Critical ReviewsTM in Eukaryotic Gene Expression, 22(3):197-203 (2012)
Recombinant Laccase: II. Medical Biosensor Nicola Luigi Bragazzi,b Eugenia Pechkova,a,b Dora Scudieri,b Tercio Bezerra Correia Terencio,b Manuela Adami,a & Claudio Nicolinia,b,c,* Nanoworld Institute Fondazione ELBA Nicolini, Pradalunga, Bergamo 24100, Italy; bBiophysics and Nanobiotechnology Laboratories, Department of Experimental Medicine, University of Genova, Genoa 16121-16167, Italy; cBiodesign Institute, Arizona State University, Tempe, AZ a
Address all correspondence to: Professor Claudio Nicolini, President Nanoworld Institute, Fondazione E.L.B.A. Nicolini and Eminent Chair of Biophysics at University of Genova, Via Antonio Pastore 3, 16132, Genova, Italy. Tel.: +39 010 353 38217; Fax: +39 010 353 38215;
[email protected].
*
ABSTRACT: Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, ks 0.57 s–1, diffusion coefficient 3 × 10–6 cm2/s, Kcat 6825.92 min–1, KM 4.1 uM, Kcat/KM 2.8 × 107 mol–1 s–1, sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor. KEY WORDS: cyclic voltammetry, chronoamperometry, UV spectrophotometry, laccase, Rigidoporus lignosus, clomipramine
I. INTRODUCTION In the last decade, LB (Langmuir-Blodgett) technology1–3 has proven successful in building and developing biosensors for a wide variety of applications, particularly because of the temporal and thermal stability and the highly ordered structure of proteins4–8 (see Ref. 9 for a systematic review). Here, our goal is to build an enzyme-based biosensor for medical purposes, in which the immobilization procedure is carried out via LB films since the LB technology allows a unique control of the thickness of the film deposited onto the surface, thus resulting in a well-suited nanomaterial for the fabrication of both mono- and multilayer structured films because of the compression of protein layers at the air-water interface. The enzyme implemented in our device is laccase (EC 1.10.3.2), which is a blue oxidase capable of oxidizing phenols and aromatic amines by reducing molecular oxygen to water by means of a full
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complement of copper atoms. Laccases belong to a large group of the multicopper enzymes, which includes among others ascorbic acid oxidase and cerulo plasmine.10,11 They catalyze the oxidation of such diverse compounds as o-, p-diphenols, aminophenols, polyphenols, polyamines, lignin, some inorganic ions, aryldiamines, benzenthiols, phenothiazines. Typical laccase reaction leads to conversion of the phenolic substrate into aryloxyradical. During the second stage of the oxidation, the active species can be converted into a quinone. In the next step, both the quinone and the free radical product undergo a nonenzymatic coupling reactions leading to the polymerization resulting in dimers, oligomers, and polymers. Moreover, it is known to demethylate lignin and methoxyphenol acids. Products of the oxidative coupling reactions result from either C-O and C-C coupling of the phenolic reactants, or N-N and C-N coupling of the aromatic amines. The particular reaction is known as
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the detoxification of phenolic contaminants. The reactions we are going to monitor are the following12,13: O2 + 4 H+ + 4 e– ↔ 2 H2O 4 RH + O2 → 4 RH· + 2 H2O Here, we used recombinant laccase from Rigidoporus lignosus14 and we briefly compare the results with those in the literature15–24 (see Ref. 25 for a review and for further references therein). Only one of these biosensors was found—to our knowledge—to be built and developed explicitly for medical purposes.26 II. MATERIALS AND METHODS A. Chemicals 1. Preparation of the LB Film of Laccase An LB thin film of recombinant laccase from Rigidoporus lignosus (obtained as previously reported in Part I) was prepared using a highly concentrated sample of laccase at 0.4 mg/ml in a solution of 50 mM sodium phosphate and 300 mM NaCL. The mixed chloroform solution in equimolar proportions has a concentration of 1 mg/ml. 50 μl of the mixture has been spread on a Milli-Q water subphase (>17 MΩ). The LB film was prepared and studied in a LB trough (MMMDT Co., Moscow, Russia) of 240 mm per 100
mm size and 300 ml volume. The instrument has two moving barriers between which films are compressed and expanded. The surface pressure measurements were made by a Wilhelmy balance with a sensitivity of 0.05 mN/m, and the monolayer has been compressed with movable barriers at a rate of 70 mm/min. The deposition is of Y-type with a dipping rate of 25 mm/min. The drainage rate (in order to remove the film) is ~3.5 mm/min. The transfer pressure to obtain the LB film is ~20 mN/m, at 22°C. 2. Characterization of the LB Thin Film of Laccase via AFM The surface morphology and topology of the LB thin film of laccase was investigated via atomic force microscope (AFM), which was a home-built instrument (Polo Nazionale Bioelettronica27,28) working in contact mode in air at a constant contact force of 3 N/m with NSC18/Cr-Au/15 MikroMasch tip, with a resonant frequency of 75 kHz. For the analysis, laccase film was deposited onto mica wafer. The acquired images have been processed and analyzed using WSXM software.29 AFM confirmed the very ordered biofilm, characterized by peaks or islands (ranging from about 20 to 70 nm, as in Fig. 1). The roughness of the film was found to be 8.22 nm and the compressibility coefficient ~37.5 m/N as determined from the LB π-A isotherm.
