Micro-Droplet Hydrodynamic Voltammetry for the Determination of Microcystin-LR Based on Protein Phosphatase

Home , Chronoamperometry, Hydrodynamic voltammetry

doi: 10.2965/jwet.18-040 Journal of Water and Environment Technology, Vol.17, No.1: 18–26, 2019

Original Article Micro-droplet Hydrodynamic Voltammetry for the Determination of Microcystin-LR Based on Protein Phosphatase

Md Saiful Islam a, Kazuto Sazawa a, Kazuharu Sugawara b, Hideki Kuramitz a

a Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Toyama, Japan b Maebashi Institute of Technology, Maebashi, Japan

ABSTRACT Microcystin-LR (MC-LR) is one of the most frequent and most lethal toxicants and a vital environmental pollutant due to its toxicity and persistence. It is hepatotoxins and have been shown to be potent tumor-promoters which pose a serious threat to human health in the form of chronic exposure through drinking water. In this study, a novel electrochemical method for the determination of MC-LR has been developed using protein phosphatase 2A (PP2A) enzyme inhibition. The rapid and simple determination of PP2A activity was accomplished by means of hydrodynamic voltammetry in a 50 µL micro-droplet with a rotating disk electrode (RDE). The chronoamperometric response were obtained from the electrochemical oxidation of p-aminophenol (PAP) following the enzymatic conversion of p-aminophenyl phosphate (PAPP) as a substrate. PP2A activity estimated by using PAPP substrate is affected by MC-LR, and this phenomenon decreases the current signal of PAP. The IC50 value was calculated as 0.08 µg/L which is well below the World Health Organization (WHO) provisional guideline value for total MC-LR of 1 µg/L in drinking water. The enzymatic reaction using RDE is possible to perform using fewer chemicals in a rapid manner. Therefore, the method can be very attractive for MC contaminated drinking water routine assessment.

Keywords: microcystin-LR, micro-droplet, electrochemical determination, rotating disk electrode, protein phosphatase 2A

INTRODUCTION sure through drinking water [1,4]. MC-LR binds with protein phosphatase type 2A (PP2A) in an irreversible approach and Cyanotoxins are produced by cyanobacteria particularly in inhibiting their enzymatic activity, results in tumor promo- freshwater and marine ecosystem where, they are capable to tions and liver cancer [5,6]. These potential risks directed the form visible blooms under definite circumstances. The bloom World Health Organization (WHO) to ascertain a guideline formations become a global environmental issue having seri- value for MC-LR in drinking water of 1 µg/L (equivalent to ous threat to ecological balance and human health problem 1.0 × 10−9 mol/L) [3]. Therefore, rapid, sensitive and reliable due to their associated toxicity [1,2]. Among the various types technique for MC-LR detection and quantification at a low of cyanotoxins, microcystins (MC) produced in freshwater concentration become very important issues to minimize the ecosystem are the most common. More than 80 variants have potential human health risk. been discovered with different structure and toxicity, where There are various analytical techniques have been devel- Microcystin-LR (MC-LR) is the most frequent and most le- oped and used for the determination of MC-LR. Despite thal toxicants having LD50 by intraperitoneal and oral route of the potential risk of other variants of MC, toxicity tests are approximately 25–150 µg/kg and 5 mg/kg, respectively [3]. usually conducted based on MC-LR. The evaluation has It is a potent hepatotoxins and tumor-promoters which pose been suggested to all the other congeners and expressing as a serious threat to human health in the form of chronic expo- MC-LR equivalents (MC-LReq). MC-LReq are usually used

Corresponding author: Md Saiful Islam, E-mail: [email protected] Received: June 29, 2018, Accepted: October 15, 2018, Published online: February 10, 2019 Copyright © 2019 Japan Society on Water Environment

