Simple leaky-waveguide devices for the detection of bacterial spores

Sensors and Actuators B 160 (2011) 1508–1513

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Short communication

Simple leaky-waveguide devices for the detection of bacterial spores John P. Hulme a,∗ , Nicholas J. Goddard b , Chen Lu a a b

Gachon Bionano Research Institute, Kyungwon University, Sujeong-Gu, Seongnam-Si Gyeonggi-Do 461-701, South Korea School of Chemical Engineering and Analytical Science, University of Manchester, M60 1QD, UK

a r t i c l e

i n f o

Article history: Received 1 August 2011 Received in revised form 21 September 2011 Accepted 23 September 2011 Available online 1 October 2011 Keywords: Label free Leaky Waveguide HEMA Low index

a b s t r a c t A variety of biocompatible optical leaky waveguide sensors were developed for bacterial detection. Divalent sensitive polymer waveguides incorporating the chelating agent nitrilotriacetic acid (NTA) were used to monitor the release of Ca2+ ions from Bacillus subtilis spores during the first stage of germination. Copolymer waveguides incorporating hydrolyzed starch were used to detect the presence of exo-enzymes released during the later stages of vegetative growth. The limit of detection for calcium, based on changes in the intensity of the TE0 mode was 60 ␮M. Changes in refractive index were demonstrated by measuring the change in the angular position of the TE1 mode resulting from the degradation of the waveguide by ␣-amylase. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The detection of bacterial spores is important to the food and water industry. Release of pathogenic compounds and toxins poses considerable risk to companies and their consumers. Unfortunately, it is only after germination that bacteria begin to exhibit their pathogenic properties [1]. Germination can be divided into two stages. The first stage involves the excretion of mono-valent cations from the interior of the spore. This is followed by the release of calcium ions and dipicolinic acid (DPA) into the surrounding medium leading to core expansion and the beginning of vegetative metabolism (stage II). Methods based on terbium dipicolinate photoluminescence [2–4], calcein fluorescence spectroscopy and mass spectrometry have primarily focused on the detection of DPA. However these methods require expensive laboratory equipment, extensive training and high power sources. Recent developments such as dye and metal clad leaky waveguides have sought to address this issue. While these devices possess adequate optical resonances they require the deposition of a metal or dye layer in addition to a sensing and protective layer [5,6]. Unlike conventional waveguides [7], leaky waveguides [8–12] confine the optical field in a low index region, permitting the integration of biocompatible materials or gels in their construction.

∗ Corresponding author. E-mail address: fl[email protected] (J.P. Hulme). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.09.076

The sensitivity of these waveguides is similar to sensors based on holography [13–15]. One of the advantages of holographic sensors is the ease with which they are fabricated. Unlike metal or dye clad leaky waveguide devices [5,6] holographic sensors can be fabricated in a single step. In this communication we will show that leaky waveguides can be constructed in a single spin coating step from a variety of biocompatible and affinity polymers. Moreover the detection of the modes is accomplished using a single polarizer without incorporating a dye or metal layer in waveguide construction. These devices can then be used in a disposable manner to monitor the germination and vegetative growth of a known concentration of Bacillus subtilis spores.

2. Experimental All reagents were of analytical grade and were supplied by either Sigma Chemical Company (Dorset, UK) or Fischer Scientific (Seoul, South Korea) unless otherwise stated. Agarose (Type II), agar, sodium phosphate buffered saline tablets (pH 7.4, 10 mM phosphate 140 mM NaCl, 3 mM KCl); tris (hydroxymethyl) aminomethane (TRIS) hydrolyzed starch, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, hydrochloric acid, and alanine, (Aldrich chemicals, South Korea). Spores (Bacillus subtilis) were purchased from NCIMB Ltd. (Aberdeen, UK) and prepared in CCY medium [15]. Nutrient broth (NB) and typtone soya broth (TSB) were purchased from Oxoid.

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Fig. 1. Optical arrangement for leaky mode detection. Insert shows three TE leaky modes on the CCD detector, (light source, 1 W white LED). Agarose/starch/agar leaky waveguide.

