Scott McNiven

Optical detection of chloramphenicol using molecularly imprinted polymers

A practical optical sensing system for the determination of chloramphenicol (CAP), utilizing molecularly imprinted polymers (MIPs) and HPLC, has been developed. The method is based on competitive displacement of a chloramphenicol-methyl red (CAP-MR) dye conjugate from specific binding cavities in an imprinted polymer by the analyte. The best of these polymers was obtained using (diethylamino)ethyl methacrylate as functional monomer at a monomer:template ratio of 2:1. HPLC with a mobile phase containing CAP-MR was used as the detection system, and injection of CAP and, to a lesser degree, thiamphenicol resulted in proportional displacement of the conjugate, which was detected at 460 nm. The detection system showed a linear response over a range of 3-1000 μg/mL and effectively detected CAP extracted from serum. This system offers a tailor-made, selective, and rapid method for CAP detection, is able to discriminate between similar molecules, and is effective below and above the therapeutic range (10-20 μg/mL serum, potentially toxic above 25 μg/mL). This technique is quite general and should enable the use of MIPs in a wide variety of applications involving the detection of families of molecules which possess a distinct arrangement of functional groups.

A mediator-type biosensor as a new approach to biochemical oxygen demand estimation

A novel biosensor for the determination of biochemical oxygen demand (BOD) was developed using potassium hexacyanoferrate(III) [HCF(III)] as a mediator. The sensor element consists of a three-electrode system, with both working and counter electrodes compactly integrated as a disposable using etching and electroplating processes. Pseudomonas fluorescens biovar V (isolated from a wastewater treatment plant) was immobilized on the surface of the working electrode using poly(vinyl alcohol)-quaternized stilbazol (PVA-SbQ) photopolymer gel. Synthetic wastewater described by the Organization for Economic Cooperation and Development (OECD) was used as a standard solution instead of glucose-glutamic acid synthetic wastewater. The conditions of amperometric measurement were optimized at +600 mV (vs. Ag/AgCl) operating potential, namely 40 mM HCF(III) in a 0.1 M phosphate buffer (pH 7.0) at 20 degrees C. The sensor response was linear from 15 up to 200 mg O l-1 BOD. The response time was 15 min at 200 mg O l-1 BOD. To demonstrate the wide metabolic range of activity of the sensor, the sensor response to 14 substances in four categories of organic compounds was investigated. Further, it was shown that the response of this BOD sensor was not influenced in samples with low concentrations of dissolved oxygen under the measuring conditions used. For real wastewaters, the BOD values were determined using the sensor and compared favorably with those determined by the conventional BOD5 method.

Improvement of a mediator-type biochemical oxygen demand sensor for on-site measurement.

We characterized a mediator-type biochemical oxygen demand (BOD) sensor with a three-electrode system using potassium ferricyanide (FC) and Pseudomonas fluorescens in our previous study. In the present study, we have utilized the advantages of a mediator-type biosensor, which does not require air-supply equipment for on-site measurements, and made a fully disposable sensor tip for a portable device. The tip consists of a two-electrode system with P. fluorescens immobilized on a cellulose acetate membrane and is packaged in polyester film to prevent it from drying out. By aeration with a 0.1 M NaCl solution of P. fluorescens (after growth), the sensor responses as well as their reproducibility and stability have been successfully improved. The responses increased more than seven times, and the calibration curve from 15 to 260 mg l(-1) also remained linear although the response decreased approximately half the original after at least 35 days in storage. The reproducibility of the sensor responses improved to 12.7% (average of relative standard deviations (RSDs)) in the calibration curve obtained by using the Organization for Economic Cooperation and Development synthetic sewage. Examination of real samples from three different sources showed that the BOD as determined by the sensor correlates well with the conventional 5-day BOD method (r(2)=0.982, 0.823, and 0.809). Consequently, the aeration process makes it possible to realize rapid, and in situ measurements without the long conditioning process that is generally required to activate the microorganisms immobilized on bio-films before use. Finally, we have designed a portable device that utilizes our disposable sensor tip.

A chemiluminescent FIA biosensor for phosphate ion monitoring using pyruvate oxidase

We have constructed an automatic phosphate ion sensing system for the quality control of drinking water. The analyte was detected using the phosphate ion-dependent pyruvate oxidase reaction and the hydrogen peroxide produced was detected by luminol chemiluminescence catalyzed by Arthromyces ramosus peroxidase. We obtained a detection limit of 0.16 microM phosphate ion (5 ppb phosphorus) and it was possible to detect 0.32 microM phosphate ion for 48 days using pyruvate oxidase immobilized on Chitopearl BCW-2601 beads. An excellent correlation (r2 = 1.00) was obtained between the results obtained using our phosphate ion sensor and those using a modified Molybdenum Blue method.

