Electrochemical detection of endotoxin using recombinant factor C zymogen

Electrochemistry Communications 12 (2010) 1066–1069

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Electrochemistry Communications j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e l e c o m

Electrochemical detection of endotoxin using recombinant factor C zymogen Kumi Y. Inoue ⁎, Kosuke Ino, Hitoshi Shiku, Tomokazu Matsue ⁎ Graduate School of Environmental Studies, Tohoku University, 6-6-11 Aoba, Aramaki, Aoba, Sendai 980-8579, Japan

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Article history: Received 20 April 2010 Received in revised form 17 May 2010 Accepted 24 May 2010 Available online 1 June 2010 Keywords: Protease zymogen p-nitroaniline Biosensor Lipopolysaccharide Electrochemical sensor

a b s t r a c t We have developed a zymogen-based electrochemical sensor. Zymogen is an inactive enzyme precursor (proenzyme) and it is necessary to transform it biochemically (e.g., by hydrolysis and conformational change) to make it an active enzyme. In this study, we demonstrated the detection of endotoxin by using recombinant Factor C (rFC), which is a protease zymogen activated by endotoxin binding. The activated rFC hydrolyzes a synthetic substrate of Boc-Val-Pro-Arg-p-nitroanilnide to generate an electrochemical active compound, p-nitroaniline (pNA). The liberated pNA was detected by differential pulse voltammetry at –0.75 V. By using this electrochemical process, 5000 endotoxin units (EU) L−1 and 1000 EU L−1 were detected in a Tris-Ac buffer with a pH of 7.5 at 37 °C for reaction times of 1 h and 3 h, respectively. The concept of zymogen-based electrochemical sensors is expected to lead to the development of new biosensors. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Endotoxins (also called lipopolysaccharides) are the constituents of the membrane surface of gram-negative bacteria. Endotoxins initiate innate immune responses, thereby inducing septic shock in mammalian cells. Thus, monitoring the biological activity of endotoxins in potentially contaminated medical supplies is important for safe medical treatment and healthcare. The Limulus amebocyte lysate (LAL) assay, a zymogen-based assay, has been the most widely used assay for the detection of endotoxins. Zymogen is an inactive enzyme precursor that requires a biochemical change to become an active enzyme. In the LAL reactions, Factor C, one of the protease zymogens found in horseshoe crab's blood, acts as the first component in the cascade of the host defense system against a Gram-negative bacterial infection [1,2]. Activated Factor C initiates the cascade reactions that finally lead to the formation of coagulin gel, which is detected photometrically. Recombinant FC (rFC) protein has also been used in assay systems for detecting endotoxins [3–6]. rFC activated by endotoxins catalyzes the hydrolysis of Boc-Val-Pro-Arg-pNA (pNA: p-nitroaniline). The absorbance of the pNA produced by the hydrolysis was used to identify the endotoxins [5]. Boc-Val-Pro-ArgMCA (MCA: 7-amido-4-methylcoumarin) is also used as a substrate for hydrolysis with activated rFC protein for fluorescence-based detection. Electrochemical sensors are effective in environmental and pharmaceutical analyses because they are small in size, field-portable, ⁎ Corresponding authors. K.Y. Inoue is to be contacted at tel.: + 81 22 795 7281; fax: + 81 22 795 6167. T. Matsue, tel./fax: + 81 22 795 7209. E-mail addresses: [email protected] (K.Y. Inoue), [email protected] (T. Matsue). 1388-2481/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2010.05.028