2.1. Spore preparation The conditions for growth and sporulation were as previously reported [16]. In short spores were prepared using sporulation agar. B. subtilis cells were then grown on agar plates for five days at 30 ◦ C. Cells (exponential phase) were collected in distilled water using a sterile loop. The suspension was centrifuged at 1400 × g for 40 min and washed in MgCl2 solution (40 mM) then rinsed several times in deionised water. Refractile spores were counted using a phase microscope. Spores were stored at 4 ◦ C before use. B. subtilis spores were added to a Tris-buffered saline solution (pH 7.5 at 30 ◦ C) containing 10 mM l-alanine. Germination was assessed by measuring the change in optical density of a buffered spore solution (108 cfu/mL) at 600 nm using a (Cole & Parmer instruments, IL, USA) UV–visible spectrophotometer. pH measurements associated with the germination and vegetative growth of spores were conducted with a hand-held micro pH electrode (Orion, USA). 2.2. Detection of extracellular enzymes A nutrient broth solution containing B. subtilis spores (107 cfu/mL) was placed in an incubator for 14 h at 37 ◦ C. A sample of the supernatant was taken every 60 min. Amylase activity was measured using an established method [17]. 2.3. Instrumentation Instrumentation consisted of a collimated white LED (1 W), SF10 prism, 1 polarizer, a 650 nm broad band filter and a 2000 linear CCD detector with processing software (K-mac, Korea). Schematic of the instrumentation is shown in Fig. 1. Glass substrates (SF10) 1 mm thick, were purchased from (Comar Optics, Canada), the substrates were soaked for 40 min in a 3% solution of 3aminopropyltriethoxysilane (APES) in toluene. The chips were then dried at 70 ◦ C in a vacuum oven for 35 min. Before polymer coating 100 ␮L of 25% of polyglutaraldehyde was dispensed on each glass substrate and left for 2 min. Slides were then rinsed with deionised water for 10 min. Agarose films for refractive index calibration were constructed as follows: a 20 mL solution of deionised water was placed in a microwave to which 0.6 g of agarose was added. The solution was gently mixed for a minute and then heated to 100 ◦ C for 10 min. Approximately 600 ␮l of solution was then spin coated onto a preheated glass substrate at 1200 rpm for 30 s. The gel was allowed to set and placed on the prism (see Fig. 1). A flow cell

Fig. 2. A multimode profile in an agarose co-polymer waveguide. Interrogation wavelength of 650 nm.

was attached and the gel irrigated with deionised water for 20 min. Samples were placed in cold storage and used when required. 2.4. Waveguide structure and modeling The sensor structure consisted of a single polymer film of known thickness and refractive index coated spin coated on a 1 mm thick SF-10 substrate. In the first instance the thickness of the poly-HEMA film was designed to support a single TE0 mode. In regard to the co-polymer films incorporating hydrolyzed starch, the thickness of the films was chosen to support three TE modes (Fig. 2). There are number of advantages of using a high index substrate such as SF10. Firstly there is a large refractive index between the substrate and the polymer core which enhances reflectivity and decreases the leakage rate at the core/substrate interface. This allows the waveguide to be fabricated in a single step and does not require the deposition of a metal or dye layer. Secondly, at resonance there are numerous peaks and dips in the reflectivity of the waveguide. These signals appear as red and green colored lines when the waveguide is illuminated with white light (see insert in Fig. 1). Using inhouse modeling software (L-pro version 4) the distribution of the modes within a 6 ␮m agarose waveguide was then modeled (see Fig. 2). The simulation shows the waveguide supporting 3 TE modes. The lowest order mode (TE0 ) is sensitive to refractive index changes throughout the waveguide. The lowest order modes will be sensitive to a smaller portion of the waveguide and at different places relative to the upper and lower core boundary. 2.5. Refractive index calibrations A 6 ␮m thick 3% agarose film was removed from cold storage and fully hydrated for 20 min with deionised water. A 4% (v/v) glycerol stock solution was prepared in deionised water (refractive index value 1.33842 (Abbe refractometer)). The distance from each mode to the surface of the prism ranged from 185 to 187 mm. With a CCD pitch of 14 ␮m the angle subtended by one pixel for the TE0 , TE1 and TE2 modes was 4.33 × 10−3 , 4.23 × 10−3 , and 4.15 × 10−3 . Using the setup in Fig. 1 the coupling angle was 48◦ for a hydrated agarose waveguide. It should be noted that the leaky modes could be generated with (coupling angle 50◦ ) or without a cylindrical lens. The data for the cylindrical is available upon request.

