- Email: [email protected]
Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori
Accepted Manuscript Title: Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori Author: W. Lonsdale D.K. Maurya M. Wajrak C.Y. Tay B.J. Marshall K. Alameh PII: DOI: Reference:
S0925-4005(16)30876-0 http://dx.doi.org/doi:10.1016/j.snb.2016.06.024 SNB 20350
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-12-2015 30-5-2016 2-6-2016
Please cite this article as: W.Lonsdale, D.K.Maurya, M.Wajrak, C.Y.Tay, B.J.Marshall, K.Alameh, Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori W. Lonsdalea, D. K. Maurya*a, M. Wajrakb, C. Y. Tayc, B. J. Marshallc, K. Alameha a
Electron Science Research Institute, Edith Cowan University, Joondalup, WA 6027, Australia School of Natural Sciences, Edith Cowan University, Joondalup, WA 6027, Australia c The Marshall Centre for Infectious Diseases Research and Training, The University of Western Australia, Nedlands, WA 6009, Australia *Corresponding author: D. K. Maurya, E-mail: [email protected], Telephone: +61-863042868 b
Highlights
Miniaturised sensor for H. pylori detection. Rapid quantification of urease activity (1 to 50 U/mL) within 30 s. In vitro detection of H. pylori bacteria in less than 1 minute. High-sensitivity potentiometric RuO2 thin film based pH sensor Stable sensor response over a wide pH range (1-10)
Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori W. Lonsdalea, D. K. Maurya*a, M. Wajrakb, C. Y. Tayc, B. J. Marshallc, K. Alameha a
Electron Science Research Institute, Edith Cowan University, Joondalup, WA 6027, Australia School of Natural Sciences, Edith Cowan University, Joondalup, WA 6027, Australia c The Marshall Centre for Infectious Diseases Research and Training, The University of Western Australia, Nedlands, WA 6009, Australia *Corresponding author: D. K. Maurya, E-mail: [email protected], Telephone: +61-863042868 b
Abstract The feasibility of using a thin-film RuO2 (ruthenium oxide) pH sensor for the detection of H. pylori is investigated. In particular, we demonstrate the ability of the sensor to measure urease activity between 1.0 and 50 U/mL in less than 30 seconds. The developed sensor exhibited a super-Nernstian response of 77.74 mV/pH and excellent reversibility. The pH sensor’s ability to detect H. pylori in real time is demonstrated, and found to be much faster than the rapid urease test. Keywords: Ruthenium oxide, Urease activity, pH sensor, R.F. sputtering, H. pylori detection 1.
Background
Helicobacter pylori (H. pylori) is a bacteria known to colonise the stomach where it can cause gastric ulcers, gastritis and potentially lead to stomach cancer [1,2]. H. pylori produces the enzyme, urease, which hydrolyses urea naturally present in the stomach to ammonia, which neutralises gastric acid; allowing the bacteria to survive long enough in gastric juice to colonise gastric mucus [1,2]. H. pylori infection is commonly diagnosed by detecting its high urease activity, as demonstrated by the Rapid Urease Test (RUT) or the Urea Breath Test (UBT) [3,4]. While the RUT is currently considered the most rapid technique for H. pylori detection, it can be inaccurate when H. pylori concentration is low or the stomach is not acidic enough; the technique is also invasive, because it requires endoscopy [3]. pH sensors have been used for the measurement of urease activity [5]. T. Sato et al. [6] developed a pH sensitive field effect transistor (pH-FET) mounted on an endoscope for H. pylori detection. Their strategy involves placing a pH-FET near the gastric mucus during endoscopy, replacing the gastric fluid around the sensor with a buffered ammonium solution, then measuring the change in pH that occurs due to the hydrolysis of urea by H. pylori present in the mucus. pH-FETs have several disadvantages compared to potentiometric pH sensors, including higher drift rate, higher hysteresis and sensitivity to ambient light [7]. On the other hand, traditional potentiometric glass pH probes are also not suitable for use in the human body due to their large size and mechanical fragility. Potentiometric pH sensors employing metal oxide thin film sensing electrodes offer an alternative to pH-FETs and traditional glass pH probes, due of their small-footprint, cost effectiveness and ease to manufacture [8]. Of the numerous metal oxides that exhibit pH sensitivity, RuO 2 has been widely investigated due to its excellent corrosion resistance, thermal stability, high sensitivity, low hysteresis and low resistivity in comparison with other metal oxides (Table 1) [8,9]. RuO2 has also demonstrated excellent biocompatibility in cell studies and has been used for the construction of neural electrodes [10,11]. An endoscopic sensor employing a RuO2 pH sensing electrode would have higher sensitivity, lower drift rate and smaller hysteresis values, in comparison with pHFET counterparts, potentially enabling faster detection of H. pylori through potential slope measurements. Table 1: comparison of RuO2 to other metal oxide pH sensors. Metal Oxide
pH Range
RuO2 Ta2O5 SnO2 WO3 IrO2
4-10 1-10 2-12 1-12 2-12
Sensitivity (mV/pH) 69.83 56.19 59 59 59.5
Response Time (s) 3 2
Hysteresis (mV) 4 5 7-11 10.5 10
Ref. [14] [12] [13] [14] [13]
This work demonstrates the viability of using a simple potentiometric thin film RuO 2 pH sensor for the rapid measurement of urease activity. The measurement of urease activity was then applied to the detection of H. pylori. The developed pH sensor can be used as a potential tool for the fast detection of H. pylori. Further work is planned to further miniaturise the pH sensor for integration into an in-vivo endoscopic sensor for H. pylori detection. 2.
