Development of a long-life capillary enzyme bioreactor for the determination of blood glucose

A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability than previous systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53 mm i.d. fused-silica capillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxide (H₂O₂). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactor into a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumption compared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 °C) stability for 20 weeks, the glucose biosensing system was prepared using an optimal GOx concentration (200 mg/mL). This exhibited an FIA peak response of 7 min and a detection limit of 10 μM (S/N = 3) with excellent reproducibility (coefficient of variation, CV < 0.75%). It also had a linear working range from 10¹ to 10⁴ μM. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samples were analyzed using this system, and these correlated favorably (correlation coefficient, r² = 0.935) with results for the same samples obtained using a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.     

Spectrofluorimetric method for measuring the activity of the enzyme α-l-fucosidase using the ion associate of 2-chloro-4-nitro phenol-rhodamine-B

A low cost and accurate method for the detection and analytical determination of the activity of the enzyme α-l-fucosidase (AFU) was developed. The method was based upon measuring the fluorescence intensity of the complex ion associate of the ion associate of rhodamine-B and the compound 2-chloro-4-nitrophenol (RB⁺ CNP⁻) at 580 nm in phosphate buffer (pH 5) against the reagent blank. The influence of the different parameters, e.g. pH, incubation time, temperature, 2-chloro-4-nitrophenol concentration, foreign ions and surfactants that control the fluorescence intensity of the produced ion associate was critically investigated. The correlation between the fluorescence activity of the enzyme AFU by the developed procedures and the standard method was positive and highly significant in patients and controls (r² = 0.99, p < 0.001). The developed method is simple and proceeds without practical artifacts compared to the standard method.     

Single-walled carbon nanohorn as new solid-phase extraction adsorbent for determination of 4-nitrophenol in water sample

Single-walled carbon nanohorn (SWCNH) was developed as new adsorbent for solid-phase extraction using 4-nitrophenol as representative. The unique exoteric structures and high surface area of SWCNH allow extracting a large amount of 4-nitrophenol over a short time. Highly sensitive determination of 4-nitrophenol was achieved by linear sweep voltammetry after only 120 s extraction. The calibration plot for 4-nitrophenol determination is linear in the range of 5.0 × 10⁻⁸ M-1.0 × 10⁻⁵ M under optimum conditions. The detection limit is 1.1 × 10⁻⁸ M. The proposed method was successfully employed to determine 4-nitrophenol in lake water samples, and the recoveries of the spiked 4-nitrophenol were excellent (92-106%).     

Determination of trace aluminium by adsorptive stripping voltammetry on a preplated bismuth-film electrode in the presence of cupferron

This work reports the use of adsorptive stripping voltammetry (AdSV) for the determination of aluminium on a rotating-disc bismuth-film electrode (BiFE). Al(III) ions in the non-deoxygenated sample were complexed with cupferron and the complex was accumulated by adsorption on the surface of the preplated BiFE. The stripping step was carried out by using a square-wave (SW) potential-time voltammetric excitation signal. The experimental variables as well as potential interferences were investigated and the figures of merit of the method were established. Using the selected conditions, the 3σ limit of detection for aluminium was 0.5 μg l⁻¹ at a preconcentration time of 240 s and the relative standard deviation was 4.2% at the 5 μg 1⁻¹ level for a preconcentration time of 120 s (n = 8). The accuracy of the method was established by analysing water and metallurgical samples.     

Carbon nanospheres enhanced electrochemiluminescence of CdS quantum dots for biosensing of hypoxanthine

This work developed a novel method to greatly enhance the electrochemiluminescence (ECL) of CdS quantum dots (QDs). The ECL amplification was achieved by the assembly of QDs on poly (diallyldimethylammonium chloride)-functionalized carbon nanospheres (PFCNSs), and successfully employed for sensitive ECL biosensing of oxidase substrates. The carbon nanospheres were prepared by a “green” method, and the high loading of QDs on carbon nanospheres led to a 4-times increased ECL intensity with dissolved O₂ as the coreactant. Using xanthine oxidase (XOD) as a model, an ECL biosensor was fabricated by immobilizing the enzyme on the mixing membrane of PFCNSs and QDs. The ECL biosensor showed a fast response to hypoxanthine with a linear concentration range from 2.5 × 10⁻⁸ to 1.4 × 10⁻⁵ M. The limit of detection was 5 nM at a signal-to-noise ratio of 3. The assay results of hypoxanthine in fish samples were in a good agreement with the reference values by amperometric technique. This facile approach to prepare the PFCNSs/QDs system for ECL biosensing could be of promising application in bioanalysis and electronic device.     

