Microfluidic conductimetric bioreactor

A microfluidic conductimetric bioreactor has been developed. Enzyme was immobilized in the microfluidic channel on poly-dimethylsiloxane (PDMS) surface via covalent binding method. The detection unit consisted of two gold electrodes and a laboratory-built conductimetric transducer to monitor the increase in the conductivity of the solution due to the change of the charges generated by the enzyme-substrate catalytic reaction. Urea–urease was used as a representative analyte-enzyme system. Under optimum conditions urea could be determined with a detection limit of 0.09 mM and linearity in the range of 0.1–10 mM (r = 0.9944). The immobilized urease on the microchannel chip provided good stability (>30 days of operation time) and good repeatability with an R.S.D. lower than 2.3%. Good agreement was obtained when urea concentrations of human serum samples determined by the microfluidic flow injection conductimetric bioreactor system were compared to those obtained using the Berthelot reaction (P < 0.05). After prolong use the immobilized enzyme could be removed from the PDMS microchannel chip enabling new active enzyme to be immobilized and the chip to be reused.     

Calorimetric biosensors with integrated microfluidic channels

A microfluidic device capable of measuring real-time enthalpy changes of biochemical reactions and thermal properties of biological fluids is presented in this paper. The device consists of a freestanding microthermopile integrated with a glass microfluidic reaction chamber. The p-type polysilicon/gold microthermopiles fabricated on a 2 μm thick thermally isolated membrane showed a sensitivity of 0.94 V/W and a thermal time constant of less than 100 ms. Although the device is not restricted to enzymatic reactions, in this paper measurements of the heat of reaction from the catalytic action of glucose oxidase, catalase, and urease on glucose, hydrogen peroxide, and urea, respectively, are reported. Reactions were performed in open air using liquid batch testing and in enclosed fluidic reaction chamber by continuous flow experiments. A sensitivity of 53.5 μV/M for glucose, 26.5 μV/M for hydrogen peroxide and 17 μV/M for urea was obtained. Detection limit for glucose in the continuous flow mode is 2 mM (30 pmol). The aim of this work is to demonstrate the potential of the integrated calorimetric microfluidic device for fundamental thermodynamic studies in biochemical reactions. Using arrays of such devices with immobilized enzymes multi-analyte detection can be accomplished and the effects of interferents from competing substrates can be compensated. This paper presents the design, fabrication and initial testing results from such a microthermopile-based thermal biosensor.     

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15–30 s), sigmoidal responses for the urea concentration working range from 1×10^−2.5 to 1×10^−1.5 M and a lifetime of 4 weeks.     

Glucose biosensor based on nano-SiO2 and "unprotected" Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose

In this paper, a novel amperometric glucose biosensor was constructed by alternative self-assembly of positively charged poly(diallydimethylammonium chloride) (PDDA) and negatively charged glucose oxidase (GOx) onto a 3D Nafion network via electrostatic adsorption. The amount of Nafion in the electrode and the number of the (PDDA/GOx)_n multilayers were optimized to develop a sensitive and selective glucose biosensor. Under optimal conditions, the glucose biosensor with (PDDA/GOx)₅ multilayers exhibited remarkable electrocatalytic activity, capable of detecting glucose with enhanced sensitivity of 9.55 μA/mM cm² and a commendably low detection limit of 20 μM (S/N = 3). A linear response range of 0.05–7 mM (a linear correlation coefficient of 0.9984, n = 20) was achieved. In addition, the glucose biosensor demonstrated superior selectivity towards glucose over some interferents, such as ascorbic acid (AA) and uric acid (UA), at an optimized detection potential of 0.6 V versus Ag/AgCl reference.     

Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: application for neurotoxins detection

The aim of the present work is to develop an evanescence wave array biosensor exploiting the “kinetic” approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO₂ layer to increase the surface area available for immobilization.

The biosensor enabled the detection of 2.5 μM paraoxon, and 10 μM DFP and parathion, respectively. Very short response time of 30 s can be achieved with a total analysis time of less than 2 min. When operated at room temperature and stored at 4 °C, the waveguide retained reasonable activity for greater than 45 days.     

