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Long-range surface plasmons for sensitive detection of bacterial analytes
Sensors and Actuators B 139 (2009) 59–63
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Long-range surface plasmons for sensitive detection of bacterial analytes M. Vala a , S. Etheridge b , J.A. Roach b , J. Homola a,∗ a b
Institute of Photonics and Electronics ASCR, Chaberská 57, 18251 Prague, Czech Republic US Food and Drug Administration, College Park, USA
a r t i c l e
i n f o
Article history: Available online 28 August 2008 Keywords: Biosensor Surface plasmon resonance Long-range surface plasmon Bacterium
a b s t r a c t We report a novel prism-coupled surface plasmon resonance (SPR) biosensor based on special surface plasmon (SP) mode referred to as long-range surface plasmon (LRSP). Utilization of LRSP in prism-coupled SPR sensor offers several advantages in comparison with SPR sensors with conventional SPs (cSP) such as extended probe depth of LRSP mode into sensed dielectric and higher sensitivity to the bulk refractive index (RI) changes. The LRSP-supporting multilayer structures and prototype of the sensor setup were prepared. Performance of the LRSP-based sensor was compared to that of the cSP-based sensor in model experiments. LRSP-based sensor was determined to be almost 8 times more sensitive to sample RI changes than the cSP-based sensor. In addition, LRSP-based sensor responses were up to 2.5 and 5.5-fold greater than cSP-based sensor responses for latex beads and the bacterium E. coli HB101P, respectively. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade numerous bacterial pathogens implicated in food safety have been targeted by surface plasmon resonance (SPR) biosensors. These include Escherichia coli O157:H7 [1,2], Salmonella sp. [3–5], Listeria monocytogenes [3,6], Campylobacter jejuni [6], and Staphylococcus aureus [7]. Although SPR biosensor technology has made significant advances both in terms of SPR sensor instrumentation and surface functionalizations [8], further improvements in detection limits for bacterial analytes are highly desired. One of the issues in detection of bacterial analytes is their size, which substantially exceeds the penetration depth of a conventional surface plasmon (cSP), which for gold as a SPR-active medium and a wavelength range 600–1000 nm ranges from 100 to 600 nm [8]. As the sensitivity of SPR sensors decreases with an increasing distance from the surface of metal supporting a SP, only a limited part of the bacterium captured at the surface contributes to response of the SPR sensor. By utilizing a special SP mode referred to as a long-range surface plasmon (LRSP) propagating along a very thin metal layer, the SP probe depth and potentially the sensitivity to large analytes can be increased. The use of LRSPs was first proposed by Matsubara et al. [9]. LRSPs have been demonstrated to propagate along a thin metal film when it is embedded between media with similar refractive indices (RI). To satisfy this condition in an SPR biosensor, a medium (buffer) with a RI close to the sensed medium (typically around 1.33 for
∗ Corresponding author. Tel.: +420 266 773 448; fax: +420 284 680 222. E-mail address: [email protected] (J. Homola). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.08.029
biosensing applications) has to be used on the opposite side of the metal film. The use of LRSPs was shown to significantly improve both the sensor sensitivity and resolution. Nenninger et al. demonstrated an SPR sensor based on spectroscopy of LRSPs capable of resolving refractive index (RI) changes as small as 3 × 10−7 RIU (RIU: refractive index units) [10]. Subsequently, Slavík and Homola demonstrated an even better RI resolution of 3 × 10−8 RIU [11]. In this contribution, we advance the use of LRSPs for the detection of large analytes. An optical multilayer structure supporting LRSPs and an optical system for spectroscopy of LRSP have been developed. In order to evaluate the potential of LRSPbased sensors for detection of large analytes, detection of dielectric microparticles (latex beads) and the bacterium E. coli HB101P was evaluated. 2. Experimental 2.1. Preparation of SPR sensing chips 2.1.1. LRSP-supporting sensor chip Polished BK7 glass substrates (from Schott, USA) were first cleaned by acetone and dried in a clean air stream. Then, the substrates were placed into a UV ozone cleaner (UVO cleaner 42, Jelight Company, USA) for 20 min, rinsed with pure ethanol, dried in a dry nitrogen gas stream, rinsed with deionized (DI) water and again dried with nitrogen. In order to improve the adhesion of buffer layer material (Teflon AF) to the glass substrate, substrates were immersed in a boiling 2% solution of silane primer (1H,1H,2H,2Hperfluorodecyltriethoxysilane, Lancaster Synthesis, USA) in 20:1 ethanol:DI water. After 5 min, substrates were removed from the
