Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H(2)O(2), and then the generated H(2)O(2) oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 x 10(-5)molL(-1) to 1.0 x 10(-3)molL(-1), and the detection limit was 5 x 10(-6)molL(-1) (3sigma). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3mmolL(-1), which was smaller than that of GOD immobilized in sol-gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platform

In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4 degrees C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

A novel chemiluminescence paper microfluidic biosensor based on enzymatic reaction for uric acid determination

In this work, chemiluminescence (CL) method was combined with microfluidic paper-based analytical device (μPAD) to establish a novel CL μPAD biosensor for the first time. This novel CL μPAD biosensor was based on enzyme reaction which produced H(2)O(2) while decomposing the substrate and the CL reaction between rhodanine derivative and generated H(2)O(2) in acid medium. Microchannels in μPAD were fabricated by cutting method. And the possible CL assay principle of this CL μPAD biosensor was explained. Rhodanine derivative system was used to reach the purpose of high sensitivity and well-defined signal for this CL μPAD biosensor. And the optimum reaction conditions were investigated. The quantitative determination of uric acid could be achieved by this CL μPAD biosensor with accurate and satisfactory result. And this biosensor could provide good reproducible results upon storage at 4°C for at least 10 weeks. The successful integration of μPAD and CL reaction made the final biosensor inexpensive, easy-to-use, low-volume, and portable for uric acid determination, which also greatly reduces the cost and increases the efficiency required for an analysis. We believe this simple, practical CL μPAD biosensor will be of interest for use in areas such as disease diagnosis.     

Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor

Together with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on-line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 muM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (+/- 4.4%). Both the bi-enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system. (c) 1993 John Wiley & Sons, Inc.     

Fabrication of a miniature CMOS-based optical biosensor

This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm × 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H₂O₂ and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 μL. Both optical H₂O₂ and glucose biosensors have an optimal operation temperature and pH of 20–25 °C and pH 8.4, respectively. The linear dynamic range of optical H₂O₂ and glucose biosensors is 0.05–20 mM H₂O₂ and 0.5–20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range.     

Photo‐induced chemiluminescent method for determination of reducing sugars

In the present study, a simple and sensitive photo‐induced chemiluminescence (CL) method for the quantitation of reducing sugars, including fructose, glucose, sucrose and lactose, was developed. This method was based on the on‐line photocatalytic reaction of the reducing sugars, using a home‐made photoreactor consisting of PTEF tube helically coiling around a high‐pressure mercury UV lamp. Reducing sugars were detected by direct CL emission resulting from the reaction between the photoproducts and luminol. To maximize the signal intensity, the effects of irradiation time, flow rate, luminol concentration, buffer pH and concentration were tested. Under optimum conditions, the linear dynamic ranges were all 0.36–18 mg/L and the relative standard deviations (RSDs) were 1.8–2.3%, with limits of detection (3σ) of 0.06 mg/L for fructose, glucose, sucrose and lactose. Finally, interference effects from ascorbic acid, amino acids (alanine, glycine, serine, leucine and methionine) and some metal ions and anions were also investigated. Copyright © 2008 John Wiley & Sons, Ltd.     

Chemiluminescence biosensor system for lactic acid using natural animal tissue as recognition element

A new method based on natural animal tissue porcine kidney as recognition element for chemiluminescence sensing of lactic acid is proposed in this paper. The principle for lactic acid sensing is that lactic acid is oxidized by oxygen under the catalysis of alpha-hydroxy acid oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of potassium ferricyanide to generate a CL signal. The experimental results show that the CL emission intensity was linear with lactic acid concentration in the range of 1-1000 micromol/L and the detection limit (3sigma) for lactic acid was 0.2 micromol/L. The biosensor could be used continuously for 6h with no significant changes in the response. More than 240 measurements were carried out during this time. A complete analysis, including sampling and washing, could be performed in 1.5 min with a relative standard deviation of 1.12% for 100 micromol/L lactic acid. The reproducibility among tissue columns was satisfactory (RSD among columns is less than 5%). The biosensor has been applied successfully to the analysis of lactic acid in plasma and milk samples.     

Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase.

