Development of a Potential-Modulated Electrochemiluminescence Measurement System for Selective and Sensitive Determination of the Controlled Drug Codeine.

Codeine is a common analgesic drug that is a pro-drug of morphine. It also has a high risk of abuse as a recreational drug because of its extensive distribution as an OTC drug. Therefore, sensitive and selective screening methods for codeine are crucial in forensic analytical chemistry. To date, a commercial analytical kit has not been developed for dedicated codeine determination, and there is a need for an analytical method to quantify codeine in the field. In the present work, potential modulation was combined with electrochemiluminescence (ECL) for sensitive determination of codeine. The potential modulated technique involved applying a signal to electrodes by superimposing an AC potential on the DC potential. When tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) was used as an ECL emitter, ECL activity was confirmed for codeine. A detailed investigation of the electrochemical reaction mechanism suggested a characteristic ECL reaction mechanism involving electrochemical oxidation of the opioid framework. Besides the usual ECL reaction derived from the amine framework, selective detection of codeine was possible under the measurement conditions, with clear luminescence observed in an acidic solution. The sensitivity of codeine detection by potential modulated-ECL was one order of magnitude higher than that obtained with the conventional potential sweep method. The proposed method was applied to codeine determination in actual prescription medications and OTC drug samples. Codeine was selectively determined from other compounds in medications and showed good linearity with a low detection limit (150 ng mL-1).

Sensitive screening of methamphetamine stimulant using potential-modulated electrochemiluminescence.

Methamphetamine (MA) is one of the most commonly abused recreational stimulants; thus, rapid and sensitive screening methods for MA are of great importance in forensic analytical chemistry. In the present work, potential modulation was combined with electrochemiluminescence (ECL) for the sensitive determination of MA. The potential modulated (PM) technique involved applying a signal to electrodes by superimposing an alternating current potential on the direct current potential. When tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) was used as an ECL emitter, the sensitivity of MA detection by PM-ECL was over 100 times that obtained with in conventional potential sweep mode. The radical on the α-carbon of the amine moiety is thought to play an important role in the ECL reaction mechanism involving amine-containing species. However, in the case of MA-type stimulants, density functional theory calculations suggest that the generated α-carbon radicals induce further intramolecular proton transfer. On the basis of the proposed ECL reaction route, we clarified the conditions under which MA could be selectively detected in the presence of the similar substance methoxyphenamine. The proposed method was applied to MA determination in a spiked human urine sample and showed good linearity with a low detection limit (100 nM, ca. 15 ng mL-1).

Electrochemiluminescence of Tris(2,2'-bipyridine)ruthenium(II)/Tri-n-propylamine with an Electric Contactless Power Transfer System.

An electrochemiluminescence (ECL) analytical device was developed using an electric contactless power transfer system. A mutually induced electromotive voltage was generated by wrapping an enameled wire around a commercial contactless charger. There was no electrical contact between the power supply and the electrochemical cell. For the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+)/tri-n-propylamine system, a weak ECL signal was observed. When an inexpensive rectifier diode was introduced between the coil and the working electrode, the ECL intensity detection sensitivity increased by more than 100 times. The relationship between the waveform of the applied voltage and the ECL response was clarified, and the optimum conditions were determined. The intensity of the induced electromotive voltage was easily controlled by changing the number of turns in the coil. The proposed method is a safe, simple, and inexpensive technique without electrical contact.

Sensitive Simultaneous Determinations of 1,2-Dihydroxynaphthalene and Catechol by an Amperometric Biosensor.

An amperometric biosensor for 1,2-dihydroxynaphthalene (DHN) and catechol (Cat) has been developed in order to monitor the biodegradaton of polycyclic aromatic hydrocarbons (PAHs). DHN is a common intermediary metabolite in naphthalene and phenanthrene degradation, while Cat is produced by further degradation. These compounds were detected by a biosensor modified with pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). The biosensor was based on signal amplification by enzyme-catalyzed redox cycling and was able to detect DHN and Cat at very low concentrations down to 10-9 M. Since the anodic waves of DHN and Cat were well separated, simultaneous determinations of these compounds were possible. Although the current signal for DHN was reduced in repeated measurements due to the oxidative polymerization of DHN, it can be avoided when the concentration of DHN was sufficiently low (<1 µM).

Sensitive electrochemical measurement of hydroxyl radical generation induced by the xanthine-xanthine oxidase system.