Experimental
2.1 Sensor fabrication Thin-film RuO2 pH sensors were fabricated using the methods detailed by A. Sardarinejad et al. [15,16]. Briefly, a RuO2 thin-film was sputtered, using an R.F. magnetron sputtering system in conjuction with shadow masking, on the platinium (Pt) sensing electrode of a rugged two-electrode cell on ceramic substrate (dimensions 15 mm × 61 mm × 0.67 mm), purchased from Pine Research Instrumentation (http://www.pineinstrument.com/). The purchased cell consisted of a screen printed Pt working electrode (2 mm diameter) and electroplated Ag/AgCl reference (1 mm diameter). Optimal sensitivity was achieved with an RuO2 pH sensing electrode of thickness 300 nm using 80:20 Ar:O2 process gas ratio at 1 mTorr process pressure. 2.2 Sensor functionality test An Agilent 34410A high performance digital multimeter was used for real-time potential recording between the sensing and reference electrode. A unity-gain impedance-matching buffer amplifier was developed in-house and used to reduce signal noise and hence improve the Signal-to-Noise Ratio (SNR) of the sensor. The sensor performance was evaluated using commercial pH buffer standards ranging from pH 1 to pH 10 (Rowe Scientific, Australia). Potential difference between the RuO2 sensing electrode and Ag/AgCl reference electrode was recorded at 2 s intervals for 60 s to 600 s in pH buffer standards. All measurements were made at 22 oC with continuous magnetic stirring three times; results presented are averages and error bars represent the 95% confidence interval. 2.3 Urease measurement and H. pylori detection To evaluate the sensor’s ability to measure urease activity the potential difference between sensing and reference electrodes were recorded in Dulbecco’s Phosphate Buffered Saline (PBS) with 20 mM added urea and between 1 and 50 U/mL urease (derived from Jack Bean); all from Sigma-Aldrich. The 20 mM of added urea was used since this was enough to exceed the buffering capacity of PBS, once hydrolysed. A baseline was recorded in the PBS-urea solution for 100 s followed by the addition of the desired concentration of urease and the reaction recorded for a further 100 s. A calibration curve was prepared by calculating the rate of change of pH (as mV/s) over the 10-24 s time period (after the addition of urease). This period was chosen since the rate of change of pH was the most linear with respect to time and urease concentration. In vitro experiments intended to demonstrate the pH sensor’s capability to detect H. pylori bacteria were carried out. A 300 µL chamber, using acrylic and commercial adhesive, was created around the pH sensor’s working area. H. pylori solutions were prepared from a culture of H. pylori strain 13-021 and pipetted into the chamber; bacteria numbers were estimated using optical density measurements [17]. The potentials of 300 µL PBS solutions with 106, 5×105 and 105 H. pylori cells were recorded for 60 s, then 40 mM of urea was added and the reaction recorded for a further 3 minutes. The rates of change of potential (which is proportional to the rate of change of pH) were calculated between the 20 s and 44 s data points, post addition of urea. 3.
Results and Discussion
3.1 pH response The sensitivity of the pH sensor was determined using pH buffer standards (pH 1, 2, 4, 7 and 10), results taken after a 60 s equilibration period are shown in Fig. 1. The sensor’s response to pH was found to be linear between pH 1 and 10 with a super-Nernstian response of 77.74 mV/pH. Sensor reversibility was examined by sequentially switching the pH at the sensor between 1, 2, 4, 7 and 10 in a forward and backward order at 300 s intervals without cleaning or drying of the electrode, whilst the potential was monitored. Results shown in Fig. 2 demonstrate excellent reversibility of the sensor and stable response over the pH range. Sensor drift was examined by measuring pH buffers (1 to 10) for 10 minutes (not shown), a pH drift of 0.02 pH/min was observed, which was deemed acceptable since the electrode stability (time taken to reach 95% of the stable reading) was less than 20 s. The sensitivity, reversibility, drift and stability of the RuO2 pH sensor manufactured
here demonstrates excellent performance which is in agreement with results reported by A. Sardarinejad et al. [15,16]; however here the sesnor was shown to perform well from pH 1 to 10 (instead of just 4 to 10).
Potential (mV)
700
y = -77.746x + 639.27 R² = 0.9931
500 300 100 -100 -300 0
2
4
6
8
10
pH Fig. 1. Measured potential versus pH for the developed RuO 2 pH sensor, where the sensitivity (slope) is 77.74 mV/pH. Measurements were made in triplicate; where not visible error bars are within the data point.