18 Journal of Water and Environment Technology, Vol. 17, No. 1, 2019 19 for the expression of total MC varieties concentration in immunoassay with rapid amperometric detection using RDE absence of other congeners information [7]. Mouse bioassay and micro-litter of droplet as a reaction vessel. The major was the first screening method to detect MC-LR 8,9[ ]. The advantage of this technique is the decrease in dilution of commonly used methods are immunoassays [10], whole cell the product of the enzymatic reaction, which results in high bioassays [11], protein phosphatase inhibition assays (PPIA) sensitivity and rapid detection for minimizing the nonen- [12–15], high-performance liquid chromatography (HPLC) zymatic hydrolysis of substrate and product. Our research [16], and liquid chromatography-mass spectrometry (LC- group previously established micro-droplet hydrodynamic MS) [17] etc. Mouse bioassay is suitable to get the sample voltammetry for a rapid and simple method for evaluating toxicity indication. However, it was not widely accepted enzyme activity and genotoxicity [28,32–34]. due to its low sensitivity, lack of consistency, and moral al- In this research work, we are proposing the development legations [5]. The protein phosphatase inhibition assays and of toxicity test based on the inhibition of protein phosphatase immunoassays are suitable substitutions for rapid detection enzyme activity using the micro-droplet hydrodynamic of MC-LR because of its potency to inhibit the activities voltammetry for the determination of MC-LR. The electro- of protein phosphatases 1 and 2A (PPl, PP2A) as a similar chemical detection has been performed by hydrodynamic manner to okadic acid [18,19], where the former methods are chronoamperometry using RDE in 50 µL of droplet. p- sensitive than later due to the provision of insight on toxicol- Aminophenyl phosphate (PAPP) was selected as a substrate ogy [2]. Like MC-LR, other MC variants are also involving for protein phosphatase 2A (PP2A). It is well-known that the potent inhibition of protein phosphatases and, therefore, the enzymatic reaction product p-aminophenol (PAP) shows PPIA has been used for the determination of MC contamina- excellent electrochemical properties, such as high sensitiv- tion [20]. These assays are considered as the ideal toxicity ity, low oxidation potential, and reversible electrochemical assays for MC determination in the environmental samples behavior [35]. The parameters for measurement of PP2A in- [21–23]. Protein phosphatase 2A (PP2A) was discovered hibition by MC-LR were optimized in this study. The results as more sensitive towards MC compare to PP1 and have were compared with other electrochemical and colorimetric higher prospective to detect MC [24]. In addition to these measurements. techniques, colorimetric enzyme inhibition assays using colorless p-nitrophenyl phosphate (p-NPP) substrate to pro- MATERIALS AND METHODS duce yellow colored p-nitrophenol (p-NP) are also developed by some researchers [25–27]. However, these techniques Reagents require expensive instrumentation, very complex sample MC-LR was purchased from Sigma-Aldrich (St. Louis, preparation, skilled operators, and are also time consuming. USA). MC-LR stock solution was prepared in methanol at Additionally, they are not suitable for determining samples a concentration of 25 µg/mL and subsequently diluted in a have suspended materials or coloring substances. buffer solution at pH 8.4 containing 30 mM Tris–HCl, 2 mM

Electrochemical detection techniques have some advan- ethylenediaminetetraacetic acid (EDTA) and 20 mM MgCl2. tages over optical detection including its relative simplicity, PP2A isolated from bovine kidney was obtained from EMD sensitivity, rapidness, an ability to detect samples containing Millipore (Billerica, USA). The activity of the purchased colored components or suspended solids. Moreover, it is PP2A solution was 800 U/mL, one unit is defined as the possible to detect a small sample volume without sensitivity amount of enzyme that will release 1.0 nmol of [32P] Pi from loss which is advantageous over optical detection techniques 32P-labeled phosphorylase per min at 30°C, pH 7.0. PAPP and such as UV/Vis spectrometry and fluorometry 28 [ ]. The components of buffers were purchased from Sigma-Aldrich. electrochemical detection using rotating disk electrode The substrate solution was prepared used immediately after (RDE) developed by Levich [29] has become a sensitive and preparation. All reagents were of analytical grade, and the popular hydrodynamic technique due to its ability to mix the solutions were prepared using Milli-Q water (resistivity sample solution effectively and at the same time acts as an ≥18.2 MΩ cm−1, Direct-Q 3 UV). electrochemical detection device. The hydrodynamic flow at the RDE results from centrifugal forces achieves a highly Apparatus sensitive determination for enzymatic activity that decreases Rotating disk electrode (RDE) hydrodynamic amperom- the incubation time for enzyme with substrate. Wijayaward- etry was performed using an electrochemical analyzer [ALS- hana et al. [30,31] developed the magnetic microbead-based 1200, Bioanalytical Systems Inc. (BAS) (West Lafayette, 20 Journal of Water and Environment Technology, Vol. 17, No. 1, 2019

Fig. 1 Schematic diagram of the PP2A inhibition bioassay with electrochemical hydrodynamic detection using RDE in a micro-droplet.