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2.6.1. Synthesis of divalent sensitive films Ion sensitive films were fabricated using a reported method [18]. A poly-HEMA solution containing 4% ethylene glycol dimethyacrylate (EDMA) as cross-linker and 6 mol% R-NTA (2-(biscarboxymethyl-amino)-6-(2-methyl-acrylamino)-hexanoic acid as the chelating agent and 0.1% (w/v) Irgacure 2959 were prepared (Ciba Specialty chemicals, Basel, Switzerland) in a 50% propanol solution. The solution was degassed with nitrogen then exposed to 20-s pulses of UV light, mixed and degassed again. Approximately 500 ␮L of the solution was dispensed onto a glass substrate pretreated with 3-(trimethoxysilyl) propyl methacrylate and spin coated at 4500 rpm for 3 min. Samples were placed in an oven saturated with CO2 and exposed to UV light for 40 min [17]. HEMA films were subsequently washed in 50% propanol solution for 10 min. The waveguide was hydrated with Tris buffered solution (pH 7.4, 10 mM alanine) for 20 min. The effect of calcium ions released from a known concentration (108 cfu/mL) of germinating B. subtilis spores on the waveguide volume was recorded at regular intervals over an hour. The sensitivity of a NTA waveguide to different salt solutions (5 mM and 10 mM) KCl, NaCl, CaCl2 and MgCl2 was also investigated.

300

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2.6. Sensor fabrication

250 200 150 100 50 0

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Time (min) Fig. 3. Effect of divalent (Ca2+ ) ions release, from a germinating culture of B. subtilis (108 cfu/mL) spores on the intensity of a TE0 mode within a RNTA waveguide (). Insert shows the change in position and of the TE0 mode as the waveguide contracts (+60 min) (n = 3).

3. Results and discussion 3.1. Stage I germination. Detection of Ca2+ from B. subtilis spores

2.6.3. Synthesis of starch/agar/agarose based films Polymer waveguides were prepared as follows. To a 50 mL Pyrex flask 0.5 g of agarose, 0.3 g agar and 20 mL of Tris buffer (0.5 M, pH 9.0) were added. The solution was gently mixed for 5 min and then micro-waved for 15 min. In a 20 mL container, 2.5 g of starch was added to 10 mL of distilled water. The solution was then autoclaved at 121 ◦ C for 5 min. Both solutions were allowed to cool to 100 ◦ C then 10 mL from each solution was dispensed into a pre-heated beaker (100 ◦ C). The solution was placed on a hotplate (135 ◦ C) to which 300 ␮L of polyglutaraldehyde was added and mixed for a further 5 min. Approximately 500 ␮L of the gel solution was spin coated at 1200 rpm onto a pretreated glass substrate for 1 min and left to set at room temperature for a further 20 min. The film was hydrated with CCY medium for 10 min at 30 ◦ C. Subsequent changes in the position and intensity of the leaky modes (refractive index) via enzymatic degradation were measured directly and indirectly. Direct measurement involved the injection of 250 ␮L of a B. subtilis spore solution (107 cfu/mL) into the flow cell. The effect on the modal intensity and position was measured at regular time intervals over a 14 h period. Cell-free supernatants were collected every hour and ␣-amylase activity was measured using a previously reported technique [16].