700 Potential (mV)
pH 1
500 pH 2
300 pH 4
100
pH 7
-100
pH 10
-300 0
1000
2000 Time (s)
3000
4000
Fig. 2. Reversibility data for pH sensor over 5 minute intervals for a loop pH 1-pH 10; pH 10-pH1; pH1-pH 10. 3.2 Urease measurement and H. pylori detection The potential of a PBS buffer solution with 20 mM added urea was measured over 100 s and found to be stable. Addition of between 1 and 50 U/mL of urease to the solution resulted in a pH change due to hydrolysis of urea (Equation 1 below), which was recorded as a change in potential (Fig. 3). It is noticed in Fig. 3 that, after a transient period of 10 s, the rate of change of potential (and therefore pH) is relatively constant over the following 14 s. The rate of change of potential during this period versus urease concentration is shown in Fig. 4. It is obvious from Fig. 4 that the change in potential is directly proportional to urease concentration over the tested range (1 to 50 U/mL). Minimum urease activity values quoted by T. Sato et al. [6] for H. pylori positive patients equate to approximately 1.5 U/mL, near the gastric mucus. This is within the linear range determined here (1-50 U/mL) and suggests the sensing protocol developed could be suitable for H. pylori detection. →
(Equation 1)
Potential (mV)
220
1 U/ml 2 U/ml
PBS+ 20mM Urea
200
4 U/ml
180 160
10 U/ml
140
20 U/ml 30 U/ml 40 U/ml 50 U/ml
120 0
50
100 Time (s)
150
200
Fig. 3. Potential versus time for different urease concentrations in PBS+20 mM urea.
Rate of Change (mV/s)
-3.5 -2.5 -1.5
y = -0.0661x R² = 0.9907
-0.5 0.0
10.0
20.0 30.0 Urease (U/mL)
40.0
50.0
Fig. 4. Rate of change of pH (as potential) versus urease concentration. Measurements were made in triplicate; where not visible error bars are within the data point. The sensor’s ability to detect H. pylori was confirmed by analysing solutions containing H. pylori cells. As can be seen in Fig. 5, a change in potential occurs after the addition of urea to the H. pylori solution. Here the transient period was found to be slightly longer than the previous experiment, likely due to the lack of mixing of the urea/H. pylori solution in the chamber. Therefore the rate of change of potential (i.e., pH) was calculated between the 20 s and 44 s data points, post addition of urea. Fig. 6 shows the rate of change of potential for solutions containing 0 to 106 H. pylori cells. It can be seen that the rate of change of potential is greater for solutions with higher bacteria numbers and that the detection of the solution containing 10 5 H. pylori cells was achieved in under 60 s, after the addition of urea. Note that a typical RUT requires approximately 10 5 cells for a positive result, however the number of H. pylori in a gastric biopsy is typically much greater than this count [18]. This demonstrates that the slope-based approach proposed in this work in conjunction with the use of a RuO2 thin-film pH sensor is more suitable for the in-vivo detection of H. pylori.‖
Potential (mV)
230 0 Cells
210
1× 105 Cells
190
5×105 Cells
170 1× 106 Cells
150 0
50
100
150 Time (s)
200
Rate of Change (mV/s)
Fig. 5. Potential versus time for different numbers of H. pylori in PBS with 40 mM of urea added at 60 s.
-0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0E+00
1E+05 5E+05 H. Pylori (n)
1E+06
Fig. 6. Rate of change of potential for different numbers of H. pylori. 4. Conclusion A RuO2 based pH sensor has been developed and its capability to accurately measure pH between pH 1 and 10 was demonstrated. The developed sensor exhibited a super Nernstian response of 77.74 mV/pH, excellent reversibility and stability. Experimental results demonstrated the ability of the sensor to quantify urease activity in PBS between 1 and 50 U/mL in less than 30 seconds. The ability of the developed pH sensor to detect H. pylori in real time has been demonstrated, and found to be much faster than the rapid urease test. The results presented here are of practical significance and allow for the development of an endoscopic sensor for in-vivo measurement of urease activity, and hence the detection of H. pylori infection and gastric ulcer diagnosis. Acknowledgement This research was supported by Edith Cowan University, Australia, and the Department of Education and Training, Australian Government. H. pylori samples and testing were provided by the Marshal Centre for Infectious Diseases Research and Training, The University of Western Australia. This study was funded in part by NHMRC grant number 572723. Funding was also received from the Western Australian Government’s Department of Commerce and Department of Health, and the University of Western Australia’s Special Infrastructure Fund. Declaration of personal interests: Barry Marshall has served as a speaker and an advisory board member, and has received funding from Vice-Chancellor of University Western Australia, NHMRC (No. 572723). Barry
