Chemiluminescence in Analytical Chemistry

Similar to the work described by Spohn et al. [34], a trienzyme sensor was developed recently for the determination of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine). Leucine dehydrogenase, NADH oxidase, and peroxidase were coimmobilized covalently on tresylate-hydrophylic vinyl polymer beads and packed into a transparent PTFE tube (20 cm 1.0 id), which was used as flow cell. The sensor was free of interferences from protein and NH4 and it was stable for 2 weeks. The sensor system was applied to the determination of branched-chain amino acids in plasma with recoveries ranging from 98 to 100% [36].

A packed bed flow microreactor containing alanine aminotransferase and glutamate oxidase immobilized on sieved porous glass beads was combined with a CL detector for the generated H2O2 [37]. To catalyze the indicator reaction between luminol and H2O2, Co(II) and immobilized peroxidase from Arthromyces ramosus (ARP) were used in a fiberoptic detection cell. L-Alanine was determined from cell cultivation media in the 2–500-µM concentration range, with a limit of detection of 1 µM using Co(II), and in the 5–800-µM concentration range with a limit of detection of 2 µM when ARP was used. L-Glutamate and α-ketoglutarate were also determined with detection limits of 5 and 1 µM, respectively.

4.1.4Choline and Acetylcholine

The detection of choline using a CL biosensor is based on the immobilization on a polymer [38] or nylon [39] of choline oxidase and fungal peroxidase. The calibration graphs were linear in the 0.1–1-µmol/L concentration range with a limit of detection of 1 µmol/L. Acetylcholine and choline were determined satisfactorily in human serum using a biosensor prepared by covalently coupling acetylcholinesterase (AchE) and choline oxidase (ChO) to the controlled-pore glass as an enzyme reactor to produce hydrogen peroxide [40]. Electrostatically immobilized luminol and copper ion exchange resin were used as a transduction element in an injection system. The analytes were injected into a continuous stream of simple medium flowing through a sequence of enzyme reactors in which hydrogen peroxide was produced. Luminol and Cu2 were eluted and reacted with H2O2 to produce CL. The complete analysis was finished within 2 min with a detection limit of 500 fmol; the stability of the sensor was for 6 months.

4.1.5Phosphate

A FIA system has been proposed for the CL detection of phosphate based on an enzymatic reaction and the application of a subsequent luminol reaction [41]. The system consists of an immobilized pyruvate oxidase column, a mixing chamber for the CL reaction, and a PMT. H2O2 is generated by the reaction of phosphate and pyruvate oxidase and then reacts with luminol and HRP, producing

CL emission. The system allows a simple determination of phosphate in 3 min with a linear range of 4.8–160 µM. Owing to its sensitivity, this method could be satisfactorily applied to the analysis of maximum permissible phosphate concentrations in natural waters [42–44]. Also, the maltose-phosphorylase, mutarose, and glucose oxidase (MP-MUT-GOD) reaction system combined with an ARP-luminol reaction system has been used in a highly sensitive CL-FIA sensor [45]. In this system, MP-MUT-GOD is immobilized on N-hydroxysuccinimide beads and packed in a column. A linear range of 10 nM–30 µM and a measuring time of 3 min were provided, yielding a limit of detection of 1.0 µM as well as a satisfactory application in the analysis of river water.

4.1.6Carbamate and Organophosphorous Compounds

The above-mentioned system has also been used for the indirect CL determination of some carbamate and organophosphorous pesticides that inhibit acetylcholinesterase. Acetylcholinesterase in solution or immobilized on methacrylate beads is coupled to immobilized choline oxidase and peroxidase [46].

Acetylcholine

↓Acetylcholinesterase Choline

↓Choline oxidase

Peroxidase

Betaine 2 H2O2 → aminophthalate anion N2 3H2O light

luminol OH

In this system, choline formed by acetylcholinesterase is oxidized by choline oxidase and the hydrogen peroxide produced is determined using the luminol/peroxidase CL reaction. The sensor has been used for the analysis of Paraoxon and Aldicarb pesticides, with detection limits of 0.75 µg/L and 4 µg/ L, respectively. Recoveries in the range of 81–108% in contaminated samples of soils and vegetables were obtained.

4.1.7Xanthine and Hypoxanthine

A fiberoptic biosensor has been used for the determination of xanthine and hypoxanthine by immobilization of xanthine oxidase and peroxidase on different preactivated membranes, which were mounted onto the tip of the fiberoptic bundle [47]. The hydrogen peroxide generated was measured using the luminol reaction. A linear calibration curve of the sensors occurred in the range of 1–316 nM hypoxanthine and of 3.1–316 nM xanthine, respectively, with a detection limit of 0.55 nM.