A sensitive electrochemical measurement system for hydroxyl radical (OH) was developed using enzyme-catalyzed signal amplification. In the presence of 2,6-xylenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) as a catalyst, the amperometric signal of the trapping adduct 2,6-dimethylhydroquinone (DMHQ) produced by the hydroxylation of 2,6-xylenol was able to be amplified and detected sensitively. The limit of detection (signal/noise [S/N]=3) for DMHQ was 1 nM. There was no significant interference from urate and other oxidizable compounds in the reaction mixture at the applied potential of 0V versus Ag/AgCl. This method was employed to observe the OH generation induced by the xanthine-xanthine oxidase (XO) system. The reaction rates of the DMHQ production induced from the xanthine-XO system in the presence and absence of various Fe(III) complexes and proteins were compared. Those with a free coordination site on the Fe atom effectively enhanced the OH generation.

A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases.

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase.

Cellulases hydrolyze cellulose to soluble sugars and this process is utilized in sustainable industries based on lignocellulosic feedstock. Better analytical tools will be necessary to understand basic cellulase mechanisms, and hence deliver rational improvements of the industrial process. In this work we describe a new electrochemical approach to the quantification of the populations of enzyme that are respectively free in the aqueous bulk, adsorbed to the insoluble substrate with an unoccupied active site or threaded with the cellulose strand in the active tunnel. Distinction of these three states appears essential to the identification of the rate-limiting step. The method is based on disposable graphene-modified screen-printed carbon electrodes, and we show how the temporal development in the concentrations of the three enzyme forms can be derived from a combination of the electrochemical data and adsorption measurements. The approach was tested for the cellobiohydrolase Cel7A from Hypocrea jecorina acting on microcrystalline cellulose, and it was found that the threaded enzyme form dominates for this system while adsorbed enzyme with an unoccupied active site constitutes less than 5% of the population.

Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain.

Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose.

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the ß-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.

Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A.

The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10-25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ~30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

Sensitive electrochemical detection of the hydroxyl radical using enzyme-catalyzed redox cycling.

Enzyme-catalyzed signal amplification was introduced to the electrochemical detection of the OH radical. In the presence of phenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-containing glucose dehydrogenase (PQQ-GDH) as a catalyst, the current signal for the trapping adducts (catechol and hydroquinone) produced by the hydroxylation of phenol could be amplified and detected sensitively. The limit of detection (S/N = 3) for catechol was 8 nM. The trapping efficiency of phenol was also estimated.

Thermal modulation voltammetry at a 1,2-dichloroethane/water microinterface using visible laser heating with optically absorbing supporting electrolyte.

Thermal modulation voltammetry (TMV) using laser heating at a liquid/liquid microinterface is a useful technique for determining the standard entropy change of ion transfer. In this work, for achieving TMV using a visible laser as a heating source at a 1,2-dichloroethane (DCE)/water microinterface, we used an optically absorbing supporting electrolyte, i.e., crystalviolet tetrakis(4-chlorophenyl)borate (CVTClPB). CVTClPB served well not only as a supporting electrolyte but also as an optical absorber in the DCE solution, and thereby, well-defined linear sweep (LS) and TM voltammograms could be obtained. On the basis of LS and thermal modulation (TM) voltammograms obtained with CVTClPB, the standard entropy changes of ion transfer for six model ions were successfully determined.

Decomposition of free chlorine with tertiary ammonium.

The reaction of free chlorine with tertiary ammonium or amine compounds in aqueous solution was studied by the amperometry at a rotating Pt-disk electrode. The amperometric method can be applied to follow the concentration of free chlorine (c(Cl)) even in the presence of chloramine species. By addition of mono- and dibutylammonium to the solution containing free chlorine, the step-like decrease in c(Cl) was observed, indicating the rapid formation of the stable chloramine species. By addition of tributylammonium, the c(Cl) was decreased exponentially to nearly zero even if the free chlorine was present initially in excess. The c(Cl)-t curves can be explained by tributylammonium-species-catalyzed decomposition of free chlorine to chloride ion. The catalytic decomposition was observed also with the tertiary-ammonium-based anion-exchange resins. Furthermore, the anion-exchange resins exhibited the decomposition of not only free chlorine but also chloramines in water.

Discoidin domain of chitosanase is required for binding to the fungal cell wall.

Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.

Activity measurement of chitosanase by an amperometric biosensor.

An amperometric biosensor for the detection of glucosamine (GlcN) and chitosan oligosaccharides ((GlcN)n) was introduced for an activity measurement of chitosanase. By using the biosensor, an increase in the anodic current due to the production of GlcN and (GlcN)n by chitosanase was measured in a chitosan solution. The maximum value of the slope of the current increase was proportional to the enzyme concentration up to 1.4 microg mL(-1). The present method had the advantages of being simple and rapid over the conventional Elson-Morgan method.