Marshall, and Chin Yen Tay are all employees of the University of Western Australia. Barry Marshall owns shares in Tri-med, a company which markets diagnostic tests for Helicobacter pylori manufactured by Kimberley Clark USA (the CLOtest™ rapid urease test and Pytest ™ C14-urea breath test). Tri-med also distributes several medications including rifabutin, tetracycline, levofloxacin, furazolidone, secnidazole and DeNol (colloidal bismuth subcitrate). Barry Marshall also owns shares in Ondek Pty Ltd, a biotechnology company developing natural immune modulatory products, patents pending to Ondek Pty Ltd: patent WO2012119203A1, patent WO2010148459A1, patent WO2010139018A1 patent WO2011160182A1, Helicobacter and immunotherapy 1, Helicobacter and immunotherapy 2, Helicobacter and immunotherapy 3, patent WO2008055316A1. In addition, patent US20070134264A1 is issued to Ondek Pty Ltd. References [1] E. Goers Sweeney, J. N. Henderson, J. Goers, C. Wreden, K. G. Hicks, J. K. Foster, R. Parthasarathy, S. J. Remington, and K. Guillemin, ―Structure and Proposed Mechanism for the pH-Sensing Helicobacter pylori Chemoreceptor TlpB,‖ Structure, vol. 20, no. 7, pp. 1177–1188, 2012. [2] J. P. Celli, B. S. Turner, N. H. Afdhal, S. Keates, I. Ghiran, C. P. Kelly, R. H. Ewoldt, G. H. McKinley, P. So, S. Erramilli, and R. Bansil, ―Helicobacter pylori moves through mucus by reducing mucin viscoelasticity.,‖ Proc. Natl. Acad. Sci. U. S. A., vol. 106, no. 34, pp. 14321–14326, 2009. [3] A. Isabel Lopez, F. F. Vale, and M. Oleastro, ―Heliobacter pylori infection - recent developments in diagnosis,‖ World J. Gastroenterol., vol. 20, no. 28, pp. 9299–9313, 2014. [4] M. L. Viiala CH, Windsor HM, Forbes GM, Chairman SO, Marshall BJ, ―Evaluation of a new formulation CLOtest.,‖ J. Gastroenterol. heptology, vol. 17, no. 2, pp. 127–130, 2002. [5] J. M. Bibby and D. W. L. Hukins, ―Measurement of pH to quantify urease activity,‖ J. Biochem. Biophys. Methods, vol. 25, pp. 231–236, 1992. [6] T. Sato, M. Fujino, Y. Kojima, H. Ohtsuka, M. Ohtaka, K. Kubo, T. Nakamura, A. Morozumi, M. Nakamura, and H. Hosaka, ―Endoscopic urease sensor system for detecting Helicobacter pylori on gastric mucosa,‖ Gastrointest. Endosc., vol. 49, no. 1, pp. 32–38, 1999. [7] M. Yuqing, C. Jianrong, and F. Keming, ―New technology for the detection of pH,‖ J. Biochem. Biophys. Methods, vol. 63, no. 1, pp. 1–9, 2005. [8] L. Manjakkal, K. Cvejin, J. Kulawik, K. Zaraska, and D. Szwagierczak, ―A Low-Cost pH Sensor Based on RuO2 Resistor Material,‖ Nano Hybrids, vol. 5, pp. 1–15, 2013. [9] A. Fog and R. P. Buck, ―Electronic semiconducting oxides as pH sensors,‖ Sensors and Actuators, vol. 5, no. 2, pp. 137–146, 1984. [10] D. Wu and E. Hoffman, ―Deposition and Characterization of Ruthenium Films for Neural Electrodes,‖ NNIN REU Accompl., pp. 112–115, 2011. [11] M. Brischwein, H. Grothe, J. Wiest, M. Zottmann, J. Ressler, and B. Wolf, ―Planar Ruthenium Oxide Sensors for Cell-on-a-Chip Metabolic Studies,‖ Chem. Analityczna, vol. 54, no. 6, pp. 1193–1201, 2009. [12] D. Maurya, A. Sardarinejad, and K. Alameh, ―Recent Developments in R.F. Magnetron Sputtered Thin Films for pH Sensing Applications—An Overview,‖ Coatings, vol. 4, no. 4, pp. 756–771, 2014. [13] T. Y. Kim and S. Yang, ―Fabrication method and characterization of electrodeposited and heat-treated iridium oxide films for pH sensing,‖ Sensors Actuators, B Chem., vol. 196, pp. 31–38, 2014. [14] P. Salazar, F. J. Garcia-Garcia, F. Yubero, J. Gil-Rostra, and A. R. González-Elipe, ―Characterization and application of a new pH sensor based on magnetron sputtered porous WO3 thin films deposited at oblique angles,‖ Electrochim. Acta, vol. 193, pp. 24–31, 2016. [15] A. Sardarinejad, D. K. Maurya, and K. Alameh, ―The effects of sensing electrode thickness on ruthenium oxide thin-film pH sensor,‖ Sensors Actuators, A Phys., vol. 214, pp. 15–19, 2014. [16] A. Sardarinejad, D. Maurya, and K. Alameh, ―The pH Sensing Properties of RF Sputtered RuO2 ThinFilm Prepared Using Different Ar/O2 Flow Ratio,‖ Materials (Basel)., vol. 8, no. 6, pp. 3352–3363, 2015. [17] W. Bell and P. Kilian, ―EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells,‖ Phytomedicine, vol. 14, no. 1, pp. 5–8, 2007. [18] T. Uotani and D. Y. Graham, ―Diagnosis of Helicobacter pylori using the rapid urease test.,‖ Ann. Transl. Med., vol. 3, no. 1, p. 9, 2015.