Detection of heavy metal toxicity using cardiac cell-based biosensor

Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner which offers insight into the physiological effect of an analyte. To investigate the potential applications of cell-based biosensors on heavy metal toxicity detection, a novel biosensor for monitoring electrophysiological activity was developed by light-addressable potentiometric sensor (LAPS). Extracellular field potentials of spontaneously beating cardiomyocytes could be recorded by LAPS in the range of 20 μV to nearly 40 μV with frequency of 0.5–3 Hz. After exposed to different heavy metal ions (Hg²⁺, Pb²⁺, Cd²⁺, Fe³⁺, Cu²⁺, Zn²⁺; in concentration of 10 μM), cardiomyocytes demonstrated characteristic changes in terms of beating frequency, amplitude and duration under the different toxic effects of ions in less than 15 min. This study suggests that, with the physiological monitoring, it is possible to use the cardiac cell-based biosensor to study acute and eventually chronic toxicities induced by heavy metal ions in a long-term and no-invasive way.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl₂ produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential (E^0′) on the solution pH (56 mV pH^− 1) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10^− 11 mol cm^− 2_, indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant (k_s) was 1.43 s^− 1, indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K_m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.     

Formaldehyde-sensitive sensor based on recombinant formaldehyde dehydrogenase using capacitance versus voltage measurements

A new formaldehyde-selective biosensor was constructed using NAD⁺- and glutathione-dependent recombinant formaldehyde dehydrogenase as a bio-recognition element immobilised on the surface of Si/SiO₂/Si₃N₄ structure. Sensor's response to formaldehyde was evaluated by capacitance measurements. The calibration curves obtained for formaldehyde concentration range from 10 μM to 20 mM showed a broad linear response with a sensitivity of 31 mV/decade and a detection limit about 10 μM. It has been shown that the output signal decreases with the increase of borate buffer concentration and the best sensitivity is observed in 2.5 mM borate buffer, pH 8.40. The response of the created formaldehyde-sensitive biosensor has also been examined in 2.5 mM Tris–HCl buffer, and the shift to the positive bias of the C(V) curves along with the potential axis has been observed, but the sensitivity of the biosensor in this buffer is decreased dramatically to the value of 2.4 mV/decade.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

A chemiluminometric method for the determination of urea in serum using a three-enzyme bioreactor

A flow injection chemiluminometric assay for urea has been developed based on a minicolumn bioreactor packed with immobilized enzyme-bearing glass beads. The reactor contains immobilized urease, L -glutamate dehydrogenase and L -glutamate oxidase, aligned in this order (upstream to the downstream). When the sample is introduced into the bioreactor, urea is first hydrolysed by urease to produce ammonia, which is then converted into L -glutamate by L -glutamate dehydrogenase. L -Glutamate is finally oxidized by L -glutamate oxidase to produce hydrogen peroxide, which is quantified by measuring chemiluminescence emitted upon admixing with luminol and potassium ferricyanide. One assay cycle is completed within 1 minute. The method is sensitive (detection limit 0.5 nmol) and is linear in the range 0–30 mmol/l. It can be readily applied to the determination of urea in human serum, and requires no blank corrections for ammonia and/or L -glutamate present in serum samples.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

Template synthesis of highly ordered Prussian blue array and its application to the glucose biosensing

In this paper, we propose a strategy to form nanoelectrode arrays by electrochemical deposition of the Prussian blue (PB) through highly ordered porous anodic alumina (PAA) membrane. The structure and morphology of the nanoarrays were characterized by scanning electron microscopy (SEM). As the highly ordered PB arrays can behave as an ensemble of closely spaced but isolated nanoelectrodes, the nanostructured PB arrays are successfully applied to improve the analytical performances of glucose by electrocatalytic reduction enzymatically liberated H₂O₂. The resulting PB based nanoelectrode arrays show a wide linear calibration range over three orders of magnitude of glucose concentrations (5.0 × 10⁻⁶ to 8.0 × 10⁻³ M) and a low detection limit of 1 μM. Moreover, the biosensor exhibits other good characteristics, such as short response time, high selectivity, excellent operation stability. In addition, effects of the glucose oxidase (GOx) loading, applied potential and pH on the biosensor performance were also discussed.     

Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.     

A preliminary study of a biosensor based on flow injection of the recognition element

A biosensor based on flow injection of the recognition element has been developed. As a model a pH-transducer was used, and urease was chosen as the recognition element. The pH-transducer was immersed in an internal flow-through chamber which was in contact with the sample solution via a semi-permeable membrane. The recognition element, urease, was injected into the buffer solution passing through the biosensor. The enzyme catalysed the hydrolysis of urea and the concomitant increase in pH was recorded. The biosensor response time was about three minutes at a constant flow rate of 0·05 ml/min. The linear range of the calibration curve of the biosensor was 0–5 mM. The observed detection limit was approximately 0.1 mM. The sample throughput was 6–12 per hour. The pH-response of the biosensor, for a sample solution containing urea (3·26 mM), showed a reproducibility (r.s.d) of 28% (n = 5) and a repeatability (r.s.d.) of 8% (n = 5). Operation at elevated temperatures (up to 50°C) was demonstrated. The presence of glucose (28 mM), acetone (6·7 mM), citric acid (0·2 mM) or sodium acetate (0·6 mM) in the sample solution did not interfere with the sensor response. A lowering of the biosensor response which was observed in the presence of copper ions (due to urease inhibition) could be completely eliminated by adding EDTA to the urease solution. Thus, this work demonstrates a new type of biosensors, based on SIRE-technology (Sensors with Injectable Recognition Elements), which show high accuracy and stability, quick response and high sample throughput. These features suggest the suitability of the system for automation. Such sensors should readily be combined with other enzymes or enzyme systems. The enzyme (urease) cost per analysis (injection) for the biosensor was estimated to be approximately US$0·02. This could be substantially reduced by further optimisation and miniaturisation.     

New series of potent delta-opioid antagonists containing the H-Dmt-Tic-NH-hexyl-NH-R motif

Heterodimeric compounds H-Dmt-Tic-NH-hexyl-NH-R (R = Dmt, Tic, and Phe) exhibited high affinity to δ- (K_iδ = 0.13–0.89 nM) and μ-opioid receptors (K_iμ = 0.38–2.81 nM) with extraordinary potent δ antagonism (pA₂ = 10.2–10.4). These compounds represent the prototype for a new class of structural homologues lacking μ-opioid receptor-associated agonism (IC₅₀ = 1.6–5.8 μM) based on the framework of bis-[H-Dmt-NH]-alkyl (Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2003, 46, 3201), which exhibited both high μ affinity and bioactivity.     

Antioxidant properties of violacein: possible relation on its biological function

Violacein, a violet pigment produced by Chromobacterium violaceum, has attracted much attention in recent literature due to its pharmacological properties. In this work, the antioxidant properties of violacein were investigated. The reactivity with oxygen and nitrogen reactive species and 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, was evaluated. EPR studies were carried out to evaluate the reactivity with the hydroxyl radical. The action of violacein against lipid peroxidation in three models of lipid membranes, including rat liver microsomes, Egg and Soy bean phosphathidylcholine liposomes were also evaluated. The compound reacted with DPPH (IC₅₀ = 30 μM), nitric oxide (IC₅₀ = 21 μM), superoxide radicals (IC₅₀ = 125 μM) and decreased the hydroxyl radical EPR signal. The compound protected the studied membranes against peroxidation induced by reactive species in the micromolar range. The reconstitution of violacein into the membranes increased its antioxidant effect. These results indicate that the compound has strong antioxidant potential. Based on these results we suggest violacein plays an important role with the microorganism membrane in defense against oxidative stress.     

Synthesis, SAR, and X-ray structure of human BACE-1 inhibitors with cyclic urea derivatives

We describe synthesis and evaluation of a series of cyclic urea derivatives with hydroxylethylamine isostere. Modification of P3, P1, and P2′ and combination of SAR display a >100-fold increase in potency with good cellular activity (IC₅₀ = 0.15 μM) relative to the previously reported compound 3.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.