easy to use, and inexpensive. In our previous study, we demonstrated the detection of endotoxin using an electrochemical device with recombinant cells [7]. This cell-based sensor is highly sensitive but has drawbacks relating to portability and the preservation and long-term storage of living sensor cells. In the present study, we demonstrate the detection of endotoxins using a zymogen-based electrochemical sensor. Fig. 1 shows the principle behind our assay system. The inactive rFC (zymogen) is first activated by binding endotoxins (analyte) to the endotoxin-binding domain of the rFC. Then, the activated rFC catalyzes the hydrolysis of VPR-pNA (substrate) to pNA. The product, pNA, is detected electrochemically using differential pulse voltammetry (DPV). The current intensity due to the produced pNA depends on the endotoxin concentration. The detection principle of the present zymogen-based sensor is different from that of conventional electrochemical enzyme sensors in which the analytes are the enzyme substrates. To the best of our knowledge, this is the first report of an electrochemical sensor that uses a zymogen as the sensing element. The combination of a zymogen-based assay system and an electrochemical-based detector can be useful in biosensing applications. 2. Experimental 2.1. Materials and reagents Escherichia coli O55:B5 control standard endotoxin (Lonza, USA) was diluted with endotoxin-free water (Lonza, USA) for obtaining standard endotoxin solutions. These standard solutions were mixed vigorously by vortexing for more than 15 min just before use. VPRpNA (Watanabe Chemical Industries, Japan) and pNA (Wako Pure Chemical Industries, Japan) were dissolved in dimethyl sulfoxide

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Fig. 1. Schematic illustration of the principle underlying the rFC-based electrochemical endotoxin assay.

(DMSO) to obtain a 10 mM stock solution, which was stored at −20 °C. rFC enzyme solution was supplied as one of the reagents in a PyroGene® kit (Lonza, USA). 100 mM Tris-Ac buffer containing 50 mM NaCl (pH 7.5) was used as reaction buffer, and it was filtered using Acrodisc® units with Mustang® membrane (Pall Corporation, USA) to remove any contaminant endotoxins. Aqueous solutions were prepared with high-purity distilled and deionized water from a MilliQ filtration system (Millipore Corporation, USA). 2.2. Equipment and method for electrochemical measurements Electrochemical measurements were carried out using a potentiostat (CompactStat; Ivium Technologies, Netherlands). A glassy carbon (GC) disc electrode (BAS Inc, Japan; diameter = 1.0 mm), an Au sheet, and Ag/AgCl/sat. KCl was used as the working electrode, counter electrode, and reference electrode, respectively. DPV was carried out with a pulse time of 70 ms, a pulse amplitude of 50 mV, a potential step of 5 mV, and a scan rate of 5 mV s−1. All the potentials were measured relative to the Ag/AgCl/sat. KCl reference electrode.

increased with the concentration of pNA. These results indicate that pNA can be separately detected by DPV even in the presence of VPR-pNA. 3.2. Electrochemical endotoxin assay using rFC We performed an electrochemical endotoxin assay using rFC. rFC, Tris-Ac buffer solutions (pH 7.5) containing different endotoxin concentrations, and 100 μM VPR-pNA were incubated in a 96-well plate. After incubation at 37 °C for 1 h or 3 h, the electrodes were inserted into the well to perform DPV measurements. Fig. 3 shows typical voltammograms between −0.66 V and −0.86 V. The reduction peak at −0.75 V increased with the endotoxin concentration. As mentioned in Section 3.1, the peak indicates the reduction of pNA, which is the product of the hydrolysis of VPR-pNA. Only a negligible decrease was observed in the current response in repeated measurements. This decrease was probably caused by the adsorption of

2.3. Endotoxin assay method 120 μL of Tris-Ac buffer, 20 μL of rFC enzyme solution, and 20 μL of 1 mM VPR-pNA solution were mixed and preincubated in a 96-well plate at 37 °C for 10 min. Then, 40 μL of a standard endotoxin solution was added and the mixture was incubated at 37 °C for 1 h or 3 h. The pNA released from the substrate was detected by DPV [8,9]. The endotoxin concentration was indicated by the final concentration of pNA in the mixture solution. 3. Results and discussion 3.1. Basic characterization of the electrochemical behavior of VPR-pNA and pNA We first characterized the basic electrochemical performance of VPR-pNA (substrate for activated rFC) and pNA (product obtained by the hydrolysis of VPR-pNA catalyzed by activated rFC). Fig. 2 shows the differential pulse voltammograms recorded for the Tris-Ac buffer solution (pH 7.5) mixed with pNA and VPR-pNA. The total concentration of pNA and VPR-pNA was 100 μM. The voltammograms show two peaks, at −0.73 V and −0.60 V, that correspond to the reduction of pNA and VPR-pNA, respectively. These reduction peaks indicate the reduction of the nitro group of pNA to a hydroxyl group or to other related groups [10]. The peak at −0.73 V for pNA was clearly distinct from the peak for VPR-pNA, and the peak height at −0.73 V