Flow injection analysis system for on-line cutinase activity assay

A FIA system based on a micellar system for a substrate (p-nitrophenylbutyrate) with low stability in aqueous phase was built to monitor cutinase activity in bioprocesses. All samples were previously diluted with 50 mM phosphate buffer pH 7.0 containing 11.6 mM sodium cholate. The cutinase activity in this diluted solution enhances about 40% in relation to phosphate buffer. Furthermore, the enzyme adsorption and consequent blocking/fouling of injector and tubes of the FIA system was eliminated due to excellent properties of sodium cholate as surface active agent.The cutinase activity is based in following the hydrolysis of p-nitrophenylbutyrate to p-nitrophenol in the reaction stream through the formation of an absorbance peak at 400 nm proportional to enzyme activity. The compositions of reaction stream as well as its stability were studied in order to minimize non-enzymatic hydrolysis of p-nitrophenylbutyrate and maximize cutinase activity assay reproducibility. An excellent correlation was obtained between the FIA system and off-line method for determination of cutinase activity in the culture media, and during separation of Saccharomyces cerevisiae cells and cutinase concentration by micro and ultrafiltration, respectively.     

On-line selective solid-phase extraction of 4-nitrophenol with β-cyclodextrin bonded silica

The selective analysis of 4-nitrophenol (4-NP) from water samples using on-line solid-phase extraction (SPE) coupled to HPLC system was studied. The β-cyclodextrin bonded silica (CDS) was utilized as the selective sorbent. Using 100 ml of sample solution spiked with 4-nitrophenol and other six phenols (Ph) in double distilled water, the sorbent showed strong capacity in adsorbing 4-nitrophenol and the recovery was 104% with the detection limit of 0.017 μg/l. The selectivity was investigated by utilizing a washing step with acetonitrile after preconcentration and only 4-nitrophenol was detected with the recovery of 99%. Donghu lake (Wuhan, China) water sample was used to test the on-line SPE-HPLC system and 4-nitrophenol was selectively extracted with the recovery obtained as 90%.     

Sequential injection monosegmented flow voltammetric determination of cadmium and lead using a bismuth film working electrode

A cost-effective sequential injection monosegmented flow analysis (SI-MSFA) with anodic stripping voltammetric (ASV) detection has been developed for determination of Cd(II) and Pb(II). The bismuth film working electrode (BiFE) was employed for accumulative preconcentration of the metals by applying a fixed potential of −1.10 V versus Ag/AgCl electrode for 90 s. The SI-MSFA provides a convenient means for preparation of a homogeneous solution zone containing sample in an acetate buffer electrolyte solution and Bi(III) solution for in situ plating of BiFE, ready for ASV measurement at a flow through thin layer electrochemical cell. Under the optimum conditions, linear calibration graphs in range of 10-100 μg L⁻¹ of both Cd(II) and Pb(II) were obtained with detection limits of 1.4 and 6.9 μg L⁻¹ of Cd(II) and Pb(II), respectively. Relative standard deviations were 2.7 and 3.1%, for 11 replicate analyses of 25 μg L⁻¹ Cd(II) and 25 μg L⁻¹ Pb(II), respectively. A sample throughput of 12 h⁻¹ was achieved with low consumption of reagent and sample solutions. The system was successfully applied for analysis of water samples collected from a draining pond of zinc mining, validating by inductively coupled plasma-optical emission spectroscopy (ICP-OES) method.     

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71-27 ng ml⁻¹ with a limit of detection (LOD) of 0.32 ng ml⁻¹, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC₅₀) was 4.2 ng ml⁻¹. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67-27 ng ml⁻¹ with a LOD of 0.38 ng ml⁻¹ and an IC₅₀ of 3.7 ng ml⁻¹. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml⁻¹ were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.     