Comparison of the dynamic transient- and steady-state measuring methods in a batch type BOD sensing system

Abstract Miniature oxygen electrodes functionalized with the yeast Trichosporon cutaneum were used for investigating the relationship between the steady-state method and the dynamic transient method in a batch type BOD sensing system. The two methods are equivalent in terms of reliability, having a high correlation (r2=−0.998) for both standard solutions and wastewater samples. The minimum dI/dt value was used as the response of the BOD sensors in the dynamic transient method. The advantage of this method is that the measuring time can be restricted to about 3 min. The next sample can be injected as soon as the slope of the current/time curve becomes constant, whereas the basic requirement for steady-state measuring is that the current itself becomes constant. This dramatically shortens the interval between each measurement. The stability of the BOD sensors was ensured by injecting a controlled amount of nutrient solution after each measurement. The standard deviation of reproducibility was 7%, not significantly different from that of the steady-state method (8%) but the lower limit of detection for the steady-state method (about 0.5 mg l−1 BOD) is half that of the dynamic transient method.

DETECTION OF PHYCOBILIN PIGMENTS AND THEIR SEASONAL CHANGE IN LAKE KASUMIGAURA USING A SENSITIVE IN SITU FLUOROMETRIC SENSOR

A two-channel fluorometric sensor system for the detection of the water bloom phytoplankton Microcystis aeruginosa has been developed. Excitation wavelengths of 620 nm and 440 nm were used; the former for detecting the cyanobacteria themselves, and the latter for subtracting the interference due to eukaryotic algae present in the sample. The fluorescence of the cyanobacteria and eukaryotic algae was measured at 645 nm and 680 nm, respectively. Applied to real samples from Lake Kasumigaura, Japan, after ultrasonication, this fluorometric method is capable of the rapid determination of phycocyanin. We used this sensor system for monitoring surface water in Lake Kasumigaura in 9 months in laboratory experiments. Moreover, an automatic device based on this system was used for the continuous in situ monitoring of phycocyanin in lakewater. As expected, higher levels of phycocyanin due to cyanobacteria were observed, compared to chlorophyll a due to eukaryotic algae in summer.

Monitoring of the composting process using a mediator-type biochemical oxygen demand sensor

The pH, electrical conductivity, ammonia-nitrogen, chemical oxygen demand, carbon-to-nitrogen ratio, total organic carbon and biochemical oxygen demand (BOD5) of compost extract solution and the moisture content and organic matter content of solid compost samples were investigated as parameters for monitoring the composting process. It was observed that the BOD5 value of the compost sample decreased dramatically from 355 to 33 mg L−1 during the process. Compared with other parameters, the BOD5 value showed the largest change. However, the conventional method of BOD5 determination is not suitable for process control owing to its time-consuming nature and the complicated procedures required. Thus, compost monitoring using a mediator-type BOD sensor consisting of Pseudomonas fluorescens biovar. V and potassium ferricyanide was investigated. In order to realize rapid and reproducible measurements, we developed a 10-channel potentiostat system incorporating screen-printed disposable chips incorporating microorganisms immobilized in sodium alginate gel. Using sludge extract solutions as standard solutions for calibration, it was found that aqueous extracts of the compost were most suitable for BOD determination. In conclusion, the relative change in BOD sensor values determined using our system corresponded well with the BOD5 values obtained using the standard 5 d method during 58 d of composting.

Molecularly Imprinted Polymers Selective for β-Estradiol

Abstract Polymers able to recognize the female steroid hormone, β-estradiol, have been synthesized using molecular imprinting based on non-covalent interactions. A chromatographic study of the polymers demonstrated a strong dependence of the selectivity and strength of their interaction with analytes on the solvents used both as porogen and chromatographic mobile phase. Hydrogen bonding between the OH-group at C-17 of β-estradiol and methacrylic acid (the functional monomer of the polymer) plays a major role in the process of molecular recognition but hydrophobic and dipole-dipole interactions also contribute.

Selective Recognition of 2,4-Dichlorophenoxyacetic Acid Using a Molecularly Imprinted Polymer

Abstract 2,4-dichlorophenoxyacetic acid (2,4-D) is a widely used herbicide, but environmental contamination by 2,4-D is problematic. Although biosensors for 2,4-D detection have been investigated, 2,4-D binding proteins and antibodies are too unstable to be used as biosensor recognition elements. In this study, a synthetic polymer selective for 2,4-D was prepared by molecular imprinting, and the binding characteristics of 2,4-D imprinted polymers were examined. Selectivity for 2,4-D was realized using methacrylic acid (MAA) as a functional monomer and the polymer may be used in a 2,4-D sensor.

A SIMPLE, MULTIPLE SIMULTANEOUS SPECTROPHOTOMETRIC METHOD FOR BOD DETERMINATION USING DCIP AS THE REDOX COLOR INDICATOR

ABSTRACT Especially in the analysis of environmental samples, it is necessary to perform rapid, simple and multiple analyses because it highly desirable to determine the results quickly as well as to analyze numerous samples simultaneously. We use 2,6-dichlorophenolindophenol (DCIP) as a redox color indicator for the Biochemical Oxygen Demand (BOD) determination in this report. The absorbance of DCIP decreases due to the metabolism of organic substances in aqueous samples by Pseudomonas fluoresens biovar V. Our technique using a microplate reader is able to measure 96 samples simultaneously. Thus real samples, blanks, standards and replicates can be determined concurrently, ensuring the accuracy of this method. The process requires approximately 20 min per determination, compared with 5 days for the standard BOD method. This method gives a linear response (r 2 = 0.943) to OECD synthetic sewage samples from 50 to 430 mg L −1.