USA)]. The electrode system consists of a glassy carbon at 37°C. (GC) disk electrode [6 mm outer diameter, 3 mm inner Chronoamperometric detection was performed by apply- diameter (BAS)] as working (RDE) electrode, a silver wire ing a potential of 350 mV at a rotation rate of 3,000 rpm. (0.7 mm diameter) as reference electrode, and a platinum After an equilibration time of 5 s, the current was measured wire (0.5 mm diameter) as counter electrode. The GC disk to establish a baseline. At 20 s, 10 µL of substrate solution electrode was polished sequentially with 0.3 and 0.05 μm (PAPP: 12.5 mM, final concentration 2.5 mM) was added to alumina paste prior to use. the droplet via micropipette, and the current recording was continued for an additional 120 s. Within the micro-droplet, Electrochemical protein phosphatase assay PP2A converts the PAPP into PAP, which can then be oxi- The electrochemical measurements were performed fol- dized to p-quinone imine (PQI) via reversible electrochemi- lowing our previous study [33] and a schematic diagram cal reaction. The slope between 80 s and 90 s was corrected of the detection system is given in Fig. 1. A micro-droplet by deducting the background slope, taken between 10 and (40 µL) of 0.05 U/mL PP2A solution was placed between 20 s, for calculating the reaction rate. the RDE and the Parafilm® covered glass slide located on a thermostatically controlled warm plate (KM-1, Kitazato Co. Optimization of electrochemical parameters Ltd., Fuji, Japan) as the reaction medium. The micro-droplet The role of rotation rate and substrate concentration on was sandwiched between the RDE and Parafilm® surface. detection of enzyme activity by using RDE has been consid- The micro-droplet formed by the strong surface tension was ered. In our previous study, we have investigated the impacts used as the reaction vessel for electrochemical detection. The of rotation on detection of enzyme activity by using RDE temperature of the micro-warm plate was always maintained [34]. The hydrodynamic chronoamperograms were recorded Journal of Water and Environment Technology, Vol. 17, No. 1, 2019 21 at 0 to 4,000 rpm with different concentrations of PAPP. In this study, PAPP has been used as a substrate for the electrochemical detection. For the optimization of substrate concentration, hydrodynamic amperometric responses of 0.016 U/mL PP2A with PAPP concentrations of 0, 0.25, 0.5, 1.0, 2.5, 4.0, and 5.0 mM were investigated at a rotation rate of 3,000 rpm. The electrochemical measurements were performed according to the previously described procedure.

