Sensors 2015, 15, 5331-5343; doi:10.3390/s150305331 OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Article Hydrodynamic Voltammetry as a Rapid and Simple Method for Evaluating Soil Enzyme Activities Kazuto Sazawa 1,* and Hideki Kuramitz 2 1 Center for Far Eastern Studies, University of Toyama, Gofuku 3190, 930-8555 Toyama, Japan 2 Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Gofuku 3190, 930-8555 Toyama, Japan; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel./Fax: +81-76-445-66-69. Academic Editor: Ki-Hyun Kim Received: 26 December 2014 / Accepted: 28 February 2015 / Published: 4 March 2015 Abstract: Soil enzymes play essential roles in catalyzing reactions necessary for nutrient cycling in the biosphere. They are also sensitive indicators of ecosystem stress, therefore their evaluation is very important in assessing soil health and quality. The standard soil enzyme assay method based on spectroscopic detection is a complicated operation that requires the removal of soil particles. The purpose of this study was to develop a new soil enzyme assay based on hydrodynamic electrochemical detection using a rotating disk electrode in a microliter droplet. The activities of enzymes were determined by measuring the electrochemical oxidation of p-aminophenol (PAP), following the enzymatic conversion of substrate-conjugated PAP. The calibration curves of β-galactosidase (β-gal), β-glucosidase (β-glu) and acid phosphatase (AcP) showed good linear correlation after being spiked in soils using chronoamperometry. We also performed electrochemical detection using real soils. Hydrodynamic chronoamperometry can be used to assess the AcP in soils, with a detection time of only 90 s. Linear sweep voltammetry was used to measure the amount of PAP released from β-gal and β-glu by enzymatic reaction after 60 min. For the assessment of soil enzymes, the results of hydrodynamic voltammetry assay compared favorably to those using a standard assay procedure, but this new procedure is more user-friendly, rapid and simple. Sensors 2015, 15 5332 Keywords: soil enzymes; hydrodynamic voltammetry; rotating disk electrode; microdroplet sample; humic acid; p-aminophenol 1. Introduction Enzymes are released in soils by microbes and plant roots. They can degrade complex substrates into low molecular weight compounds in soils [1,2]. The enzymes can be affected by many factors, biological (microbial populations, fauna, etc.), chemical (pH, organic matter contents, etc.) and, human activities (fire treatment, pesticides, heavy metals, etc.) [3–5], all of which change the biogeochemical cycles in an ecosystem. Therefore, the evaluation of enzyme activity in the soil is very important in order to assess the biochemical function, organic matter fraction, nutrient cycle, and decomposition of xenobiotics. In general, regulatory soil enzyme assays are based on the colorimetric determination of p-nitro-phenol (PNP) that is released by enzyme reactions when a soil sample is incubated in an optimum buffer solution containing substrate-conjugated PNP at optimum temperature [6–8]. However, regulatory assays are not suitable to evaluate a sample that contains a high concentration of colored dissolved organic matter, because these assays are based on spectroscopic detection. Furthermore, regulatory assays require long enzymatic reaction times (1 to 24 h) with substrates and involve complicated methods for soil particle removal by filtration or centrifugation. The aim of the present study was to develop a new rapid and simple soil enzyme assay based on hydrodynamic electrochemical detection using a rotating disk electrode (RDE) in a microliter (ca. 50 μL) droplet. Soil enzyme activities are determined by measuring the electrochemical oxidation of PAP, which is converted from the substrate by the enzyme reaction in soil. Compared with spectroscopic detection, the advantage of electrochemical detection is that it is much simpler and does not interfere with the colored components and soil particles in a sample solution. Furthermore, hydrodynamic voltammetry in a droplet can achieve the effective mixing of a sample solution and convective mass transport of an analyte to the electrode surface, which results in rapid detection [9–11]. Previously, we developed a new genotoxicity test based on hydrodynamic electrochemical detection using a RDE in a microliter droplet, which evaluates the DNA damage caused by chemicals based on the β-gal activity of a bacteria strain [12]. This methods can be used for a rapid assessment of the β-gal activity of a bacteria strain in sediments. The two main objectives for this study were: (1) to use commercial