dialysate collected in the sample loop by perfusing Ringer’s solution through the microdialysis probe is automatically injected into the flow-injection line with a dual enzyme electrode arranged in parallel for the flow direction. The dual enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film to two sensing parts, which respond selectively to l-lactate and pyruvate, respectively, without any cross-reactivity. Both the sensing parts respond linearly to the concentrations of both analytes between 0.01 and 5mM, without any interference from oxidizable species and low-molecular weight proteins present in the dialysate. The proposed flow-injection analysis (FIA) method can be successfully applied to the simultaneous in vitro and in vivo assays of both analytes in serum and rat brain, respectively. The system can be automatically processed at an analytical speed of 19dialysatesh−1 over a period of 5h.

Simultaneous in vivo monitoring of glucose, l-lactate, and pyruvate concentrations in rat brain by a flow-injection biosensor system with an on-line microdialysis sampling

A flow-injection biosensor system with an on-line microdialysis sampling is proposed for the simultaneous monitoring of glucose, l-lactate, and pyruvate in rat brain. In the first stage of the operation, the dialysate from the microdialysis probe is delivered to a sample loop of the six-way autoinjector by perfusing Ringer’s solution for 120 s at 2 μl min −1. In the second stage, the dialysate collected in the sample loop is automatically injected for 10 s into the flow-injection line with a triple enzyme electrode arranged in perpendicular for the flow direction. The triple enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film to three sensing parts which respond selectively to glucose, l-lactate, and pyruvate, respectively, without any cross-reactivity. The three sensing parts of the triple enzyme electrode respond linearly to the concentrations of glucose, l-lactate, and pyruvate between 0.1 and 10 mM, without any interference from oxidizable species present in the dialysate. The proposed flow-injection analysis method can be successfully applied to the simultaneous in vitro and in vivo assays of three analytes in serum and in rat brain, respectively. The system can be automatically processed at an analytical speed of 28 dialysates h −1.

Flow Injection Biosensor System for in vivo Analysis of Biomolecules in the Extracellular Space of Brain