The effects of divalent ions particularly calcium from a germinating spore solution (108 cfu/mL) on the intensity of the TE0 or the first leaky mode is shown in Fig. 3. The intensity of the TE0 mode increased by 300 pixels, which is an order of magnitude greater than the shift in the modal position, assuming that an increase of 1 intensity unit equals a shift of 1 pixel. The position of the TE0 mode remained constant over the next 24 h and the change in intensity was fully reversible. The change in optical density and the release of calcium ions occur simultaneously [15]. Fig. 4 shows the rate of change in the intensity of the TE0 mode is similar to the rate of spore germination observed at 600 nm (r2 = 98). Subsequent measurements of the waveguide response to varying concentrations Ca2+ –DPA (dipicolinic acid) showed that an increase intensity of 300 pixels corresponded to a Ca2+ ion of concentration of 60 ␮M released by 108 spores/mL (insert in Fig. 4). For the next part of the investigation we looked at the sensitivity of a NTA waveguide to various ionic (aq) interferents. It is 1.2 1.1

A 600 (t) / A 600 (init).

2.6.2. Synthesis of pH sensitive films An additional poly-HEMA solution was prepared in a similar manner however 5% methacrylic acid (MAA) monomer was used instead of DTA. For both types of film the preparation and exposure times were identical. Methacrylic films were hydrated with nutrient broth for 20 min. A known concentration of B. subtilis (108 cfu/mL) spores prepared in Tris-buffered solution (pH, 7.5, 10 mM alanine) was injected into the flow cell. Changes in waveguide thickness associated with spore germination and vegetative growth were recorded by measuring the change in intensity of the TE0 mode at regular intervals over a 2 h period. The pH response was measured in parallel with a hand held micro-pH electrode.

1 0.9 0.8 0.7 0.6 0.5 0.4 0

10

20

30

40

50

Time (min) Fig. 4. Changes in the absorbance value associated with germination of a B. subtilis spores (108 cfu/mL) in TBS buffer (10 mM alanine, pH 7.5). Insert shows the correlation between the TE0 mode and changes in A600 (n = 3).

J.P. Hulme et al. / Sensors and Actuators B 160 (2011) 1508–1513 Table 1 The response of the DTA waveguide to different metal salts at milli-molar concentrations. Standard error ± 2 pixels. Metal salt

Change in TE0 Intensity/a.u. (5 mM)

Change in TE0 Intensity/a.u. (10 mM)

CaCl2 MgCl2 KCl NaCl

48 ± 4 41 ± 3 N/A N/A

90 ± 4 80 ± 3 N/A N/A

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electrode were consistent with changes in modal intensity producing a linear correlation (see insert) (r2 = 0.96). Absorbance measurements (600 nm) revealed that the pH of the extracellular medium decreases when germination is complete and vegetative growth has begun. Therefore real time monitoring of the pH change associated with bacterial metabolism has been shown. In theory such a device may be able to detect the vegetative growth of individual spores. 3.3. Standard curves: ˛-amylase detection

well known that sodium and potassium as well as calcium are released from the spore casing during the first stage of germination, although it is calcium that is the predominant ion [1]. Table 1 shows the sensitivity of the waveguide to a variety of divalent and monovalent cations. The addition of KCl or NaCl did not significantly affect the mode at the stated concentrations. However, there was marked change in the position and intensity of the modes when MgCl2 or CaCl2 solution was added to the waveguide. These divalent ions can shield the repulsive forces within the polymer but also bind to several ionic groups, resulting in contraction of the polymer matrix [18–21]. According to the values in Table 1 the selectivity of the sensor towards the different metal ions was as follows Ca2+ > Mg2+ > Na+ > K+ . The sensitivity of the device at the stated concentrations is low but this maybe improved by increasing the starting concentration of the chelating monomer. 3.2. Leaky mode detection of pH flux associated with vegetative growth (stage II) Changes in pH of the extracellular medium associated with B. subtilis metabolism were investigated using a poly [HEMA-coEDMA (5 mol%)-co-MAA (4 mol%)] waveguide; pH changes in the medium were measured directly using a standardize hand held pH meter. Fig. 5 shows the decrease in extracellular pH (7–5.7) during the germination and vegetative growth of B. subtilis spores. As the pH of the medium decreases so do the repulsive forces between the ionisable groups within the polymer waveguide. This in turn causes a decrease in the volume of the waveguide and an increase in the intensity of the mode. Changes in the pH of the extracellular medium detected by the standard