S0925-4005(16)30876-0 http://dx.doi.org/doi:10.1016/j.snb.2016.06.024 SNB 20350
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-12-2015 30-5-2016 2-6-2016
Please cite this article as: W.Lonsdale, D.K.Maurya, M.Wajrak, C.Y.Tay, B.J.Marshall, K.Alameh, Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori W. Lonsdalea, D. K. Maurya*a, M. Wajrakb, C. Y. Tayc, B. J. Marshallc, K. Alameha a
Electron Science Research Institute, Edith Cowan University, Joondalup, WA 6027, Australia School of Natural Sciences, Edith Cowan University, Joondalup, WA 6027, Australia c The Marshall Centre for Infectious Diseases Research and Training, The University of Western Australia, Nedlands, WA 6009, Australia *Corresponding author: D. K. Maurya, E-mail: [email protected], Telephone: +61-863042868 b
Highlights
Miniaturised sensor for H. pylori detection. Rapid quantification of urease activity (1 to 50 U/mL) within 30 s. In vitro detection of H. pylori bacteria in less than 1 minute. High-sensitivity potentiometric RuO2 thin film based pH sensor Stable sensor response over a wide pH range (1-10)
Rapid measurement of urease activity using a potentiometric RuO2 pH sensor for detection of Helicobacter pylori W. Lonsdalea, D. K. Maurya*a, M. Wajrakb, C. Y. Tayc, B. J. Marshallc, K. Alameha a
Electron Science Research Institute, Edith Cowan University, Joondalup, WA 6027, Australia School of Natural Sciences, Edith Cowan University, Joondalup, WA 6027, Australia c The Marshall Centre for Infectious Diseases Research and Training, The University of Western Australia, Nedlands, WA 6009, Australia *Corresponding author: D. K. Maurya, E-mail: [email protected], Telephone: +61-863042868 b
Abstract The feasibility of using a thin-film RuO2 (ruthenium oxide) pH sensor for the detection of H. pylori is investigated. In particular, we demonstrate the ability of the sensor to measure urease activity between 1.0 and 50 U/mL in less than 30 seconds. The developed sensor exhibited a super-Nernstian response of 77.74 mV/pH and excellent reversibility. The pH sensor’s ability to detect H. pylori in real time is demonstrated, and found to be much faster than the rapid urease test. Keywords: Ruthenium oxide, Urease activity, pH sensor, R.F. sputtering, H. pylori detection 1.
Background
Helicobacter pylori (H. pylori) is a bacteria known to colonise the stomach where it can cause gastric ulcers, gastritis and potentially lead to stomach cancer [1,2]. H. pylori produces the enzyme, urease, which hydrolyses urea naturally present in the stomach to ammonia, which neutralises gastric acid; allowing the bacteria to survive long enough in gastric juice to colonise gastric mucus [1,2]. H. pylori infection is commonly diagnosed by detecting its high urease activity, as demonstrated by the Rapid Urease Test (RUT) or the Urea Breath Test (UBT) [3,4]. While the RUT is currently considered the most rapid technique for H. pylori detection, it can be inaccurate when H. pylori concentration is low or the stomach is not acidic enough; the technique is also invasive, because it requires endoscopy [3]. pH sensors have been used for the measurement of urease activity [5]. T. Sato et al. [6] developed a pH sensitive field effect transistor (pH-FET) mounted on an endoscope for H. pylori detection. Their strategy involves placing a pH-FET near the gastric mucus during endoscopy, replacing the gastric fluid around the sensor with a buffered ammonium solution, then measuring the change in pH that occurs due to the hydrolysis of urea by H. pylori present in the mucus. pH-FETs have several disadvantages compared to potentiometric pH sensors, including higher drift rate, higher hysteresis and sensitivity to ambient light [7]. On the other hand, traditional potentiometric glass pH probes are also not suitable for use in the human body due to their large size and mechanical fragility. Potentiometric pH sensors employing metal oxide thin film sensing electrodes offer an alternative to pH-FETs and traditional glass pH probes, due of their small-footprint, cost effectiveness and ease to manufacture [8]. Of the numerous metal oxides that exhibit pH sensitivity, RuO 2 has been widely investigated due to its excellent corrosion resistance, thermal stability, high sensitivity, low hysteresis and low resistivity in comparison with other metal oxides (Table 1) [8,9]. RuO2 has also demonstrated excellent biocompatibility in cell studies and has been used for the construction of neural electrodes [10,11]. An endoscopic sensor employing a RuO2 pH sensing electrode would have higher sensitivity, lower drift rate and smaller hysteresis values, in comparison with pHFET counterparts, potentially enabling faster detection of H. pylori through potential slope measurements. Table 1: comparison of RuO2 to other metal oxide pH sensors. Metal Oxide
pH Range
RuO2 Ta2O5 SnO2 WO3 IrO2
4-10 1-10 2-12 1-12 2-12
Sensitivity (mV/pH) 69.83 56.19 59 59 59.5
Response Time (s) 3 2
Hysteresis (mV) 4 5 7-11 10.5 10
Ref. [14] [12] [13] [14] [13]
This work demonstrates the viability of using a simple potentiometric thin film RuO 2 pH sensor for the rapid measurement of urease activity. The measurement of urease activity was then applied to the detection of H. pylori. The developed pH sensor can be used as a potential tool for the fast detection of H. pylori. Further work is planned to further miniaturise the pH sensor for integration into an in-vivo endoscopic sensor for H. pylori detection. 2.