Anionic sweet potato peroxidase (SPP; Ipomoea batatas) was shown to efficiently catalyse luminol oxidation by hydrogen peroxide, forming a long-term chemiluminescence (CL) signal. Like other anionic plant peroxidases, SPP is able to catalyse this enzymatic reaction efficiently in the absence of any enhancer. Maximum intensity produced in SPP-catalysed oxidation of luminol was detected at pH 7.8-7.9 to be lower than that characteristic of other peroxidases (8.4-8.6). Varying the concentrations of luminol, hydrogen peroxide and Tris buffer in the reaction medium, we determined favourable conditions for SPP catalysis (100 mmol/L Tris-HCl buffer, pH 7.8, containing 5 mmol/L hydrogen peroxide and 8 mmol/L luminol). The SPP detection limit in luminol oxidation was 1.0 x 10(-14) mol/L. High sensitivity in combination with the long-term CL signal and high stability is indicative of good promise for the application of SPP in CL enzyme immunoassay.     

Enhanced chemiluminescence of the luminol–AgNO3 system by Ag nanoparticles

The oxidation reaction of luminol with AgNO₃ can produce chemiluminescence (CL) in the presence of silver nanoparticles (NPs) in alkaline solution. Based on the studies of UV‐vis absorption spectra, photoluminescence (PL) spectra and CL spectra, a CL enhancement mechanism is proposed. The CL emission spectrum of the luminol–AgNO₃–Ag NPs system indicated that the luminophore was still 3‐aminophthalate. On injection of silver nanoparticles into the mixture of luminol and AgNO₃, they catalysed the reduction of AgNO₃ by luminol. The product luminol radicals reacted with the dissolved oxygen, to produce a strong CL emission. As a result, the CL intensity was substantially increased. Moreover, the influences of 18 amino acids, e.g. cystine, tyrosine and asparagine, and 25 organic compounds, including gallic acid, tannic acid and hydroquinone, on the luminol–AgNO₃–Ag NPs CL system were studied by a flow‐injection procedure, which led to an effective method for detecting these compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Influence of the storage time and the method of stimulation on whole blood chemiluminescence

The ultra‐weak light, chemiluminescence (CL), of stimulated leukocytes is a well‐known phenomenon. Parameters of this CL are modified by many factors including laboratory procedures. The order of stimulation and enhancement (two possibilities) and two concentrations of luminol create four types of procedure, which were accomplished in five sample storage ‘time points’. We received the strongest signals of CL using higher concentrations of luminol (and DMSO), but only when stimulation (FMLP) was used before enhancement (luminol); luminol used before FMLP strongly inhibited CL. For lower luminol concentration (and DMSO), the order of stimulation and enhancement was of no importance. There were comparable but weaker signals of CL in this case. We received stronger signals with storage time for all procedures. It may be dependent on the priming of phagocytes by releasing cell factors. Stimulation (FMLP) before enhancement (luminol) eliminates the inhibitory effect of DMSO on CL. Copyright © 2002 John Wiley & Sons, Ltd.     

Plant tissue-based chemiluminescence flow biosensor for determination of unbound dopamine in rabbit blood with on-line microdialysis sampling

A novel plant tissue-based chemiluminescence (CL) biosensor for dopamine combined with flow injection analysis is presented in this paper. The potato roots act as molecular recognition elements. Dopamine is oxidized by oxygen under the catalysis of polyphenol oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of potato tissue to generate CL signal. The CL emission intensity was linear with dopamine concentration in the range of 1x10(-5)-1x10(-7) g/ml and the detection limit was 5.3x10(-8) g/ml (3sigma) with a relative standard deviation of 1.7%. Combined with microdialysis sampling, the biosensor was applied to monitor the variation of dopamine level in the blood of rabbit after the administration of dopamine to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.     

The effect of pH on chemiluminescence of different probes exposed to superoxide and singlet oxygen generators

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO₂ and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO₂ and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H₂O₂ did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H₂O₂, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H₂O₂ also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H₂O₂ had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H₂O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used. © 1997 John Wiley & Sons, Ltd.     