4.1.8L-Lactate

A bienzyme fiberoptic sensor for the CL-FIA of L-lactate was developed by immobilizing lactate oxidase and peroxidase covalently on preactivated polyamide membrane. Hydrogen peroxide generated by the lactate oxidase reaction in the presence of L-lactate was the substrate of the second reaction catalyzed by peroxidase, in which light was produced in the presence of luminol [48]. Compartmentalization of the enzyme layer was obtained by stacking a peroxidase membrane on a lactate oxidase membrane at the sensing tip of the fiberoptic sensor. The detection limit was 250 pmol and the method was satisfactorily applied to lactate determinations in reconstituted whey solutions. Enzyme-modified silica and graphite paste were used to construct another CL biosensor for L-lactate [49]. L-Lactate oxidase was coupled with luminol/Na2CO3 (pH 9.2) to generate the CL. The system is very sensitive and selective when it is used in clinical analysis.

4.1.9Oxalate

An oxalate sensor by immobilizing spinach tissue as the source of oxalate oxidase was developed by Li [50]. The sensor responds linearly to oxalate concentration in the range of 1.0–100 µM with a detection limit of 0.6 µM. The sensor was stable for 30 days when stored at 4°C and a complete analysis for the determination of oxalate could be performed in 1 min including sampling and washing. Considering the low cost of the plant tissue and simple procedure for plant tissue immobilization, this report is most valuable.

4.1.10 Nicotinamide Adenine Dinucleotide (NADH)

In recent years, dehydrogenase-based biosensors have been extensively reported. NAD is required to catalyze the enzyme reaction of dehydrogenase. NADH produced by the enzyme reaction is related to the substrate concentration, and therefore, the substrate concentration can be determined by measuring NADH concentration. Usually, an electrochemical method is applied as sensing technique for NADH. Because high overpotential is necessary to oxidize NADH directly at an electrode, an electron transfer mediator is normally employed for electrochemical oxidation of NADH, such as ruthenium tris(2,2′-bipyridine). In this sense, a regenerable ECL biosensor for NADH based on dehydrogenase and tris(2,2′-bipyridyl) ruthenium(II) complex immobilized on Eastman AQ and Nafion polymer films was reported [51]. The biosensor design places an enzymeloaded polymer film adjacent to a tris(2,2′-bipyridyl)ruthenium(II)-loaded polymer film covering a platinum electrode, which is used in a FIA system as an ECL detector. The CL response to samples containing enzyme substrate and cofactor NAD results from the Ru(bpy)32 ECL reaction with NADH produced by the enzyme. Similar results were obtained with both kinds of films, providing

a detection range of 0.01–100 µM. Another bioluminescence biosensor is based on the detection on NADH by coupling NADH-flavin mononucleotide (FMN) oxidoreductase and bacterial luciferase [52]. A bioactive layer associated with the transducer was designed using a commercial preactivated polyamide membrane to which bacterial luciferase and oxidoreductase were covalently bound. The calibration graph was linear in the 10 pmol/L–0.5 nmol/L concentration range.

4.1.11Ethanol

Alcohol oxidase was used to generate H2O2 followed by its reaction with luminol in the presence of K3[Fe(CN)6] as a catalyst [53]. The luminescence was transmitted from the flow cell to the detector via optical fibers. Ethanol can be determined in the 3–750-µmol/L concentration range, with a detection limit of 3 µmol/L. Also, using an immobilized alcohol dehydrogenase reactor in glass beads, a FIA sensor for a reduced form of NADH was constructed by the ECL using the abovementioned ruthenium tris(2,2′-biryridine) complex. The sensor was satisfactorily applied to the determination of ethanol concentration [54].

4.1.12Cholesterol

Cholesterol oxidase was recently immobilized onto amine-modified silica gel via glutaraldehyde activation, and packed in a column [55]. The analytical reagents, including luminol and ferricyanide, were electrostatically coimmobilized on an anion-exchange column. Cholesterol was detected by the CL reaction between H2O2 generated in the enzymatic reaction and luminol and ferricyanide, which were released from a column by elution with immobilized reagents. The method was satisfactorily applied to the determination of cholesterol in human serum in a linear range of 5–100 ppm with a relative standard deviation less than 5%.

Some other typical examples of the enzyme-based CL sensors [56–62] are also included in Table 1.

4.2 Non-Enzyme-Based CL Sensors

4.2.1Chlorine

CL sensors based on immobilization of nonenzyme reagents have been extensively studied in recent years. Nakagama et al. [63] developed a CL sensor for monitoring free chlorine in tap water. This sensor consisted of a Pyrex tube, packed with the uranine (fluoresceine disodium) complex immobilized on IRA93 anion-exchange resin, and a PMT placed close to the Pyrex tube. It was used for monitoring the concentration of free chlorine (as HClO) in tap water, up to 1 mmol/L, with a detection limit of 2 µmol/L. The coefficient of variation (n

HRP, horseradish peroxidase; ARP, Arthromyces ramosus peroxidase.