Experimental
2.1 Sensor fabrication Thin-film RuO2 pH sensors were fabricated using the methods detailed by A. Sardarinejad et al. [15,16]. Briefly, a RuO2 thin-film was sputtered, using an R.F. magnetron sputtering system in conjuction with shadow masking, on the platinium (Pt) sensing electrode of a rugged two-electrode cell on ceramic substrate (dimensions 15 mm × 61 mm × 0.67 mm), purchased from Pine Research Instrumentation (http://www.pineinstrument.com/). The purchased cell consisted of a screen printed Pt working electrode (2 mm diameter) and electroplated Ag/AgCl reference (1 mm diameter). Optimal sensitivity was achieved with an RuO2 pH sensing electrode of thickness 300 nm using 80:20 Ar:O2 process gas ratio at 1 mTorr process pressure. 2.2 Sensor functionality test An Agilent 34410A high performance digital multimeter was used for real-time potential recording between the sensing and reference electrode. A unity-gain impedance-matching buffer amplifier was developed in-house and used to reduce signal noise and hence improve the Signal-to-Noise Ratio (SNR) of the sensor. The sensor performance was evaluated using commercial pH buffer standards ranging from pH 1 to pH 10 (Rowe Scientific, Australia). Potential difference between the RuO2 sensing electrode and Ag/AgCl reference electrode was recorded at 2 s intervals for 60 s to 600 s in pH buffer standards. All measurements were made at 22 oC with continuous magnetic stirring three times; results presented are averages and error bars represent the 95% confidence interval. 2.3 Urease measurement and H. pylori detection To evaluate the sensor’s ability to measure urease activity the potential difference between sensing and reference electrodes were recorded in Dulbecco’s Phosphate Buffered Saline (PBS) with 20 mM added urea and between 1 and 50 U/mL urease (derived from Jack Bean); all from Sigma-Aldrich. The 20 mM of added urea was used since this was enough to exceed the buffering capacity of PBS, once hydrolysed. A baseline was recorded in the PBS-urea solution for 100 s followed by the addition of the desired concentration of urease and the reaction recorded for a further 100 s. A calibration curve was prepared by calculating the rate of change of pH (as mV/s) over the 10-24 s time period (after the addition of urease). This period was chosen since the rate of change of pH was the most linear with respect to time and urease concentration. In vitro experiments intended to demonstrate the pH sensor’s capability to detect H. pylori bacteria were carried out. A 300 µL chamber, using acrylic and commercial adhesive, was created around the pH sensor’s working area. H. pylori solutions were prepared from a culture of H. pylori strain 13-021 and pipetted into the chamber; bacteria numbers were estimated using optical density measurements [17]. The potentials of 300 µL PBS solutions with 106, 5×105 and 105 H. pylori cells were recorded for 60 s, then 40 mM of urea was added and the reaction recorded for a further 3 minutes. The rates of change of potential (which is proportional to the rate of change of pH) were calculated between the 20 s and 44 s data points, post addition of urea. 3.
Results and Discussion
3.1 pH response The sensitivity of the pH sensor was determined using pH buffer standards (pH 1, 2, 4, 7 and 10), results taken after a 60 s equilibration period are shown in Fig. 1. The sensor’s response to pH was found to be linear between pH 1 and 10 with a super-Nernstian response of 77.74 mV/pH. Sensor reversibility was examined by sequentially switching the pH at the sensor between 1, 2, 4, 7 and 10 in a forward and backward order at 300 s intervals without cleaning or drying of the electrode, whilst the potential was monitored. Results shown in Fig. 2 demonstrate excellent reversibility of the sensor and stable response over the pH range. Sensor drift was examined by measuring pH buffers (1 to 10) for 10 minutes (not shown), a pH drift of 0.02 pH/min was observed, which was deemed acceptable since the electrode stability (time taken to reach 95% of the stable reading) was less than 20 s. The sensitivity, reversibility, drift and stability of the RuO2 pH sensor manufactured
here demonstrates excellent performance which is in agreement with results reported by A. Sardarinejad et al. [15,16]; however here the sesnor was shown to perform well from pH 1 to 10 (instead of just 4 to 10).
Potential (mV)
700
y = -77.746x + 639.27 R² = 0.9931
500 300 100 -100 -300 0
2
4
6
8
10
pH Fig. 1. Measured potential versus pH for the developed RuO 2 pH sensor, where the sensitivity (slope) is 77.74 mV/pH. Measurements were made in triplicate; where not visible error bars are within the data point.