The in vivo monitoring of the concentration changes of biomolecules at a variety of sites of the living systems including blood stream, subcutaneous tissue, muscle, internal organs, and brain, has been desired for the pharmacological, clinical, and biological studies. The most general approach to achieve such goals was the use of implanted biosensors, but it was limited because of the variations in sensitivity during the prolonged in vivo monitoring. Recently, microdialysis has been used as a sampling technique for in vivo analysis. This article reviews novel flow-injection biosensor systems which make possible on-line monitoring of glucose metabolism and pharmacological behavior of neurotransmitters in the extracellular space of brain. This approach was developed by coupling microdialysate sampling to the biosensor system using a flow-injection interface. The advances in these researches are reviewed with the results obtained in our recent works. element Microdialysis is a sampling technique for in vivo analysis (l) 2.1. On-line amperometric assay of glucose, Lglutamate, and and has successfully been applied in pharmacological and acetylcholine using immobilized enzyme reactors (14) biological studies of biomolecules in dialysate. In many cases, Here, we describe an on-line amperometric monitoring system however, the dialysate was collected and usually assayed by of glucose, L-glutamate, and acetylcholine. These analytes are not liquid chromatography. detectable by direct electrochemical oxidation. Therefore, we The use of miniaturized biosensors was the most simple chose to use the glucose oxidase (GOD), L-glutamate oxidase approach for the in vivo monitoring, but it was limited because of (GlOD), and acetylcholine esterase (ACE) + choline oxidase the variations in sensitivity during the prolonged in vivo (ChO), to convert each of these analytes to hydrogen peroxide monitoring. In recent years, sensor systems combined which can be detected amperometrically, according to microdialysis sampling have been developed for in vivo analysis /3 -D-glucae + O2 gluconolactone + &02, and continuous monitoring of concentration changes of L-glutamate + O2 2-0xoglutarate + NH3 + H202, and biomolecules in blood (2), subcutaneous tissue (3), and brain (4). acetylcholine + O2 + 2 bo-betaine + acetate + 2 HA. For such researches, some miniaturized enzyme-based Each of the enzymes was loaded or coloaded onto reactor (PEFE amperometric biosensors have been proposed as specific coil; 20 mm X 0.5 mm i.d) packed with aminopropyl-CPG using detectors for the in vivo monitoring of glucose (3, 5-71, L- glutaraldehyde as a cross-linking agent The microdialysis system glutamate (8-101, acetylcholine(ll), and L-lactate (3, 121. A used in this work is outlined in Fig. 1. similar attempt was also achieved by using an enzyme reactor as . The apparatus consisted of a two-channel microsyringe pump, a recognition element of a particular analyte in dialysate, e.g., potentiostat, and a strip chart recorder. Each of the immobilized glucose (13). enzyme reactors was positioned after a microdialysis probe Glucose is a main nutrient in the brain, while L-lactate and shown schematically in Fig. 2 and in front of a poly(l,2- pyruvate are formed from glucose in an actively glycolyzing diaminobenzene) film-coated platinum electrode. A constant system, and are of interest in metabolic disorders. Also, L- potential (+ 0.6 V vs. AgIAgCl) was applied to the polymer film- glutamate and acetylcholine are the most important excitatory coated electrode and the current was recorded. The Ringer's neurotransmitters in the brain. Therefore, the in vivo monitoring of concentration changes in such analytes that take place rapidly in brain cells has been especially desired in the research field of brain chemistry. This article reviews the in vivo flow-injection biosensor systems with an on-line microdialysis sampling which make possible the specific detection of a single analyte or the simultaneous monitoring of plural analytes in brain. This approach has been developed by coupling microdialysis sampling Fig. 1 Schematic diagram of microdialysis system with to the biosensor system using a flow-injection interface. The immobilized enzyme reactor. A: two-channel microsyringe pump, advances in these researches are reviewed with the results B: microdialysis probe, C: sample, D immobilized enzyme obtained in our recent works.

A flow injection biosensor system for highly sensitive detection of 2,4,6-trichlorophenol based on preoxidation by ceric sulfate.

A flow injection biosensor system was proposed for the highly sensitive detection of 2,4,6-trichlorophenol (2,4,6-TCP). The system is based on the preoxidation by ceric sulfate to the corresponding benzoquinone (2,6-dichloro-1,4-benzoquinone: 2,6-DC-1,4-BQ), which was characterized using cyclic voltammetry, hydrodynamic voltammetry, and UV-vis spectrophotometry. The laccase-based biosensor used in this analytical system responded sensitively to 2,4,6-TCP after the preoxidation by ceric sulfate. The response could be based on the bioelectrocatalytic recycling of oxidation product (2,6-DC-1,4-BQ) between laccase membrane and the electrode, because the oxidation product (2,6-DC-1,4-BQ) of 2,4,6-TCP was an electrochemically reversible redox species. The signal current was linearly related to the 2,4,6-TCP concentrations in a dynamic range of 2 nM - 2 microM; the slope and the y-intercept of the straight line were 1150 nA microM(-1) and 0.88 nA, respectively. The detection limit was 1.2 nM (S/N = 3) for a 20 microl injection. Among a variety of chlorophenols and some phenolic compounds, the only interferent was 2,4-dichlorophenol.

Highly Sensitive Detection of 4-Chlorophenol by Flow Injection Biosensor System Based on Substrate Recycling

Highly Sensitive Detection of 4-Chlorophenol by Flow Injection Biosensor System Based on Substrate Recycling

A Flow Injection (FI) Biosensor System for Pentachlorophenol (PCP) Using a Substrate Recycling Scheme

