Huifeng Ren

Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt-Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt-Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl, and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.

Biosensor system for continuous glucose monitoring in fish

A biosensor system was developed for continuous estimation of blood glucose in fish. Because it is difficult to measure blood components in real-time due to decreased sensor output resulting from blood coagulation and coalescing blood proteins at the sensor placement site, we used the eyeball scleral interstitial fluid (EISF) as the site of sensor implantation. Evaluation of the relationship between EISF and blood glucose concentrations revealed that the blood glucose concentration correlated closely with the EISF glucose concentration (y=2.2996+0.9438x, R=0.960, n=112). To take advantage of the close correlation between blood and EISF glucose, we prepared a needle-type enzyme sensor for implantation in the fish sclera using a flexible wire electrode. The sensor provided a rapid response, good linearity, and reproducibility. Continuous glucose monitoring could be carried out by implanting this needle-type glucose sensor onto the eye. The findings indicated that the glucose concentration increased with sensor output current over time, and that changes in the blood glucose were continuously reflected in the EISF. The glucose concentration was estimated based on the one-point or two-point calibration methods. The two-point calibration method yielded the most accurate glucose monitoring (blood glucose range of 70-420 mg dL(-1)) over 160 min. Sensor-estimated glucose and whole blood glucose values were highly correlated (y=0.4401+0.8656x, R=0.958).

The superiority of organically cultivated vegetables to general ones regarding antimutagenic activities

We found organically cultivated (OC) vegetables, using a water-soluble chitosan as a soil improvement agent and leaf surface spray, had much longer shelf life and better taste than that of generally cultivated (GC) vegetables. The purpose of this study is to determine the relative antimutagenic activity between OC and GC vegetables. Eleven OC vegetables were harvested in March and April in 1999 and 2000, and GC ones were supplied as a control from nearby farms on the same date. The former vegetables were planted on the field where no pesticide had been used for the last 3 years. Forward mutation test with Salmonella typhimurium TM677 and 8-azaguanine as a detection agent was used to determine the antimutagenic activity of juices prepared from OC and GC vegetables against authentic mutagens, such as 4-nitroquinoline oxide (4NQO), benzo(a)pyrene (BaP), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2). This microbiological test is a convenient method to use for the food samples containing free histidine. Antimutagenic activity was evaluated by the difference of mutagenic activities between mutagenecity of authentic compounds and that observed upon incubation at 37 degrees C for 2h with each vegetable juice. OC Chinese cabbage, carrot, Welsh onion, and Qing-gen-cai suppressed 37-93% of the mutagenic activity of 4NQO, while the GC ones were held down to 11-65%. Against BaP, three species of OC vegetables showed 30-57% antimutagenecity, while GC ones did only 5-30%. Similarly, the OC spinach decreased the activity of Trp-P-2 to 78%, and the GC suppressed it by 49%.

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.

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.

Wireless Biosensor System for Real-Time L-Lactic Acid Monitoring in Fish

We have developed a wireless biosensor system to continuously monitor l-lactic acid concentrations in fish. The blood l-lactic acid level of fish is a barometer of stress. The biosensor comprised Pt-Ir wire (φ0.178 mm) as the working electrode and Ag/AgCl paste as the reference electrode. Lactate oxidase was immobilized on the working electrode using glutaraldehyde. The sensor calibration was linear and good correlated with l-lactic acid levels (R = 0.9959) in the range of 0.04 to 6.0 mg·dL−1. We used the eyeball interstitial sclera fluid (EISF) as the site of sensor implantation. The blood l-lactic acid levels correlated closely with the EISF l-lactic acid levels in the range of 3 to 13 mg·dL−1 (R = 0.8173, n = 26). Wireless monitoring of l-lactic acid was performed using the sensor system in free-swimming fish in an aquarium. The sensor response was stable for over 60 h. Thus, our biosensor provided a rapid and convenient method for real-time monitoring of l-lactic acid levels in fish.

Removal of cadmium from scallop processing waste by washing with weak acid solution and utilization of useful constituents for organic fertilizer manufacturing

Hepatopancreases were washed four times with 2% acetic acid solution. In both the washed hepatopancreases and the supernatant of the washing solution, the residual cadmium (Cd) concentration was in accordance with the required standard for organic fertilizers. Hepatopancreases do not naturally contain high levels of nitrogen, but they do contain several amino acids which are useful for cultivation, and these were retained during the washing process. Although the condensed precipitate of the washing solution contained a considerable amount of Cd, Cd elution levels at the time of final disposal met judgment standard for special management industrial waste as a result of kneading and immobilizing with chelating adsorbent for treatment of contaminated soil. The volume of the precipitate of the washing solution was one-fifth of the original hepatopancreas, and the solid waste was suitable for burial at a final disposal site without further treatment.