700 Potential (mV)
pH 1
500 pH 2
300 pH 4
100
pH 7
-100
pH 10
-300 0
1000
2000 Time (s)
3000
4000
Fig. 2. Reversibility data for pH sensor over 5 minute intervals for a loop pH 1-pH 10; pH 10-pH1; pH1-pH 10. 3.2 Urease measurement and H. pylori detection The potential of a PBS buffer solution with 20 mM added urea was measured over 100 s and found to be stable. Addition of between 1 and 50 U/mL of urease to the solution resulted in a pH change due to hydrolysis of urea (Equation 1 below), which was recorded as a change in potential (Fig. 3). It is noticed in Fig. 3 that, after a transient period of 10 s, the rate of change of potential (and therefore pH) is relatively constant over the following 14 s. The rate of change of potential during this period versus urease concentration is shown in Fig. 4. It is obvious from Fig. 4 that the change in potential is directly proportional to urease concentration over the tested range (1 to 50 U/mL). Minimum urease activity values quoted by T. Sato et al. [6] for H. pylori positive patients equate to approximately 1.5 U/mL, near the gastric mucus. This is within the linear range determined here (1-50 U/mL) and suggests the sensing protocol developed could be suitable for H. pylori detection. →
(Equation 1)
Potential (mV)
220
1 U/ml 2 U/ml
PBS+ 20mM Urea
200
4 U/ml
180 160
10 U/ml
140
20 U/ml 30 U/ml 40 U/ml 50 U/ml
120 0
50
100 Time (s)
150
200
Fig. 3. Potential versus time for different urease concentrations in PBS+20 mM urea.
Rate of Change (mV/s)
-3.5 -2.5 -1.5
y = -0.0661x R² = 0.9907
-0.5 0.0
10.0
20.0 30.0 Urease (U/mL)
40.0
50.0
Fig. 4. Rate of change of pH (as potential) versus urease concentration. Measurements were made in triplicate; where not visible error bars are within the data point. The sensor’s ability to detect H. pylori was confirmed by analysing solutions containing H. pylori cells. As can be seen in Fig. 5, a change in potential occurs after the addition of urea to the H. pylori solution. Here the transient period was found to be slightly longer than the previous experiment, likely due to the lack of mixing of the urea/H. pylori solution in the chamber. Therefore the rate of change of potential (i.e., pH) was calculated between the 20 s and 44 s data points, post addition of urea. Fig. 6 shows the rate of change of potential for solutions containing 0 to 106 H. pylori cells. It can be seen that the rate of change of potential is greater for solutions with higher bacteria numbers and that the detection of the solution containing 10 5 H. pylori cells was achieved in under 60 s, after the addition of urea. Note that a typical RUT requires approximately 10 5 cells for a positive result, however the number of H. pylori in a gastric biopsy is typically much greater than this count [18]. This demonstrates that the slope-based approach proposed in this work in conjunction with the use of a RuO2 thin-film pH sensor is more suitable for the in-vivo detection of H. pylori.‖
Potential (mV)
230 0 Cells
210
1× 105 Cells
190
5×105 Cells
170 1× 106 Cells
150 0
50
100
150 Time (s)
200
Rate of Change (mV/s)
Fig. 5. Potential versus time for different numbers of H. pylori in PBS with 40 mM of urea added at 60 s.
-0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0E+00
1E+05 5E+05 H. Pylori (n)
1E+06
Fig. 6. Rate of change of potential for different numbers of H. pylori. 4. Conclusion A RuO2 based pH sensor has been developed and its capability to accurately measure pH between pH 1 and 10 was demonstrated. The developed sensor exhibited a super Nernstian response of 77.74 mV/pH, excellent reversibility and stability. Experimental results demonstrated the ability of the sensor to quantify urease activity in PBS between 1 and 50 U/mL in less than 30 seconds. The ability of the developed pH sensor to detect H. pylori in real time has been demonstrated, and found to be much faster than the rapid urease test. The results presented here are of practical significance and allow for the development of an endoscopic sensor for in-vivo measurement of urease activity, and hence the detection of H. pylori infection and gastric ulcer diagnosis. Acknowledgement This research was supported by Edith Cowan University, Australia, and the Department of Education and Training, Australian Government. H. pylori samples and testing were provided by the Marshal Centre for Infectious Diseases Research and Training, The University of Western Australia. This study was funded in part by NHMRC grant number 572723. Funding was also received from the Western Australian Government’s Department of Commerce and Department of Health, and the University of Western Australia’s Special Infrastructure Fund. Declaration of personal interests: Barry Marshall has served as a speaker and an advisory board member, and has received funding from Vice-Chancellor of University Western Australia, NHMRC (No. 572723). Barry