Fig. 2. Differential pulse voltammograms of mixtures of VPR-pNA and pNA. The total concentration of the VPR–pNA and pNA was set at 100 μM. The ratios of VPR-pNA to pNA concentrations were 100:0 (a), 90:10 (b), 75:25 (c), 50:50 (d), 25:75 (e), and 0:100 (f). For DPV we used a pulse time of 70 ms, a pulse amplitude of 50 mV, a potential step of 5 mV, and a scan rate of 5 mV s−1.

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Fig. 4. Calibration curve for the quantitative detection of endotoxin using the rFC-based electrochemical assay. Incubation times were 1 h (○) and 3 h (□). Error bars represent standard deviations (n = 5).

the detection limit of the commonly used LAL assay [12]. Presently, we have almost achieved a detection limit of 1 EU L−1 for reaction times of 1 h using the LAL cascade reaction. In this cascade reaction, Factor C is activated by endotoxin binding and the activated Factor C activates Factor B. This activated Factor B subsequently activates the proclotting enzyme, leading to the hydrolysis of substrates such as Boc-Leu-GlyArg-pNA. Signal amplification rate of over 1000 can be expected in this cascade reaction. Zymogen-based electrochemical sensing systems can be used to detect other analytes including the molecules related to coagulation systems, complement systems, digestive systems, and apoptosis signaling systems. Zymogen-based sensors, which are distinct from common enzyme-based sensors, have unique characteristics, especially analyte-recognition capability. Therefore, they offer the possibility of detecting analytes that cannot be detected by common enzyme-based sensors. Protein engineering can help increase the number of detectable analytes by producing tailor-made biorecognition zymogens that can be used with biosensing platforms. Our zymogen-based assay system is expected to act as a model for the development of zymogen-based electrochemical biosensors in the fields of environment, food, and pharmacy. Fig. 3. Typical differential pulse voltammograms 1 h (A) and 3 h (B) after the incubation of the rFC in Tris-Ac buffer solution (pH 7.5) containing endotoxin and 100 μM VPRpNA in a 96-well plate. The concentrations of endotoxin: (a) 0, (b) 10, (c) 100, (d) 1000, (e) 5000, and (f) 10,000 EU L−1.

endotoxin or other coexisting species in the sample, on the electrode surface. The difference in the current at −0.75 V from that in the absence of endotoxin was used as the electrochemical signal for detection and plotted against the endotoxin concentration (Fig. 4). The DPV response depends on the incubation time and increases with the endotoxin concentration. Each point on the calibration plot corresponds to a mean value, and the error bar indicates the standard deviation obtained from five independent measurements. The detection limits are 5000 EU L−1 and 1000 EU L−1 for reaction times of 1 h and 3 h, respectively. Further studies for achieving high sensitivity, which is required for the practical use of the sensor, are in progress. The upper limit for the endotoxin concentration allowed in an ultrapure dialysate has been set at 30 EU L−1 by the Association for the Advancement of Medical Instrumentation (AAMI) [11]. This concentration level is the same as

4. Conclusion In this paper, we presented the basic concept of a zymogen-based electrochemical sensor. We detected endotoxin using a protease zymogen of rFC as the sensing element. When the endotoxin was added to a mixture solution containing rFC and VPR-pNA, rFC was activated by endotoxin binding; subsequently, VPR-pNA was hydrolyzed to yield pNA. The liberated pNA was detected by DPV at −0.75 V. The detection limit for endotoxin in Tris-Ac buffer with a pH of 7.5 at 37 °C was 5000 EU L−1 and 1000 EU L−1 for reaction times of 1 h and 3 h, respectively. Acknowledgements This work was partly supported by a Grant-in-Aid for Scientific Research (S) (no. 18101006) from the Japan Society for the Promotion of Science (JSPS) and by Special Coordination Funds for Promoting Science and Technology, Formation of Innovation Center for Fusion of Advanced Technologies from the Japan Science and Technology Agency.

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