Effect of Chinese parsley Coriandrum sativum and chitosan on inhibiting the accumulation of cadmium in cultured rainbow trout Oncorhynchus mykiss

Toxic heavy metals such as cadmium (Cd) accuulate in cultured fish from solid-form feed mixtures. In this paper, two natural ingredients, supposed to inhibit accumulation of heavy metals in the fish body, were investigated to develop a functional feed mixture for healthy fish. Three test diets: (i) #1, regular diet as negative control; (ii) #2, #1 +2% lyophilized Chinese parsley Coriandrum sativum (CP); and (iii) #3, #1 +3% chitosan (CT), were each fed to 50 rainbow trout Oncorhynchus mykiss once a day, at the constantrate of 1.0–1.2% of body weight for 12 weeks. No undesirable effect on fish growth was observed by an addition of either CP or CT to regular feed. Then, 10 mg Cd was added to 1 kg each of the three test diets and they were named as: (i) #4, #1 +Cd; (ii) #5, #2 +Cd; and (iii) #6, #3 +Cd, respectively. The fish given positive a control feed (diet #4) for 3 weeks accumulated Cd in the liver and kidney at approximately 0.52 mg/kg. However, Cd in ordinary muscle was found to be just above the detection limit. Upon accumulation of Cd, three groups of 50 fish were each fed test diets #4, #5, and #6 for 12 weeks at the same feed rate described above. The concentrations of Cd accumulated in the liver, kidney, and ordinary muscle of the fish were measured every 3 weeks. Contrary to the positive control fish fed diet #4, those fish fed test diets #5 and #6 accumulated 20–30% and 25–40% less Cd in the liver, respectively. Test diets were a little less effective for inhibition of Cd uptake in the kidney than that observed in the liver. These results suggest that addition of CP or CT to fish feed might be used to decrease toxic heavy metal accumulation in fish.

Development of Functional Fish Feed with Natural Ingredients to Control Heavy Metals

The effects of two natural ingredients, Chinese parsley (CP) and chitosan (CT), on growth, accumulation, and excretion of cadmium in fish body and preservation of essential trace metals in the body were investigated by using rainbow trout that had been fed cadmium-added diet, low and high concentration, for 3 weeks. This pretest confirmed that cadmium was accumulated in the liver, kidney, and intestine of the test fish. The cadmium level of the fish, fed diet with CP or CT, was decreased by 18% and 24%, respectively, compared to that of the fish given the control diet. But CP and CT did not have an influence on normal growth of test fish and the levels of essential trace metals in the body. In addition, the level of cadmium was higher in liver than kidney in the high-cadmium dietary group, indicating the Cd level in kidney follows that of liver as kidney lies in the final stage of metabolism. The cadmium accumulation in the fish body was supposed to be reduced, by giving CP to increase the solubility of Cd to body fluid by conjugation into metallothioneins (MTs), while CT was supposed to be responsible for the physical adsorption of cadmium ions by glucosamine groups.

Electrochemical flow injection immunoassay for cortisol using magnetic microbeads

We developed a novel flow injection assay for cortisol based on competitive immunologic reactions, magnetic separation, and electrochemical measurement. The proposed flow assay system was composed of two reaction units. An anti-cortisol antibody was immobilised on magnetic beads and injected into the reaction coil of a competitive reaction unit with a blood sample and a specific quantity of acetylcholinesterase-labelled cortisol (cort-AChE). 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. The performance of the proposed flow assay system was optimised under the following conditions: pH 7.5, temperature 25°C, flow rate 170 µl min−1, ATCh concentration in the substrate buffer 5 mmol L−1. The output current was well correlated with the concentration of the cortisol standard solution (range: 7.8–500 pg mL−1). The results obtained using the proposed flow method were compared with those obtained using conventional ELISA (correlation coefficient 0.9585 [y = −0.9797 + 1.173(x), n = 11]). These findings suggest that the EFIIA system can be used to analyse cortisol in fish plasma samples.