Marshall, and Chin Yen Tay are all employees of the University of Western Australia. Barry Marshall owns shares in Tri-med, a company which markets diagnostic tests for Helicobacter pylori manufactured by Kimberley Clark USA (the CLOtest™ rapid urease test and Pytest ™ C14-urea breath test). Tri-med also distributes several medications including rifabutin, tetracycline, levofloxacin, furazolidone, secnidazole and DeNol (colloidal bismuth subcitrate). Barry Marshall also owns shares in Ondek Pty Ltd, a biotechnology company developing natural immune modulatory products, patents pending to Ondek Pty Ltd: patent WO2012119203A1, patent WO2010148459A1, patent WO2010139018A1 patent WO2011160182A1, Helicobacter and immunotherapy 1, Helicobacter and immunotherapy 2, Helicobacter and immunotherapy 3, patent WO2008055316A1. In addition, patent US20070134264A1 is issued to Ondek Pty Ltd. References [1] E. Goers Sweeney, J. N. Henderson, J. Goers, C. Wreden, K. G. Hicks, J. K. Foster, R. Parthasarathy, S. J. Remington, and K. Guillemin, ―Structure and Proposed Mechanism for the pH-Sensing Helicobacter pylori Chemoreceptor TlpB,‖ Structure, vol. 20, no. 7, pp. 1177–1188, 2012. [2] J. P. Celli, B. S. Turner, N. H. Afdhal, S. Keates, I. Ghiran, C. P. Kelly, R. H. Ewoldt, G. H. McKinley, P. So, S. Erramilli, and R. Bansil, ―Helicobacter pylori moves through mucus by reducing mucin viscoelasticity.,‖ Proc. Natl. Acad. Sci. U. S. A., vol. 106, no. 34, pp. 14321–14326, 2009. [3] A. Isabel Lopez, F. F. Vale, and M. Oleastro, ―Heliobacter pylori infection - recent developments in diagnosis,‖ World J. Gastroenterol., vol. 20, no. 28, pp. 9299–9313, 2014. [4] M. L. Viiala CH, Windsor HM, Forbes GM, Chairman SO, Marshall BJ, ―Evaluation of a new formulation CLOtest.,‖ J. Gastroenterol. heptology, vol. 17, no. 2, pp. 127–130, 2002. [5] J. M. Bibby and D. W. L. Hukins, ―Measurement of pH to quantify urease activity,‖ J. Biochem. Biophys. Methods, vol. 25, pp. 231–236, 1992. [6] T. Sato, M. Fujino, Y. Kojima, H. Ohtsuka, M. Ohtaka, K. Kubo, T. Nakamura, A. Morozumi, M. Nakamura, and H. Hosaka, ―Endoscopic urease sensor system for detecting Helicobacter pylori on gastric mucosa,‖ Gastrointest. Endosc., vol. 49, no. 1, pp. 32–38, 1999. [7] M. Yuqing, C. Jianrong, and F. Keming, ―New technology for the detection of pH,‖ J. Biochem. Biophys. Methods, vol. 63, no. 1, pp. 1–9, 2005. [8] L. Manjakkal, K. Cvejin, J. Kulawik, K. Zaraska, and D. Szwagierczak, ―A Low-Cost pH Sensor Based on RuO2 Resistor Material,‖ Nano Hybrids, vol. 5, pp. 1–15, 2013. [9] A. Fog and R. P. Buck, ―Electronic semiconducting oxides as pH sensors,‖ Sensors and Actuators, vol. 5, no. 2, pp. 137–146, 1984. [10] D. Wu and E. Hoffman, ―Deposition and Characterization of Ruthenium Films for Neural Electrodes,‖ NNIN REU Accompl., pp. 112–115, 2011. [11] M. Brischwein, H. Grothe, J. Wiest, M. Zottmann, J. Ressler, and B. Wolf, ―Planar Ruthenium Oxide Sensors for Cell-on-a-Chip Metabolic Studies,‖ Chem. Analityczna, vol. 54, no. 6, pp. 1193–1201, 2009. [12] D. Maurya, A. Sardarinejad, and K. Alameh, ―Recent Developments in R.F. Magnetron Sputtered Thin Films for pH Sensing Applications—An Overview,‖ Coatings, vol. 4, no. 4, pp. 756–771, 2014. [13] T. Y. Kim and S. Yang, ―Fabrication method and characterization of electrodeposited and heat-treated iridium oxide films for pH sensing,‖ Sensors Actuators, B Chem., vol. 196, pp. 31–38, 2014. [14] P. Salazar, F. J. Garcia-Garcia, F. Yubero, J. Gil-Rostra, and A. R. González-Elipe, ―Characterization and application of a new pH sensor based on magnetron sputtered porous WO3 thin films deposited at oblique angles,‖ Electrochim. Acta, vol. 193, pp. 24–31, 2016. [15] A. Sardarinejad, D. K. Maurya, and K. Alameh, ―The effects of sensing electrode thickness on ruthenium oxide thin-film pH sensor,‖ Sensors Actuators, A Phys., vol. 214, pp. 15–19, 2014. [16] A. Sardarinejad, D. Maurya, and K. Alameh, ―The pH Sensing Properties of RF Sputtered RuO2 ThinFilm Prepared Using Different Ar/O2 Flow Ratio,‖ Materials (Basel)., vol. 8, no. 6, pp. 3352–3363, 2015. [17] W. Bell and P. Kilian, ―EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells,‖ Phytomedicine, vol. 14, no. 1, pp. 5–8, 2007. [18] T. Uotani and D. Y. Graham, ―Diagnosis of Helicobacter pylori using the rapid urease test.,‖ Ann. Transl. Med., vol. 3, no. 1, p. 9, 2015.