Effect of methanol on quantitation of MC-LR Since methanol was used to solubilize the MC-LR, the effect of different concentrations of methanol on the PP2A activity was studied. Methanol was added with the enzymatic reaction buffer (30 mM Tris/HCl, 20 mM MgCl2, 2 mM EDTA, pH 8.4). The electrochemical measurements were done according to the previously described procedure. Fig. 2 The limiting current of PAP measured by hydrodynamic linear sweep voltammograms (inset) at concentrations MC-LR exposure procedure with PP2A ranging from 0 to 0.1 mM. Error bar represents standard deviation (n = 3). To investigate the enzyme inhibition from MC-LR, 0.05 U/mL PP2A (final concentration) was exposed with MC-LR solutions at concentrations ranging from 0 to 500 µg/L. Hun- at concentrations ranging from 0 to 0.1 mM. The measure- dred µL of PP2A and MC-LR solution was mixed at 50:50 ments were conducted in a 50 µL micro-droplet at a scan ratio (vol./vol.) by gentle shaking at room temperature (23 ± rate of 100 mV/s with a rotation rate of 3,000 rpm. A linear 3°C) for 30 minutes to ensure that the enzyme is fully mixed relationship exists between the mass-transfer limiting cur- with toxicant. Subsequently, the rate of enzyme inhibition rent and the corresponding PAP concentrations. The value of was compared with that from a blank test by using the hydro- detection limit (3σ) calculated at 2.0 × 10−7 M (R2 = 0.999). dynamic chronoamperometry data described in the previous This value of detection limit is comparatively lower than that section. obtained for RDE detection excluding rotation (10.4 × 10−7 M). Thus, it can be said that the application of RDE system RESULTS AND DISCUSSION based on hydrodynamic voltammetry is useful strategy for sensitive measuring a small volume of PAP solution. PAP detection by micro-droplet hydrodynamic voltammetry Optimization of electrochemical detection param- A sensitive determination for PP2A activity can be eters for RDE measurements performed from the oxidation current of PAP by means of In RDE measurements, the optimization of electrochemi- hydrodynamic chronoamperometry. The electrochemical cal detection parameters such as rotation rate and substrate behaviors of PAP and PAPP were investigated by hydrody- concentration are important. High rotation rate gives high namic linear sweep voltammetry using RDE according to current value and above 3,000 rpm the droplet sometimes our previous research work [34]. The study was conducted to was broken. Based on that result, 3,000 rpm was considered obtain the best applied potential for PAP detection where the as the optimum for the whole measurement process. oxidation of PAP should not be influenced from the effect of The hydrodynamic amperometric responses of 0.016 U/ PAPP oxidation. The mass-transfer limited current i.e., the mL PP2A with different concentrations of PAPP ranges from Levich current was apparent potentials over 200 and 550 mV 0 to 5 mM were investigated for the optimization of substrate for PAP and PAPP oxidation, respectively. For chronoam- concentration and shown in Fig. 3 (a). The responses were perometric measurements throughout the study, a potential fitted according to the Michaelis-Menten equation v( = vmax of 350 mV was selected based on the study result. [PAPP]/ (KM + [PAPP])). The result shows that velocity Figure 2 shows the results of PAP electrochemical re- become close to steady-state above a PAPP concentration sponse obtained by hydrodynamic linear sweep voltammetry of 2.5 mM. Therefore, the optimal PAPP concentration for 22 Journal of Water and Environment Technology, Vol. 17, No. 1, 2019

Fig. 3 (a) Michaelis–Menten saturation curve, (b) Hanes-Woolf plots of PP2A. The measurements were done at 350 mV applied potential with a rotation rate of 3,000 rpm. Error bar represents standard deviation (n = 3). hydrodynamic chronoamperometry was determined to be 2.5 mM. Figure 3 (b) illustrates the Hanes-Woolf plot of PP2A obtained by the hydrodynamic voltammetry. The

Michaelis-Menten constant (KM) and the maximum velocity (vmax) obtained from the Hanes-Woolf plots were 0.78 mM and 2.02 nA/s, respectively.

Optimization of exposure time to MC-LR To elucidate the optimal exposure time, we then investigated the effect of the length of exposure to MC-LR on the PP2A activity. This investigation was conducted using 1 µg/ mL of MC-LR and 0.016 U/mL of PP2A. The solutions were mixing at 50:50 ratio (vol./vol.) by gentle shaking at room temperature (23 ± 3°C) for up to 50 min. The electrochemical measurements were done according to the procedure Fig. 4 Effect of MC-LR exposure time on the inhibition of described in the section of electrochemical protein phospha- enzyme activity. PP2A was exposed to MC-LR at a concen- tase assay, and the relative PP2A activity was calculated. tration of 1 µg/ml for different time periods. Hydrodynamic As presented in Fig. 4, PP2A activity was inhibited by the chronoamperometric measurements were performed for added MC-LR with increasing incubation time. For the given 0.016 U/mL of PP2A at an applied potential of 350 mV and concentration, a steady-state inhibition value was obtained a rotation rate of 3,000 rpm with 2.5 mM PAPP. Error bar above 20 min and the time might be required for MC-LR and represents standard deviation (n = 3). PP2A binding.