Enhancement of chemiluminescence of the KIO4–luminol system by gallic acid,acetaldehyde and Mn2+: application for the determination of catecholamines

Chemiluminescence (CL) from the oxidation of luminol with potassium periodate in strong alkaline solutions was greatly enhanced by the combined effect of gallic acid, acetaldehyde and Mn²⁺. The CL spectra exhibited only one emission band at 425 nm, indicating 3‐aminophthalate as the emitting species. Various scavengers for superoxide anion, hydroxyl radical and singlet oxygen quenched the CL emission very efficiently (74–100%), suggesting the possible involvement of these reactive oxygen species (ROS) in the CL reactions. It is postulated that oxidation of gallic acid and acetaldehyde by periodate catalyzed by Mn²⁺ generates these ROS, which then react with luminol to enhance the CL emission. We also found that the enhanced CL emission was strongly inhibited by catecholamines, probably because of their effective scavenging of ROS. Based on this observation, a simple, rapid and sensitive new CL method was developed for the determination of catecholamines. The detection limits (3σ) for dopamine, l‐ dopa, norepinephrine and epinephrine were 0.63, 1.37, 0.56 and 14.3 nmol/L, respectively. The linear range was 1–10 nmol/L; relative standard deviations were 0.71–1.34% for 0.1 µmol/mL catecholamines. This CL method was applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

A highly sensitive and temporal visualization system for gaseous ethanol with chemiluminescence enhancer

A two‐dimensional gaseous ethanol visualization system has been developed and demonstrated using a horseradish peroxidase–luminol–hydrogen peroxide system with high‐purity luminol solution and a chemiluminescence (CL) enhancer. This system measures ethanol concentrations as intensities of CL via the luminol reaction. CL was emitted when the gaseous ethanol was injected onto an enzyme‐immobilized membrane, which was employed as a screen for two‐dimensional gas visualization. The average intensity of CL on the substrate was linearly related to the concentration of standard ethanol gas. These results were compared with the CL intensity of the CCD camera recording image in the visualization system. This system is available for gas components not only for spatial but also for temporal analysis in real time. A high‐purity sodium salt HG solution (L‐HG) instead of standard luminol solution and an enhancer, eosin Y (EY) solution, were adapted for improvement of CL intensity of the system. The visualization of gaseous ethanol was achieved at a detection limit of 3 ppm at optimized concentrations of L‐HG solution and EY. Copyright © 2011 John Wiley & Sons, Ltd.     

An amperometric biosensor for polyphenolic compounds in red wine

In the present work, a biosensor was developed with Laccase Coriolus Versicolor as the biological reconnaissance element immobilized on derivatized polyethersulphone membranes and applied to a Pt-Ag, AgCl US electrode base. Its application to several polyphenols usually found in red wine (caffeic acid, gallic acid, catechin, rutin, trans-resveratrol, quercetin and malvidin) was tested. It was observed that an amperometric response was obtained for catechin at +100 mV (versus Ag, AgCl) and caffeic acid at -50 mV in acetate buffer solutions (pH 4.5) having 12% ethanol. At pH 3.5 and +100 mV the biosensor was sensitive to both substrates and their response was additive. A limit of detection of 1.0 x 10(-6) M, linearity ranging from 2.0 to 14.0 x 10(-6) M, high sensitivity (0.0566 mAM(-1)) and reproducibility (R.S.D. <10%) were achieved for equimolar mixed solutions of catechin and caffeic acid. Under the same experimental conditions the other polyphenols tested individually did not yield any biosensor response. The application of the biosensor to red wine samples required a previous solid phase extraction for polyphenols enrichment. In fact, attempts to apply the biosensor in red wine using the "standard addition" methodology showed that large interferences occurred, as was to be expected. Reduction currents of -0.33 +/- 0.03 nA were obtained when the biosensor was used with the wine extract at +100 mV. This current could be ascribed to catechin and caffeic acid, although some interference by other polyphenols at the matrix level seemed to persist. The present biosensor showed promising applications for the wine analysis in future.     

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.     

Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate

A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.     

A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment

A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrile-co-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (< 30s) with a linear range of 5 x 10(-6) to 3 x 10(-3)M and a sensitivity of 6.82 mAM(-1)cm(-2). The apparent Michaelis-Menten constant (K(M)(app)) was 7.3mM.     

Investigation on the chemiluminescence reaction of the phenylhydrazine‐luminol–peroxide system

We studied the chemiluminescence (CL) oxidation of phenyl hydrazine–luminol with various organic and inorganic peroxides. Maximum CL intensity for this system was obtained for t‐butylhydroperoxide. The enhancement in CL depended strongly on pH and was greatest at pH 12.5. The solvent drastically enhanced the CL intensity. DMSO was found to increase the CL intensity many‐fold as compared to acetonitrile and water. The effect of temperature on CL intensity has also been studied. The CL spectra revealed a broad peak at 425 nm, which suggests excited 3‐aminophthalate ion as the luminophor. A mechanism to explain the reactions is suggested. Copyright © 2012 John Wiley & Sons, Ltd.