10) obtained for the free chlorine assay is 1.6%, for a concentration of 10 mol/ L. The main disadvantage is the short lifetime of the sensor.

4.2.2Copper

The copper flow-through CL sensor comprised an anion-exchange column having luminol and cyanide coimmobilized on the resin, while copper was temporarily retained by electrochemical preconcentration on a Au electrode placed in an anodic stripping voltammetric cell [64]. Injection of 0.1 mol/L NaOH through the column eluted the reagents, which then reacted with copper, stripped from the electrode to produce a CL signal. The response was linear in the 0.01–10- g/L

Cu(II) concentration range in solution, with a detection limit of 8 ng/L. The RSD value at the 40 ng/L concentration level was 7.4%, for the assay of Cu(II) in natural waters and human serum.

4.2.3Epinephrine

By immobilizing Mn(III)-tetrakis(4-sulfonatophenyl)-porphyrin on dioctadecyldimethyl ammonium chloride bilayer membranes incorporated into a PVC film, Kuniyoshi et al. [65] developed an epinephrine CL sensor, which allowed determination of epinephrine down to 3 M with an RSD of 1.0% for 50 M of this biological compound. Compared with the previously reported epinephrine CL sensor [66], the present authors noted that the alkaline carrier solution, at high concentration levels, caused gradual deterioration of the immobilized catalyst, and this problem could be solved by the use of immobilization techniques other than ion exchange, e.g., solubilization of the catalyst that has octadecyl groups in the bilayer molecules.

4.2.4Hydrogen Peroxide, Ions, and Related Compounds

Zhang’s group proposed a type of so-called bleeding CL sensors for determination of inorganic and some organic analytes. The sensors were prepared by electrostatically immobilizing the reagents, luminol and some catalysts such as metal ions Co(II), Cu(II), or Fe(CN)63 , etc., on anion/cation-exchange columns. The analytes, e.g., H2O2, were sensed by the CL reaction of luminol and metal ions bleeding from the ion-exchange columns by hydrolysis. A flow injection system was used throughout for the measurements. The sensors could be used for determination of ClO [67]; CN [68]; Co2 [69]; V(V) [70]; Fe2 and Fe3 [71]; Fe3 [72]; SO32 [73]; H2O2 [74] and its monitoring in rain water [75], etc.

By immobilization of luminol and Co(II) on a strongly basic anion-ex-

change resin and a weakly acid cation-exchange resin, H2O2 can be determined in the 40 nmol/L–10 mol/L concentration range with a limit of detection of 12

nmol/L [76]. This system was combined with FIA and its high sensitivity made possible the analysis of hydrogen peroxide in water and the assay of glucose in serum by measuring the formation of H2O2 from a packed bed reactor with immobilized glucose oxidase. By in situ electrogenerated H2O2 on an electrode, a CL sensor for vitamin B12 determinations was developed, based on immobilizing luminol on an ion-exchange resin [77].

Also, a selective sensor for hydrogen peroxide based on FIA was proposed by Janasek et al. [78]. H2O2 can be detected in the presence of luminol and Co(II) and Cu(II) foils, obtaining linear determination ranges of 0.1–200 mol/L and 5–200 mol/L under FI conditions, respectively. To improve the selectivity, the CL detector was combined with a thin-layer gas dialysis cell.

4.2.5Sulfur-Containing Compounds

A reagentless flow sensor for sulfite was developed by electrostatically immobilizing the oxidizer permanganate and the sensitizer riboflavin phosphate on an anion-exchange column, allowing a sensitive determination of sulfite in beverage [79]. In this sensor, both the oxidation and the subsequent energy transfer process proceed directly with the immobilized reagent and there is no need to supply an eluent, thus permitting the flow sensor to operate in a reagentless way. As compared to the use of continuously delivered reagents in conventional CL flow systems, this CL sensor shows some advantages in terms of operational convenience, instrumental simplification, reducing reagent consumption, and decreasing analyte dilution, achieving a lifetime much longer than with eluting reagent. Sulfite can be assayed in beer and wine in the 0.1–100-mg/L concentration range with a detection limit of 0.06 mg/L, and an RSD of 3.7% for 1.0 mg/L of sulfite. A sensitive CL sensor was recently proposed for the determination of sulfite with FIA [80]. It is based on the weak CL produced by auto-oxidation of sulfite in the presence of rhodamine 6G immobilized electrostatically on a cation exchange column. A strong enhancement of the weak CL signal was observed in the presence of micelles of the Tween 80 surfactant, showing a linear calibration range of 0.01–5 ppm with a detection limit of 0.01 ppm. Interfering metal ions coexisting in the sample solution can be eliminated online by an upstream cation exchanger.