In this part of the investigation we examined the degradation of a multimode waveguide by ␣-amylase. A multimode was used for several reasons. Firstly by virtue of the Kretschman configuration the higher order modes are more sensitive to refractive index changes than the lower order modes. Secondly by measuring changes in the intermodal distance and intensity of the modes one can distinguish between changes in the refractive index of the waveguide and changes in its thickness; thereby minimizing false positive or negative refractive index measurements (this occurs with smart waveguides). Given these factors it was prudent to use a multimode waveguide for the next part of the investigation. An example of a false positive refractive index measurement can be found in the additional information (the effect of flow pressure). Several copies of a multimode leaky waveguides consisting of starch/agar/agarose polymers were then fabricated and used to detect different concentrations of ␣-amylase in solution; changes in the position of the modes were used to measure the rate of waveguide degradation in the presence of the enzyme. Fig. 6a shows the real time degradation of a waveguide and the subsequent change in position of the modes. All the modes move to a lower angle of incidence (negative pixel shift) indicating the refractive index of the film is decreasing. It took <5 min for the position of the TE2 mode to reach equilibrium and more than 20 and 30 min for the TE1 and TE0 modes respectively. Using the graphical software package (origin 7, USA) the change in the position of the modes was analyzed with a nonlinear regression tool. It was found that the waveguide degraded in an exponential manner with the higher order modes exhibiting second order rate kinetics (R2 = 0.99 for the TE1 and TE2 modes). However the TE0 exhibited first order rate kinetics (R2 = 0.98). The primary reason for the different rates of decay is the sensitivity of the modes to different parts of the waveguide. The higher order modes are principally governed by refractive index changes at the polymer/solution boundary. Here the diffusion distant is minimal and the substrate is quickly degraded by the amylase. On the other-hand the TE0 mode is sensitive to refractive index changes within the core of the polymer, thus the initial rate of degradation is limited by the diffusion distance. The positions of the TE1 and TE0 modes mode were least sensitive to film expansion demonstrating a measurement error ±5%. Quantitative analysis was performed by measuring the gradients of the straight line portion of each mode at different ␣-amylase concentrations. This data was then used to construct ␣-amylase curves for each of the modes (see Fig. 6b). 3.4. Release of ˛-amylase from a growing culture

Fig. 5. The effect of pH change associated with the germination and vegetative growth of B. subtilis (108 cfu/mL) spores on the intensity of the TE0 mode within a MAA co-polymer waveguide. Insert shows the correlation between the modal intensity and extracellular pH measurements using a standard pH meter (n = 3).

The effects of ␣-amylase release from an active B. subtilis culture can be seen in Fig. 7a. The results of the TE2 and TE0 mode have been excluded. The independent [17] ␣-amylase assay is shown for comparison. For the first 5 h during the lag phase the position of the TE1 mode fluctuates from 0 to −3 pixels. At the onset of vegetative growth the position of the mode begins to fall indicating the refractive index of the film was decreasing. After 13 h the position of the mode then stabilizes. Even when the film was repeatedly

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Fig. 6. (a) Real time measurement of a known ␣-amylase concentration (1000 units/mL) on the position of the leaky modes in an agarose/agar/starch film. (b) Calibration graph showing the effects of ␣-amylase concentrations on the rate of decrease in modal positions using a starch/agar/agarose based films (n = 3).

150

TE 0

TE 1 - 40

TE2

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- 15

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Fig. 7. (a) Effect of ␣-amylase release from an active B. subtilis culture (107 cfu/mL) on the position of the TE1 leaky mode using a starch/hybrid waveguide ( ). Control measurement of ␣-amylase activity using independent assay (). (b) The effect of amylase rich containing supernatant (cell culture time + 12 h) on the position of the leaky modes using starch co-polymer waveguides (n = 3).