Effect of methanol content in the MC-LR quantita- cal measurements have been carried out. In a study conducted tion by Tubaro et al. [36] for the determination of Okadic acid The effect of methanol on the PP2A activity was studied using PP2A obtained almost no impact up to the ethanol con- since methanol was used to solubilize the MC-LR. Methanol centration of 0.5%. In this study, we have tested the effect of was added with the enzymatic reaction buffer (30 mM Tris/ 0.5% methanol on PP2A and the results are presented in the

HCl, 20 mM MgCl2, 2 mM EDTA pH 8.4) and electrochemi- Table 1. The experiments have been carried out thrice. The Journal of Water and Environment Technology, Vol. 17, No. 1, 2019 23

Table 1 Effect of methanol content in the MC-LR concen- Table 2 Comparison of the proposed assay with other elec- tration on the activity of PP2A. trochemical and colorimetric techniques.

PP2A activity Detection method IC50 (µg/L) References Methanol (%) Mean SD Electrochemical 0.08 Proposed method 0 100.0 0.19 Electrochemical 83.0 [6] 0.5 98.2 0.28 Colorimetric 1.40 [27] Colorimetric 0.25 [26]

in Table 2. Comparison among these studies revealed that the PP2A inhibition assay has a sensitive response to MC- LR. The present assay and the study conducted by Campàs et al. [6] both measured PP2A activity inhibition. The lower

IC50 values for our study settled its greater sensitivity than the immobilized PP2A. This might be due to the reduction in reaction rates for immobilized enzyme when enzymes cannot mix properly with the substrate. In case of RDE, rotation causes effective mixing of enzymes and substrates which produces sensitive response. The colorimetric determinations are showing high sensitivity towards MC-LR.

However, the lower IC50 value obtained in the proposed study established it as more sensitive method than those Fig. 5 Dose–response curve for MC-LR. Inhibition is ex- colorimetric determination. Our experiments demonstrated pressed as percentage of the control (no MC). Error bar rep- that the proposed enzyme inhibition assay using hydrody- resents standard deviation (n = 3). namic chronoamperometry has advantages over the reported methods in terms of both sensitivity and measurement time. In our previous study, the effects of the conditions com- results revealed that the mean PP2A inhibition for 0.5% of monly found in drinking water supply systems on the methanol concentration was 1.8% which is considerable and immunoassay for ovalbumin based on the hydrodynamic therefore, in all succeeding measurements the final methanol amperometric detection of PAP were investigated [32]. The concentration was maintained below 0.5%. assay was unaffected for pH 6.5 and 9.5, water hardness (558 mg/L Ca2+ and 550 mg/L Mg2+), 10 mg/mL total iron, MC-LR assay 14 mg/L phosphate, and total organic content (20 mg/L Electrochemical MC-LR assay based on protein phospha- fulvic acid and humic acid) in water. Therefore, the MC-LR tase inhibition was demonstrated. The activity inhibition of assay might be applicable for both environmental waters and 0.05 U/mL PP2A was measured using hydrodynamic chro- drinking water. noamperometry. The dose-response curve exhibits a typical sigmoidal response, is given in Fig. 5. The IC50 and the limit CONCLUSIONS of detection (LOD) was calculated as 0.08 µg/L and 0.045 µg/L, respectively. Those values are lower than the values We have developed electrochemical protein phosphatase (1.40 µg/L and 0.90 µg/L, respectively) reported by Campàs 2A enzyme inhibition assay to quantify low levels of MC-LR et al. [27] using colorimetric inhibition assay. The value in water. The electrochemical determination was conducted is well correlated with the LD50 value measured by mouse by hydrodynamic chronoamperometry in a 50 µL micro- bioassays [37]. Thus, the PP2A inhibition assay could be a droplet using rotating disk electrode. The PAPP substrate suitable alternative of mouse bioassay to evaluate MC-LR used in the study was converted into PAP by the enzymatic toxicity in water samples. activity of PP2A. Chronoamperometric detection was used The IC50 value was calculated and compared with other to measure the oxidation current of PAP. The results con- electrochemical and colorimetric determinations are given firmed that the proposed method is suitable to detect very 24 Journal of Water and Environment Technology, Vol. 17, No. 1, 2019 low concentration of MC-LR. 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