Toward a Practical Impedimetric Biosensor: A Micro-Gap Parallel Plate Electrode Structure That Suppresses Unexpected Device-to-Device Variations.

We propose a rational electrode design concept for affinity biosensors based on electrochemical impedance spectroscopy to substantially suppress unexpected device-to-device variations. On the basis that the uniformity of the current distribution affects the variation, a novel micro-gap parallel plate electrode (PPE) was developed, where two planar electrodes with edges covered with a SiO2 layer were placed face to face. The structure provides a uniform current distribution over the planar electrode surface and maximizes the contribution of the planar electrode surface to sensing. For a comparative study, we also fabricated a micro-structured interdigitated electrode (IDE) that has been widely adopted for high-sensitivity measurement, although its current is highly concentrated on the electrode edge corner. Protein G (PrG) molecules were immobilized on both electrodes to prepare an immunoglobulin G (IgG) biosensor on which the specific binding of PrG-IgG can occur. We demonstrated that the IgG sensor with the PPE has small device-to-device variations, in strong contrast to the sensor with the IDE having large device-to-device variations. The results indicate that the current distribution on the electrode surface is important to fabricating electrochemical impedance spectroscopy biosensors with small device-to-device variations. Furthermore, it was found that the PPE allows ultrasensitive detection, that is, the sensor exhibited a linear range from 1 × 10-13 to 1 × 10-7 mol/L with a detection limit of 1 × 10-14 mol/L, which is a record sensitivity at low concentrations for EIS-based IgG sensors.

Development of a Novel Enhanced Biosensor System for Real-Time Monitoring of Fish Stress Using a Self-Assembled Monolayer.

Wireless biosensor systems were developed in our lab for monitoring blood glucose concentrations in fish as an indicator of fish stress. However, uniform immobilization of the enzyme on the surface of the electrode is difficult, so the sensor response is typically reduced at a range of high glucose concentrations during the stress monitoring. In this study, we attempted to enhance sensor response by using a self-assembled monolayer-immobilized enzyme. Glucose oxidase was immobilized on a working electrode modified with a self-assembled monolayer. The proposed biosensor showed a good correlation between the output current and the glucose concentration range of 10⁻3500 mg dL-1 under an optimized working condition. The dynamic measurement range of this newly developed sensor is significantly improved, especially over a high concentration range, which helps the sensor to achieve better performance in dramatic changes in the stress response of fish. In addition, we used biological samples from test fish and obtained a good correlation coefficient between the sensor output current and the glucose concentration using a conventional method. The proposed wireless biosensor system was also applied to monitor fish stress responses in real time through different stressors and to obtain some precise data that reflect real fish stress responses.

Real-time fish stress visualization came true：A novel multi-stage color-switching wireless biosensor system.

An optical communication type biosensor system has been developed which can measure blood glucose concentration, which is a stress indicator of fish, in real-time while fish swimming freely. However, this system is hard to make instant acknowledgment of fish stress level which has to contain an unavoidable delay in the judgment. In this research, we aimed to develop a novel stress visualization system which can quickly judge the levels for fish stress response instantly based on a color changeable LED while another LED was designed to send data. The present system is based on the principle of converting the output current value measured by the glucose biosensor corresponding to the stress response into a voltage value. Then, the color and stress switching points of the LED (Red, Yellow, Green) were decided based on the voltage value gained from the biosensor which mentioned above. Furthermore, we attempted to use our biosensor system to make real-time monitoring of fish stress in vivo. As results, the proposed sensor can make real-time measurement of glucose and shows a great response to those of actual fish sample in the range from 35.36 to 300 mg dl-1 (R = 0.9899). When the glucose concentration in the collected sample was switched to the concentration pre-sett, it was successful to switch the LED color according to the gained voltage value both in vitro and in vivo. Furthermore, when monitoring the stress responses of the fish in vivo, color switching corresponding to the sensor output current value was observed successfully.

New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish.