Multi-syringe flow injection solid-phase extraction system for on-line simultaneous spectrophotometric determination of nitro-substituted phenol isomers

In this paper, a time-based multi-syringe flow injection (MSFI) approach is proposed for automated disk-based sorbent extraction of three nitro-substituted phenol isomers (2-, 3-, and 4-nitrophenol) followed by on-line simultaneous determination of individual species by diode-array spectrophotometry. The method involves the on-line enrichment of the targeted analytes from an acidic medium containing 0.1 mol L⁻¹ HCl onto a co-polymeric sorbent material, and the concurrent removal of potentially interfering matrix components. The nitrophenol isomers are subsequently eluted with an alkaline solution (0.7 mol L⁻¹ NaOH), whereupon the eluate is delivered to a diode-array spectrophotometer for recording of the spectral data in the UV-vis region. Deconvolution of strongly overlapped spectra was conducted with multivariate regression models based on multiple linear regression calibration. The analytical performance of the chemometric algorithm was characterized by relative prediction errors and recoveries.The MSFI manifold was coupled to a multiposition selection valve to set a rugged analyzer that ensures minimum operational maintenance via exploitation of membrane switching protocols. As compared with earlier methods for isolation/pre-concentration of nitro-substituted phenols based on liquid-liquid extraction, the proposed flow-through disk-based system should be regarded as an environmentally friendly approach because the use of harmful organic solvents is circumvented. Under the optimized chemical and physical variables, the 3σ_blank detection limits for 2-, 3-, and 4-nitrophenol were 1.2, 3.2 and 0.3 μmol L⁻¹ for a sample loading volume of 1.5 mL, and the relative standard deviations were ≤5.0%. The flowing system, which is able to handle up to 135 samples automatically, was proven suitable for monitoring trace levels of the target isomers in mineral, tap, and seawater.     

Preparation of gold nanoparticles on eggshell membrane and their biosensing application

A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25 ± 7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl₄ without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl₄) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH ≤3.0 and ≥7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO_x) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO_x. The linear response range of the glucose biosensor is 20 μM to 0.80 mM glucose with a detection limit of 17 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field.     

Zinc oxide-potassium ferricyanide composite thin film matrix for biosensing applications

Thin film of zinc oxide-potassium ferricyanide (ZnO-KFCN) composite has been deposited on indium tin oxide (ITO) coated corning glass using pulsed laser deposition (PLD). The composite thin film electrode has been exploited for amperometric biosensing in a mediator-free electrolyte. The composite matrix has the advantages of high iso-electric point of ZnO along with enhanced electron communication due to the presence of a redox species in the matrix itself. Glucose oxidase (GOx) has been chosen as the model enzyme for studying the application of the developed matrix to biosensing. The sensing response of the bio-electrode, GOx/ZnO-KFCN/ITO/glass, towards glucose was studied using cylic voltammetry (CV) and photometric assay. The bio-electrode exhibits good linearity from 2.78 mM to 11.11 mM glucose concentration. The low value of Michaelis-Menten constant (1.69 mM) indicates an enhanced affinity of the immobilized enzyme towards its substrate. A quassireversible system is obtained with the composite matrix. The results confirm promising application of the ZnO-KFCN composite matrix for amperometric biosensing applications in a mediator-less electrolyte that could lead to the realization of an integrated lab-on-chip device.     

Visible light induced photoelectrochemical biosensing based on oxygen-sensitive quantum dots

A visible light induced photoelectrochemical biosensing platform based on oxygen-sensitive near-infrared quantum dots (NIR QDs) was developed for detection of glucose. The NIR QDs were synthesized in an aqueous solution, and characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. The as-prepared NIR QDs were employed to construct oxygen-sensitive photoelectrochemical biosensor on a fluorine-doped tin oxide (FTO) electrode. The oxygen dependency of the photocurrent was investigated at as-prepared electrode, which demonstrated the signal of photocurrent is suppressed with the decreasing of oxygen. Coupling with the consumption of oxygen during enzymatic reaction, a photoelectrochemical strategy was proposed for the detection of substrate. Using glucose oxidase (GOx) as a model enzyme, that is, GOx was covalently attached to the surface of CdTe QDs, the resulting biosensor showed the sensitive response to glucose. Under the irradiation of visible light of a wavelength at 505 nm, the proposed photoelectrochemical method could detect glucose ranging from 0.1 mM to 11 mM with a detection limit of 0.04 mM. The photoelectrochemical biosensor showed a good performance with high upper detection limit, acceptable stability and accuracy, providing an alternative method for monitoring biomolecules and extending the application of near-infrared QDs.     