FIGURE 1: Atomic force microscope (AFM) characterization of the topological properties of the LB laccase film previously deposited onto mica wafers at high (2 mm) (a) and at low (10 mm) (b) resolution. Laccase islands and peaks are clearly visible.
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3. Clomipramine Clomipramine (Anafranil30,31), a drug belonging to tricylic tertiary amine antidepressants, is widely used for the therapy of depressive and obsessive disorders. Because of its clinical importance, many analytical methods have been developed to monitor its level—above all, chromatographic techniques (gas chromatography, high-performance liquid chromatography), eventually coupled with tandem mass spectrometry, but all these techniques are time consuming and laborious. Moreover, the AGNP-TDM panel of experts has emphasized the importance of therapeutic drug monitoring.32 In our experiment, clomipramine was added at varying concentrations in the micromolar range. The therapeutic dose is from 75 to 200 mg/day; the pharmacokinetics is extremely variable among the patients (see Ref. 33 for a detailed review). Generally, the therapeutic concentration in the human blood of psychiatric patients is usually in the low micromolar range. The side effects of the drugs, especially in the case of overdose, are seizures, and hematological, cardiological, and neurological adverse effects up to the coma (tricyclic antidepressant syndrome34). For a review of the analytical chemistry of tricyclic antidepressants, the reader is referred to Ref. 34. 4. UV Characterization We performed an experiment with laccase and drug in real blood samples. Blood was collected from
healthy volunteers at San Martino Hospital (Genoa, Italy) and analyzed immediately after. Blood plus laccase and drug at varying concentrations in the low micromolar range versus only blood samples were investigated via a Jasco J-710 spectrophotomer (Jasco, Japan) equipped with a Peltier thermostatic cell holder (Model PTC-343) (Fig. 2). Peaks in the 300 and 600 nm bands were found, confirming the catalytical activity of the enzyme, and linearity was found in the dynamic response range of the optical biosensor at 400 nm (as shown in Fig. 3). 5. Potentiometric and Amperometric Apparatus The apparatus for the potentiometric sensor (in part shown in Fig. 4) is made up with an EG&G PARC model 263A potentiometer, equipped with dedicated software (by Riccardo Galletti and Marco Sartore, Elbatech SRL). The enzyme has been deposited onto the electrode via the LangmuirSchaefer technique,36 and a protocol of immobilization overnight has been followed (after depositing the film, the electrodes have been kept at 4°C up to a maximum of 16 h). The electrodes that have been used are rhotenium and graphite ones, while the counterelectrode is of silver. The electrode is ~0.75 mm per 1 mm. Cyclic voltammetry has been recorded from –300 to +500 mV, at varying concentration of the drug in the micromolar range (Fig. 2), the sweep rate is 20 mV/s, the instrument range has been gradually decreased to 1 uA.
FIGURE 2: Potentiometric measurement at increasing drug concentration (curve a no drug, curve b 5 uM, curve c 7 uM, curve d 10 uM). The measurements have been made from -500 to +300 mV with a voltage scan of 20 mV/s (a). (b) Potentiometric linearity response in the dynamic range in the blood.
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FIGURE 3: UV characterization of the biosensor at increasing drug concentration (from the bottom to the top, curve a no drug, curve b 0.4 uM, curve c 2 uM, curve d 5 uM, curve e 7 uM, curve f 10 uM, curve g 20 uM) (a) and calibration plot of the UV linearity response in the dynamic range at 400 nm in the blood (b).
FIGURE 4: A picture of a part of the experimental setup for the potentiometric and chronoamperometric measurements. Clearly visible are the electrodes and the counterelectrode and the blood sample taken from a healthy volunteer to which Clomipramine has been added.
Solving the well-known Randles-Sevcik equation, we found an electron transfer of at least one electron, α coefficient about 0.45, ks 0.57 s–1,
i = 0.4463⋅ n ⋅ F ⋅ A⋅C j ⋅
n ⋅ F ⋅ v ⋅ Dj R ⋅T
where i is the intensity current, n is the number of electrons transferred during the enzyme kinetics, F is the Faraday constant, A is the electrode area, Cj is the analyte concentration, Dj is the diffusion coefficient of the enzyme, v is the voltage scan, R is the gas constant, and T is the temperature. After having found an optimal potential at –35 mV, amperometric measurement [Fig. 5(a)] was
performed and linearity was found in the dynamic response range [Fig. 5(b)]. The data have been subsequently modeled according to the MichaelisMenten formalism (using the Lineweaver-Burk method, Fig. 6) and we calculated KM 4.1 uM, Kcat 6825.92 min–1, resulting in a quite good Kcat/KM ratio (2.8 × 107 mol–1 s–1). The time response was