enzyme activities with or without the soil samples to investigate the use of hydrodynamic chronoamperometry to evaluate the effect of dissolved organic matter and soil particles on the amperometric response of PAP; and (2) to demonstrate the use of the new method in assaying for β-gal, β-glu and AcP activities in real soils and compare the results with the standard assay based on spectroscopic detection. These enzymes are widely distributed in the environment and have been evaluated by several soil scientists. β-gal and β-glu play significant roles in the breakdown of low molecular-weight carbohydrates and cellulose in the soil, and are excreted mainly by microorganisms, animals and plants [13–15]. AcP is a major product of plant roots, and breaks down organic P for release in soils [16–19]. Sensors 2015, 15 5333 2. Experimental Section 2.1. Chemicals Modified universal buffer (MUB) with the desired pH for each of the enzymes was prepared according to previous reports [20,21]. Calcium chloride, PNP, toluene, NaOH, kaolin and quartz sand were purchased from Wako Pure Chemical Industry Ltd. (Osaka, Japan). p-Nitrophenyl-β-D-galactopyranoside (PNPGal), p-nitrophenyl-β-D-glucopyranoside (PNPGlu), p-nitrophenyl phosphate disodium salt hexahydrate (PNPP), p-aminophenyl-β-D-galactopyranoside (PAPGal), p-aminophenyl-β-D-glucopyranoside (PAPGlu), Aspergillus oryzae β-galactosidase (β-Gal; EC 3.2.1.23), almond β-glucosidase (β-Glu; EC 3.2.1.21), potato acid phosphatase (AcP; EC 3.1.3.2), PAP, and humic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Humic acid represents a main fraction of soil organic matter. An Aldrich humic acid (AHA) was dissolved in 0.1 M NaOH, and the solution was then treated with a mixture of HF and HCl. The resultant precipitate was transferred to a dialysis tube (molecular weight cutoff 500 Da). After dialysis, the slurry was freeze dried. The purified AHA stock solution (500 mg·L−1) was dissolved in 1 M NaOH solution (pH > 10) by stirring for 30 min under a gentle N2-stream. The acidity of the solutions was adjusted to pH 8.0 using 1 M HCl. p-Aminophenyl phosphate monosodium salt hydrate (PAPP) was obtained from Apollo scientific Ltd. (Stockport, UK). Tris (hydroxymethyl) aminomethane (TMAH) was purchased from MP Biomedicals (Aurora, OH, USA). Sphagnum moss peat was purchased from a local market in Toyama, Japan. Kaolin and quartz sand were ground into a fine powder using a mortar and pestle, and passed through a 0.025 mm mesh sieve. 2.2. Soil Samples Two soils were used in the present study. Soil A was collected from agricultural land at the University of Toyama. Soil B was collected from a forest in Toyama, Japan. The soils were sampled from a depth of 0–10 cm, and were air-dried at room temperature, ground for passage through a 2 mm mesh, and stored at 4 °C prior to use. The soils characteristics are presented in Table 1. Table 1. Chemical and physical characteristics of soils used in this study. EC * Moisture Ignition Abs. at 400 nm ** Abs. at 400 nm ** Land Use pH (μS/cm) Content (%) Loss (%) (TMAH) (NaOH) Soil A Agricultural land 6.22 23.2 20.8 8.4 0.105 0.264 Soil B Forest land 4.30 53.7 28.8 29.0 0.704 2.66 * EC: electrical conductivity; ** Abs. at 400 nm (TMAH and NaOH): absorbance values for 400 nm of alkaline soil extracts were obtained by mixing 1 g of soil, 4 mL of MUB, at pH 6.0, 0.25 mL of toluene, 1 mL of 0.5 M CaCl2, and either 4 mL of 0.1 M TMAH or 0.5 M NaOH, and the soil particles were removed. The pH and electrical conductivity (EC) values for the soils were measured in slurries (1:2.5 air-dried soil/distilled water). The moisture content was determined from the loss in weight after drying at 105 °C for 48 h. These samples were also combusted at 600 °C for 2 h to determine the organic content by weight loss. Absorbance values for 400 nm of alkaline soil extracts were obtained by mixing 1 g of air-dried soil, 4 mL of MUB, at pH 6.0, 0.25 mL of toluene, 1 mL of 0.5 M CaCl2, and either 4 mL of 0.1 M TMAH or 0.5 M NaOH, and the soil particles were removed, and centrifuged at Sensors 2015, 15 5334 4000 rpm (about 2000 g) for 5 min [22]. The soil particle-size fractions were separated by the sieving machine (MSV-1, AsOne, Tokyo, Japan). 2.3. Procedure for the Regulatory Soil Enzyme Assay A regulatory assay for β-gal, β-glu and AcP was modified as reported by Tabatabai et al. and Eivazi et al. [6,8]. A soil sample (0.1 g) in a test tube was mixed with toluene (0.025 mL), and MUB (0.4 mL) at pH 6.0. After shaking the test tube, the substrate solutions (0.1 mL, 25 mM of PNPGal, PNPGlu and 115 mM of PNPP) was added to the test tube and incubated at 37 °C for 60 min.