Amperometric enzyme electrodes of glucose and lactate based on poly(diallyldimethylammonium)-alginate-metal ion-enzyme biocomposites

Sodium alginate (AlgNa) and poly(diallyldimethylammonium chloride) (PDDA) were mixed to obtain an interpenetrating polymer composite via electrostatic interaction and then cast on an Au electrode surface, followed by incorporation of metal ions (e.g. Fe³⁺ or Ca²⁺, to form AlgFe or AlgCa hydrogel) and glucose oxidase (GOx) (or lactate oxidase (LOx)), to prepare amperometric enzyme electrodes. The interactions of PDDA, Alg, and Fe³⁺ are studied by visual inspection as well as microscopic and electrochemical methods. Under optimized conditions, the PDDA-AlgFe-enzyme/Au and PDDA-AlgCa-enzyme/Au electrodes can give good analytical performance (e.g. nM-scale limit of detection of glucose or lactate, and sensitivities > 50 μA cm⁻² mM⁻¹) in the first-generation biosensing mode, which are better than the reported analogs using typical polysaccharide biopolymers as enzyme-immobilization matrices. The enzyme electrodes also worked well in the second-generation biosensing mode in the coexistence of p-benzoquione or ferrocene monocarboxylic acid artificial mediator. Biofuel cells (BFCs) with the enzyme electrodes as the bioanodes and glucose (or lactate) as the biofuel were also fabricated with satisfactory results. The proposed protocols for preparation of high performance Alg-based biocomposites may find wide applications in bioanalysis.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Validation of bismuth film electrode for determination of cobalt and cadmium in soil extracts using ICP-MS

A study is presented on the use of the bismuth film electrode (BiFE) operated in the anodic stripping and the cathodic adsorptive stripping voltammetry (ASV, CAdSV) modes, for the determination of two trace heavy metals (Cd and Co, respectively), in soil extract samples. Two types of BiFE were examined in this study: the in situ prepared BiFE, which was employed in ASV determination of Cd, and the ex situ prepared BiFE, which was used in CAdSV of Co with dimethylglyoxime (DMG) as complexing agent. A series of unpretreated soil extracts with varying Cd and Co concentrations were analyzed, and the results obtained compared to those determined using inductively coupled plasma-mass spectrometry (ICP-MS). The results revealed the suitability of stripping analysis at the BiFE for determination of μg l⁻¹ levels of heavy metals in soil extracts. The promising results obtained here, coupled with the non-toxic nature of bismuth (in comparison to commonly used mercury electrodes employed in stripping analysis), offer great promise in centralized and decentralized analysis of trace heavy metals in complex environmental matrices.     

Preliminary study of a microbeads based histamine detection for food analysis using thermostable recombinant histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

We designed and prepared a micro biosensing system consisting of a flow through system with a sub-micro liter injection valve and a sub-micro liter volume bioreactor. An electrochemical detector was combined with the reactor for immediate detections. The volumes of the reactor and the sample loop for the injection were 850 nl and 320 nl, respectively. This paper described about the characteristics of the sensing system in the case of histamine detection for food analysis. Histamine oxidase from KAIT-B-007 was prepared by using a gene recombination technique and they were immobilized with chitosan beads (? = 70-105 μm). The detection less than one minute after injection made possible fast analysis for histamine. The biosensing system also showed a high performance for histamine detection in wide range of 1 μM-1 mM. In addition, we practically measured histamine content in raw tuna stored at room temperature and 35 °C up to 96 h. As a result of the comparison between our sensing system and HPLC method, there was good agreement. These results show that our microfluidic biosensing system has the potential to assist miniaturization with small sample volume and short determination time for a sequential food analysis.     