A flow injection (FI) biosensor system has been designed for the analysis of pentachlorophenol (PCP) using a substrate recycling scheme comprising immobilized bilirubin oxidase (BOX) in the presence of excess of NADH. PCP was efficiently converted to tetrachloro-p-benzoquinone (1,4-TCBQ) and then tetrachloro-p-hydroquinone (1,4-TCHQ) by bis(trifluoroacetoxy)iodobenzene (BTFAIB) and zinc powder, respectively. BOX immobilized on aminopropyl glass beads rapidly oxidized 1,4-TCHQ to 1,4-TCBQ, which in turn was readily reduced back to 1,4-TCHQ in the presence of excess NADH. This recycling scheme enabled one molecule of PCP to consume several NADH molecules leading to enhanced sensitivity. Under optimized conditions the rate of NADH uptake measured as the absorbance decrease at 340 nm yielded a detection limit for 1,4-TCHQ or oxidized PCP of 250 nM. The detection limit was improved to 25 nM for both analytes using a fluorescence detector with excitation and emission wavelengths of 345 and 450 nm, respectively. The PCP level in contaminated soil samples was measured using the FI biosensor system, and the results obtained compared well with capillary zone electrophoresis (CZE) analysis.

A Flow-Injection Biosensor System for the Amperometric Determination of Creatinine: Simultaneous Compensation of Endogenous Interferents

A flow-injection biosensor system was developed for the amperometric determination of creatinine based on coupled reactions of three sequentially aligned enzyme reactors, creatinine deiminase, glutamate dehydrogenase, and glutamate oxidase, using an oxygen electrode as the detector. To overcome the problem of endogenous ammonia and glutamate, the flow was split into two channels after the injector and rejoined before the glutamate dehydrogenase reactor. Double peak recording was obtained by setting a delay coil and a reference column in one of the two channels. The first peak gave the sum response of creatinine, endogenous ammonia, and glutamate, and the second that of endogenous ammonia and glutamate. By this method compensation for endogenous ammonia and glutamate, as well as for interfering ascorbic acid, was achieved simultaneously. The system gave linear calibrations up to 2 mM for the first peak and 3 mM for the second one. The lower detection limits were 0.1 and 0.02 mM for 35- and 100-μl injection of sample, respectively. One run was completed within 2 min. The system showed good reproducibility (<3%) and long operational stability (>1300 runs). The assay results of creatinine in urine showed good correlation with those obtained from the chemical method of Jaffe.

Multifunctional Flow-injection Biosensor for the Simultaneous Measurement of Creatinine, Glucose, and Urea.

An amperometric flow-injection biosensor system was developed for the simultaneous measurement of creatinine, glucose, and urea, with a single sample injection and one detector. The principle for the amperometric detection of urea and creatinine was based on coupled reactions of three sequentially aligned enzyme reactors, urease or creatinine deiminase/glutamate dehydrogenase/L-glutamate oxidase. Inclusion of a split point and two confluence points between the injector and detector (oxygen electrode) resulted in a flow-injection system composed of three channels. Triple peak recording was obtained by putting two delay coils of different lengths at the channels involving urease and glucose oxidase. The system gave linear calibrations for creatinine, glucose, and urea in the ranges of 0.2-5, 0.2-10, and 0.5-20mM, respectively. The assay procedure was simple and one run was completed within 4 min. The system was reproducible within 5-8% of relative standard deviation.

Amperometric Flow-Injection Biosensor System for the Simultaneous Determination of Urea and Creatinine

A flow-injection biosensor system was developed for the simultaneous assay of urea and creatinine, with a single injection and one detector. The amperometric detection of urea or creatinine was based on coupled reactions of three sequentially aligned enzyme reactors, urease or creatinine deiminase, glutamate dehydrogenase and glutamate oxidase. Ammonia produced by the enzymatic hydrolysis of urea or creatinine was converted to glutamate, and the oxygen consumption due to the oxidation of glutamate by glutamate oxidase was detected with an oxygen electrode. A split and confluence of the flow stream between the injector and the glutamate dehydrogenase reactor resulted in a two-channel system. Double-peak recording was achieved by putting a delay coil at one of the two channels. The system gave linear calibration curves over a range of 0.1-5.0mM for both urea and creatinine. The assay procedure is simple and one run can be completed within 3min. The system was reproducible within 5% of the relative standard deviation.