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. As a well-known indicator of fish stress, a simple and rapid method for detecting cortisol changes especially sudden increases is desired. In this study, we describe an enzyme-functionalized label-free immunosensor system for detecting fish cortisol levels. Detection of cortisol using amperometry was achieved by immobilizing both anti-cortisol antibody (selective detection of cortisol) and glucose oxidase (signal amplification and non-toxic measurement) on an Au electrode surface with a self-assembled monolayer. This system is based on the maximum glucose oxidation output current change induced by the generation of a non-conductive antigen-antibody complex, which depends on the levels of cortisol in the sample. The immunosensor responded to cortisol levels with a linear decrease in the current in the range of 1.25-200ngml-1 (R=0.964). Since the dynamic range of the sensor can cover the normal range of plasma cortisol in fish, the samples obtained from the fish did not need to be diluted. Further, electrochemical measurement of one sample required only ~30min. The sensor system was applied to determine the cortisol levels in plasma sampled from Nile tilapia (Oreochromis niloticus), which were then compared with levels of the same samples determined using the conventional method (ELISA). Values determined using both methods were well correlated. These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish without sample dilution. We also believe that the proposed system could be integrated in a miniaturized potentiostat for point-of-care cortisol detection and useful as a portable diagnostic in fish farms in the future.

Development of a label-free immunosensor system for detecting plasma cortisol levels in fish.

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen-antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20ß-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from -0.32 to 0.51 µA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples.

Fish stress become visible: a new attempt to use biosensor for real-time monitoring fish stress.

To avoid fish mortality and improve productivity, the physiological conditions including stress state of the cultured fish must be monitored. As an important indicator of stress, glucose concentrations are monitored using in vitro blood analysis. The physiological processes of fish under environmental conditions are harsher in many ways than those experienced by terrestrial animals. Moreover, the process of anaesthetizing and capturing the fish prior to analysis may produce inaccurate results. To solve these problems, we developed wireless biosensor system to monitor the physiological condition of fish. This system enables artificial stress-free and non-lethal analysis, and allows for reliable real-time monitoring of fish stress. The biosensor comprised Pt-Ir wire as the working electrode and Ag/AgCl paste as the reference electrode. Glucose oxidase was immobilized on the working electrode using glutaraldehyde. We used the eyeball interstitial sclera fluid (EISF) as the in vivo implantation site of the sensor, which component concentration correlates well with that of blood component concentration. In the present study, we investigated stress due to alterations in water chemistry, including dissolved oxygen, pH, and ammonia-nitrogen compounds. Stress perceived from behavioural interactions, including attacking behaviour and visual irritation, was also monitored. Water chemistry alterations induced increases in the glucose concentration (stress) that decreased with removal of the stimulus. For behavioural interactions, stress levels change with avoidance, sensory behaviour and activity. We believe that the proposed biosensor system could be useful for rapid, reliable, and convenient analysis of the fish physiological condition and accurately reflects the stress experienced by fish.

Impedimetric and amperometric bifunctional glucose biosensor based on hybrid organic-inorganic thin films.

A novel glucose biosensor with an immobilized mediator was studied using electrochemical impedance spectroscopy (EIS) and amperometry measurements. The biosensor has a characteristic ultrathin form and is composed of a self-assembled monolayer anchoring glucose oxidase (GOx) covered with Langmuir-Blodgett (LB) films of Prussian blue (PB). The immobilized PB in the LB films acts as a mediator and enables the biosensor to work under a low potential (0.0V vs. Ag/AgCl). In the EIS measurements, a dramatic decrease in charge transfer resistance (Rct) was observed with sequential addition of glucose, which can be attributed to enzymatic activity. The linearity of the biosensor response was observed by the variation of the sensor response (1/Rct) as a function of glucose concentration in the range 0 to 25mM. The sensor also showed linear amperometric response below 130mM glucose. The organic-inorganic system of GOx and PB nanoclusters demonstrated bifunctional sensing action, both amperometry and EIS modes, as well as long sensing stability for 4 days.

Electrochemical impedance spectroscopy biosensor with interdigitated electrode for detection of human immunoglobulin A.

Interdigitated electrodes (IDEs) that have a series of parallel microband electrodes with alternating microbands connected together were utilized in electrochemical impedance spectroscopy (EIS) to build a label-free human immunoglobulin A (IgA) immunosensor. Anti-human IgA (anti-IgA) was employed as an IgA receptor and was covalently immobilized on the IDE surface through a self-assembled monolayer, as confirmed by atomic force microscopy. EIS measurements revealed that the specific adsorption of IgA onto the immobilized anti-IgA gave rise to a clear increase in the value of interfacial charge transfer resistance (R(ct)). A linear relationship between ΔR(ct) and the logarithm of IgA concentration was found for the concentration range of 0.01-100 ng/mL. No modulation of R(ct) was detected by immersing the sensor in solutions of other proteins such as human immunoglobulin G or bovine serum albumin, which confirmed a high selectivity of this immunosensor for IgA. These results demonstrated that the anti-IgA receptor simply immobilized on the IDE surface can provide a sensitive biosensor.

Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish.

Maintaining high-quality fish eggs stably and efficiently is important for aquaculture. We developed a label-free immunosensor system for measuring 17,20ß-dihydroxy-4-pregnen-3-one (DHP). DHP is suddenly secreted before ovulation as a maturation-inducing hormone in fish, and therefore, DHP levels are an indicator for predicting ovulation. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry (CV). For biomolecular immobilization on the surface of sensing electrode, Au electrode, we used self-assembled monolayers of thiol-containing compounds to fix anti-DHP immunoglobulin. In addition, we used a single-walled carbon nanotube to improve sensitivity. Using this electrode, we were able to determine the CV signal change caused by the antigen-antibody complex. The proposed immunosensor system showed a linear correlation (correlation coefficient: 0.9827) between the anodic peak current of the CV and the DHP level in range from 15.6 to 50,000 pg ml(-1). The sensor system was then applied to monitor DHP of goldfish (Carassius auratus). Blood plasma of fish was collected every 3 h after administering a DHP inducer. In the measurement, the anodic peak current of the CV showed distinct changes depending on DHP levels in the blood plasma. A good relationship was observed between DHP levels determined by our proposed system and the conventional method (correlation coefficient: 0.9351).

Wireless biosensor system for real-time cholesterol monitoring in fish "Nile tilapia".

The rapidly increasing demand for cultured fish as a food resource requires simple, effective methods for controlling fish health in culture conditions. Plasma total cholesterol levels are significantly related to fish mortality following bacterial challenge, and are thus a good indicator of the general health of fish. We developed a wireless biosensor system to continuously monitor the total cholesterol concentration in fish (Nile tilapia, Oreochromis niloticus). The biosensor was constructed with Pt-Ir wire (phi0.178 mm) as the working electrode and Ag/AgCl paste as the reference electrode. Cholesterol oxidase and cholesterol esterase were immobilized on the working electrode using glutaraldehyde. The sensor output was linear and strongly correlated with the cholesterol level (R=0.9970) in the range of 2.65-403 mg dl(-1). This range covers the range of total cholesterol levels in fish. To avoid blood coagulation and proteins coalescing on the sensor, we implanted the sensor in the fluid under the scleral surface of the eyeball (EISF). The EISF is presumed to reflect the levels of most blood components and does not include the substances contained in blood that inhibit sensor measurement. Total cholesterol concentrations in blood and EISF were strongly correlated (R=0.8818, n=72) in the blood total cholesterol range of 74-480 mg dl(-1). Therefore, we used EISF as an alternative to blood and performed continuous in vivo-monitoring of the total cholesterol concentration in fish. We also investigated the application of the calibration method and wireless monitoring system. These applications enabled us to securely monitor total cholesterol levels in free-swimming fish in an aquarium for over 40 h. Thus, our newly developed sensor provided a rapid and convenient method for real-time monitoring of total cholesterol concentrations in free-swimming fish.

Incorporation of glucose oxidase into Langmuir-Blodgett films based on Prussian blue applied to amperometric glucose biosensor.

Glucose oxidase (GOx) was immobilized in the organic-inorganic Langmuir-Bldogett (LB) films consisting of octadecyltrimethylammonium (ODTA) and nanosized Prussian blue (PB) clusters. The amperometric glucose biosensors based on the LB films were fabricated and tested. It was found that the sensors exhibited a clear response current under an applied voltage of 0.0 V (vs Ag/AgCl). The linearity of current density versus glucose concentration was confirmed below 15 mmol/L concentration. This is the first observation of biosensor function of the hybrid organic-inorganic LB films. The successful preparation of glucose sensors operating at the very low potential indicates that the adsorbed PB clusters in the LB films act as an electrocatalyst for the electrochemical reduction of hydrogen peroxide, which is the final product of the enzymatic reaction sequence. The observed low potential applicability is estimated to inhibit the responses of interferants such as ascorbic acid, uric acid, and acetominophen. It was also found that an electrostatic interaction between positively charged ODTA+ and the adsorbed species of both GOx and PB provided a stabilized adsorption state in the LB films. Such stable immobilization contributes to the steady amperometric response current observed in the present ODTA/PB/GOx LB films.