Application of polyammonium cations to enzyme-immobilized electrode: voltammetric behavior of polycation-hexacyanoferrate anion complexes and applicability as electron-transfer mediator.

Cyclic voltammograms of the aqueous solution containing polyammonium cations and the [Fe(CN)6]4-. anion at a plastic-formed carbon or platinum electrode were recorded. Polyammonium salts comprising quaternary ammonium in the main chain or sub chain and those comprising primary ammonium were tested. Under certain conditions, well-developed anodic and cathodic peak currents with the midpoint potentials different from that when the polyammonium cation is absent, that is, the midpoint potential of [Fe(CN)6]3- + e- = [Fe(CN)6]4- were exhibited, indicating that the [Fe(CN)6]4-/3- anionic species associate with the polyammonium cations tested to form the polycation-[Fe(CN)6]4-/3- complex species. The polycation complex species can be easily localized around the electrode surface with a dialysis membrane. An application of the polycation complex species as an electron-transfer mediator in the catalytic electrode with a redox enzyme was examined.

Application of polyammonium cations to enzyme-immobilized electrode: application as enzyme stabilizer for bilirubin oxidase.

Bilirubin oxidase was stored in the solutions containing polyammonium salts for a given time at 30 degrees C, and the activity was assayed. The enzyme catalyzes the reaction: 4[Fe(CN)6]4- + 4H+ + O2 --> 4[Fe(CN)6]3- + 2H2O, and the activity can be measured by the absorbance at the wavelength for the absorption maxima of [Fe(CN)6)]3-. The results show that polyammonium cations comprising quaternary ammonium in the main chain and hydrophilic groups, such as hydroxyl and amide groups, stabilize the enzyme in solution. These polyammonium cations may act like a protective colloid. The membrane-covered electrode containing the polyammonium cations, the enzyme, and [Fe(CN)6]4-/3- in the internal solution phase was constructed. The electrode gave a well-defined current-potential curve with a steady state limiting current due to the polycation-[Fe(CN)6]4-/3- complex-mediated bioelectrocatalytic current for the reduction of O2. The time-dependent decrease of the limiting current indicates again the stabilizing effect of the polyammonium cations on the enzyme.

A bioelectrocatalysis method for the kinetic measurement of thermal inactivation of a redox enzyme, bilirubin oxidase.

The thermal stability of a redox enzyme, bilirubin oxidase (BOD), has been quantitatively evaluated by measuring the inactivation kinetics of BOD at several temperatures. The enzyme activity is directly related to the mediated bioelectrocatalytic current for the BOD-catalyzed reduction of O(2). Thus, the inactivation process is measured by the time-dependent decrease in the bioelectrocatalytic current. The results reveal that the inactivation obeys first-order kinetics, whose rate constants (k) are determined at pH 7.0 and at 50 - 70 degrees C. The half life of BOD activity, calculated from the k value at 50 degrees C is 114 min, which is in harmony with the thermal-stability data given in a catalog by Amano Enzyme Inc. The bioelectrocatalysis method allows in situ measurements of the inactivation kinetics in the period of a few minutes at relatively high temperatures. The rate constants show a large temperature dependence, leading to a large Arrhenius activation energy (E(A)) of 221 kJ mol(-1). The activation Gibbs energy (DeltaG(not equal)), activation enthalpy (DeltaH(not equal)), and activation entropy (DeltaS(not equal)) are also determined.

Cyclic voltammetry of the electron transfer reaction between bis(cyclopentadienyl)iron in 1,2-dichloroethane and hexacyanoferrate in water.

The electron transfer (ET) reaction between bis(cyclopentadienyl)iron(II) ([Fe(II)(C(5)H(5))2]) in 1,2-dichloroethane (1,2-DCE) and hexacyanoferrate redox couple ([Fe(II/III)(CN)6](4-/3-)) in water (W) at the interface has been studied by using cyclic voltammetry. The voltammetric results can be explained well by a theoretical equation for the so-called IT-mechanism, in which a homogeneous ET reaction between [Fe(C(5)H(5))2] (partially distributed from 1,2-DCE) and [Fe(CN)6](3-) takes place in the W phase and the resultant [Fe(C(5)H(5))2]+ ion is responsible for current passage across the interface. The forward rate constant of the homogeneous ET reaction, [Fe(C(5)H(5))2] + [Fe(CN)6](3-) = [Fe(C(5)H(5))2]+ + [Fe(CN)6](4-) in W phase, k(f)(IT), was determined to be (2.9 +/- 2.2)x 10(10) M(-1) s(-1), which was in good agreement with k(f)(IT) = (3.2 +/- 2.0)x 10(10) M(-1) s(-1), which had been determined by using normal-pulse voltammetry.