On-line stripping voltammetry of trace metals at a flow-through bismuth-film electrode by means of a hybrid flow-injection/sequential-injection system

In this work, we describe an automated stripping analyzer operating on a hybrid flow-injection/sequential-injection (FIA/SIA) mode and utilizing a bismuth-film electrode (BiFE) as a flow-through sensor for on-line stripping voltammetry of trace metals. The instrument combines the advantages of FIA and SIA and is characterised by simplicity, low-cost, rapidity, versatility and low consumption of solutions. The proposed analytical flow methodology was applied to the determination of Cd(II) and Pb(II) by anodic stripping voltammetry (ASV) and of Ni(II) and Co(II) by adsorptive stripping voltammetry (AdSV). The steps of the rather complex experimental sequence (i.e. the bismuth-film formation, the analyte accumulation, the voltammetric stripping and the electrode cleaning/regeneration) were conducted on-line and the critical parameters related to the respective analytical procedures were investigated. In ASV, for a accumulation time of 180 s the limits of detection for Cd(II) and Pb(II) were 2 and 1 μg l⁻¹, respectively (S/N = 3) and the relative standard deviations were 5.3% and 4.7%, respectively (n = 8). In AdSV, for a total sample volume of 1000 μl, the limits of detection for Ni(II) and Co(II) were 1 μg l⁻¹ (S/N = 3) and the relative standard deviations were 5.5% and 6.2%, respectively (n = 8). The measurement frequency ranged between 15 and 20 stripping cycles h⁻¹. The results indicate that the BiFE is well suited as a flow-through detector for on-line stripping analysis and, by virtue of its low toxicity, can serve as a viable alternative to mercury-based flow-through electrodes.     

An introduction to bismuth film electrode for use in cathodic electrochemical detection

A new electrode surface design, the bismuth film electrode (BiFE), is presented as a promising alternative to mercury and other solid electrodes for direct cathodic electrochemical detection of organic compounds. The preparation of the BiFE, involving an ex situ electroplating of metallic bismuth onto a glassy carbon (GC) substrate electrode, was optimised. The useful negative potential windows of the BiFE in the pH range 1 (−0.2 to −0.8 V vs Ag/AgCl) to 10 (−0.2 to −1.5 V) were determined. The reproducibility of measuring 2-nitrophenol as a model compound (relative standard deviation, r.s.d., n=10) was found to be 0.5% at the same BiFE, and 1.0% at successive newly prepared BiFEs. No polishing or any other pre-treatment of the substrate GC surface was required prior to re-plating of a new Bi film. The BiFE showed similar or even favourable voltammetric behaviour when compared to mercury and bare GC electrodes, and was successfully tested for amperometric detection under hydrodynamic conditions. The results revealed that BiFE is an attractive new non-mercury metallic electrode particularly suitable for cathodic electrochemical detection in flow analytical systems.     

A sensitive enzymatic method for paraoxon detection based on enzyme inhibition and fluorescence quenching

A sensitive and selective method for the paraoxon detection based on enzyme inhibition and fluorescence quenching was presented in this study. Under the catalytic effect of acetylcholinesterase (AChE), acetylthiocholine (ATCh) hydrolysis released thiocholine (TCh) which could react with N-(7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM) to produce a blue fluorescence compound. Subsequently, AChE catalytic activity was inhibited with the addition of paraoxon, which caused TCh decreased, leading to a significant decrease of the blue fluorescent compound. Meanwhile, p-nitrophenol, the hydrolysis product of paraoxon, would lead to a quenching of the fluorescence. Therefore, fluorescence intensity of the system would decrease dramatically by a combined effect of enzyme inhibition and fluorescence quenching. Under optimal experimental conditions, an excellent linear relationship between the decrease of fluorescence intensity and paraoxon concentration over the range from 5.5 × 10⁻¹² to 1.8 × 10⁻¹⁰ mol L⁻¹ was obtained. Fluorescence background caused by nonenzymatic hydrolysis of ATCh or other matters was relatively low, the proposed approach offered adequate sensitivity for the detection of paraoxon at 3.5 × 10⁻¹² mol L⁻¹.