4. Conclusion

washed in deionised water the original position of the mode could not be restored. Moreover the degradation of the waveguide begins approximately 1-h before there is detectable amylase activity. A possible explanation for this anomaly is that the agarose film is sensitive to other factors such as changes in the ionic strength of the culture medium. An alternative explanation is that polymer is sensitive other degradative enzymes which maybe released during the final stage of germination from the spores germ cell wall [1]. The assay was then repeated (3×) using only amylase rich supernatant from a stationary phase B. subtilis culture (t = 12 h). The effect of the supernatant on a multimode waveguide is shown in Fig. 7b. The TE2 mode was the first mode to reach equilibrium this was followed by the TE1 and TE0 modes. The rate of change in the position of the TE0 TE1 and TE2 modes was 0.66 pixels/min, 0.76 pixels/min and 1.1 pixels/min which corresponds to amylase activities of 148, 83 and 30 units/mL using the standard curves from Fig. 6. If we compare these values with the activity value obtained with the commercial assay in Fig. 7a (90 units/mL) the value of TE1 mode produces a response with a degree error of less than 10%. The activity value for the TE0 mode is appreciably lower and the time its takes for the mode to reach equilibrium significantly longer suggesting that the value for the TE0 mode can be improved by reducing the starting thickness of the waveguide.

A variety of leaky waveguide sensors have been investigated for the detection of various extracellular products released from germinating and growing cultures of B. subtilis spores. Divalent waveguides were used to monitor the release of Ca2+ ions within minutes of germination. Vegetative growth was monitored using pH sensitive MAA co-polymer waveguides. Both types of waveguides could be re-used over a 1 month period without significant loss of response. The degradation of multimode waveguides by bacterial enzyme ␣-amylase was used to measure the vegetative outgrowth of B. subtilis spores. Acknowledgements This research was supported by the Kyungwon University Research Fund 2011 (KWU-2001-R271). References [1] P. Setlow, Spore germination, Curr. Opin. Microbiol. 6 (2003) 550–556. [2] D.L. Rosen, Bacterial spore detection and determination by use of terbium dipicolinate and photoluminescence, Anal. Chem. 69 (1997) 1082–1085. [3] L.C. Taylor, M.B. Tabacco, J.B. Gillespie, Sensors for detection of calcium associated with bacterial endospore suspensions, Anal. Chim. Acta 435 (2001) 239–246.

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Biographies John P. Hulme is a lecturer at the College of Bionanotechnology at Kyungwon University. He received his PhD and MSc from Manchester University in 1998 and 2002. Nicholas J. Goddard is professor of analytical science in the School of Chemical Engineering and Analytical Science (SCEAS) at the University of Manchester. Nick Goddard studied chemistry at Oxford, where he also obtained a D.Phil, using computer-controlled spectroscopy and electrochemistry to research the properties of viologen-based electrochromic materials. He then became a research officer at Imperial College, in the microprocessor unit established by Professor W.J. Albery. After a spell in industry with Fisons (Griffin and George Ltd.) and his own computer consultancy (Valewells Ltd.), he returned to university research as a Senior Research Associate at the Institute of Biotechnology at Cambridge. He has worked on optical and acoustic biosensors since 1988, and was the Senior Research Associate in the Group at the Institute of Biotechnology, Cambridge, that developed the Resonant Mirror (IaSys) sensor. Since moving to UMIST (1992), he has worked on chemical sensing applications of optical waveguide sensors, including the Resonant Mirror, image processing for automatic recognition of braille, optical pressure sensing and integration of optical waveguide sensors with electrokinetic sample transport and separations. More recently, he has pioneered the use of leaky waveguides for chemical and biochemical sensing. He also collaborates with Dstl Porton Down on the rapid and specific detection of bacteria, viruses and small molecules and with Dstl Fort Halstead on inorganic explosives detection. He has also played a major role in developing miniaturised chemical and biochemical instrumentation based on polymer fabrication, especially by injection moulding and lamination. Chen Lu received his BSc in chemical engineering from Shandong University of Technology in 2010. He is currently studying for his MSc at Kyungwon University in the department of Bionanotechnology.