Using ECL, an assay for S2O82 was reported by application of a microring electrode [81]. A gold-coated fiber was polished to a flat surface, such that the gold formed a micro-ring electrode around the optical fiber. Tris(2,2′-bipyri- dyl)ruthenium(II) was used as reagent for CL generation. The detection limit was 4 nmol/L.

4.2.6Ammonium Ion

Zhang’s group [82] recently presented a novel CL sensor combined with FIA for ammonium ion determination. It is based on reaction between luminol, immobilized electrostatically on an anion-exchange column, and chlorine, electrochemically generated online via a Pt electrode from hydrochloric acid in a coulometric cell. Ammonium ion reacts with the chlorine and decreases the produced CL intensity. The system responds linearly to ammonium ion concentration in a range of 1.0–100 µM, with a detection limit of 0.4 µM. A complete analysis can be performed in 1 min, being satisfactorily applied to the analysis of rainwater.

4.2.7Ascorbic Acid

Three sensors based on luminol and different cations immobilized on a resin are proposed for ascorbic acid assay, in the following way: (1) D-201 type anion-

exchange resin containing luminol and permanganate immobilized [83]; (2) D- 201 7 anion-exchange resin was used for immobilization of luminol and 732 cation-exchange resin (Na form) was used for Fe(II)-immobilization [84]; (3) Amberlite A-27 anion-exchange resin containing immobilized luminol and potassium ferricyanide [85]. Using these types of flow-through sensors, the CL signal produced by the reaction with luminol was decreased in the presence of ascorbic acid. This fact allows the indirect determination of ascorbic acid on: (1) 10 µg/ L–4 mg/L; (2) 1 nmol/L–1 µmol/L; and (3) 0.01–0.8 µg/mL concentration ranges, with the following detection limits: (1) 5 µg/L; (2) 0.4 nmol/L; (3) 5.5 ng/ mL, respectively. While the first proposed sensor is free of interferences, Cu(II), thiourea, uric acid, and vitamin B1 seriously interfere with the third sensor.

4.2.8Ethanol and Organic Molecules

CL sensors are described for the assay of ethanol and organic molecules in water, comprising a γ-Al2O3 layer as catalyst, which can be coated with a Pt thin film [86]. When a mixture of air and organic molecules (e.g., ethanol and acetone) vaporized from a solution flows around the sensor, CL is emitted during the catalytic oxidation. The CL spectra consist of subbands with peak wavelengths independent of the type of vapors, the CL intensity depending on the concentration of these organic compounds, and the temperature of the sensor. The limit of detection is in the mg/L-magnitude order.

4.2.9Trichloroethylene

An optical-fiber CL sensor is reported for trichlorethylene assay [87]. The sensor consists of a glass fiber bundle and a transducer consisting of three components:

(i) a gas-permeable membrane to separate trichlorethylene from water, (ii) H2SO4- NaNO3 mixture as oxidizing agent, and (iii) a luminol solution. The assay of trichloroethylene can be done in the 0.05–0.6-µg/mL concentration range with a detection limit of 0.03 µg/mL.

4.2.10Uric Acid

For the assay of uric acid, a sensor based on KMnO4–octylphenyl polyglycol ether is proposed [88]. Uric acid can be assayed directly in urine in the 0.10– 600-µg/mL concentration range with a detection limit of 55 ng/mL. The system is free of interferences.

4.2.11Oxalate, Alkylamine, and NADH

The development of an ECL sensor based on Ru(bpy)32 immobilized in a Nafion film coated on an electrode was discussed by Downey and Nieman [89]. The

sensor was used to determine oxalate, alkylamine, and NADH. Detection limits were of 1 M, 10 nM, and 1 M, respectively, with working ranges extending over four magnitudes in concentration. The sensor remains stable for several days under suitable storage conditions, being the first research regarding NADH detection using Ru(bpy)32 ECL. Using the same system, NADH detection was optimized in a FIA sensor, providing a linear concentration range of 10–250 M [54].

The substrate selectivity of the Ru(bpy)32 ECL was changed by coating a Pt working electrode with Ru(bpy)32 -modified chitosan membrane and successively with a silica gel membrane that was prepared by the sol-gel method using tetramethoxysilane as a precursor [90]. The double coating resulted in a high selectivity toward oxalic acid at a pH 6 allowing development of an ECL sensor for oxalate [91]. This high selectivity in the presence of trimethylamine can be explained partly on the basis of the electrostatic repulsion of the chitosan membrane having a positive charge with trimethylamine, as being supported by cyclic voltametry. A linear calibration range was established of 0.1–10 mM with a detection limit of 0.03 mM [92].

4.2.12 Primary Alcohols

Another ECL sensor suitable for primary alcohols based on ECL of hydroxyl compounds by cyclic square-wave electrolysis was developed by Egashira et al.

Table 2 Some Typical Examples of Non-Enzyme-Based CL Sensors

EG, electrogenerated.