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M. Vala et al. / Sensors and Actuators B 139 (2009) 59–63
solution, rinsed with ethanol and dried with a stream of nitrogen. A solution of 6% Teflon AF fluropolymer (Teflon AF 1601S40, DuPont, USA) dissolved in fluorinated solvent FC-40 (3M, USA) was spincoated onto silane-pretreated substrates. Substrates were spun for 120 s at 600 rpm to obtain homogeneous layer thickness. Coated substrates were left for more than 1 h at a room temperature and then baked for 1 h at 160 ◦ C. The thicknesses of prepared Teflon AF layers were between 1150 and 1200 nm and were measured by profilometer (Tencor Alpha Step, USA). SPR-active gold films (with thicknesses ranging from 25 to 27 nm) were prepared by thermal evaporation in a PFEIFFER PLS 570 deposition facility at a vacuum greater than 10−7 mbar. About 1.5 nm of titanium was deposited prior to the deposition of the gold film to improve the gold adhesion to the Teflon AF surface. Substrate temperature during both deposition steps was kept at 150 ◦ C. Thicknesses of the deposited gold and titanium layers were controlled by a quartz crystal oscillator.
total reflection (ATR), see Fig. 1. Temperature stabilization of both the sensing chip and liquid samples flowed through the flow-cell was available in both types of SPR sensors.
2.1.2. cSP-supporting sensor chip Sensor chips supporting conventional SPs consisted of BK7 glass substrate coated with 1.5 nm of titanium layer and 50 nm of gold layer. Procedures for substrate cleaning and deposition of titanium and gold layers were similar to those described in the previous section for LRSP chips.
2.2.2. LRSP-based sensor Due to the high angular sensitivity of the resonant position of LRSP, a high degree of collimation of the incident optical beam is needed to avoid degradation of the SPR dip. For this reason, a broadband superluminescent diode (SLD) coupled to a single mode (SM) optical fiber (EXS8505-1411, Exalos, Switzerland) was chosen as a light source. Light emitted from the SM fiber was collimated by a diffraction-limited collimator (OZ Optics, Canada) prior to total reflection on the LRSP-supporting chip optically matched with the same ATR coupling prism as in the case of cSP-based sensor. Reflected light was coupled into two output optical fibers (M25L02, Thorlabs, USA) using GRIN lenses (GRINTECH, Germany) and deliv-
2.2. SPR sensor instrumentation In this work, we used two in-house-built SPR sensors based on spectral interrogation of surface plasmons. SPR conditions were investigated in Kretschmann configuration [12] of the attenuated
2.2.1. cSP-based sensor Light from a broadband halogen lamp was transmitted by a multimode optical fiber (M28L02, Thorlabs, USA) and then collimated by means of a home-made collimator consisting of two cylindrical lenses with 20 and 50 mm effective focal lengths (CLB1515-20P and CLB-2020-50P, Sigma Koki, Japan) and coupled in the ATR coupling prism (BK7 glass, custom-made) in optical contact with the SPR chip. Light reflected from four distinct sensing spots where cSPs were excited was coupled into four output optical fibers (M25L02, Thorlabs, USA) using graded-index (GRIN) focusing lenses (GRINTECH, Germany) and delivered into a 4-channel spectrometer (model S2000 with 0.37 nm/pixel, Ocean Optics, USA).
Fig. 1. Scheme of the sensor setup and layered sensing structures supporting (a) LRSP and (b) cSP.
M. Vala et al. / Sensors and Actuators B 139 (2009) 59–63
ered to a 2-channel spectrometer (model S2000 with 0.12 nm/pixel, Ocean Optics, USA). 2.3. Materials 11-mercapto-1-ethyleneglycolundecanol (HSC11 (EG)OH) and 16-mercapto-tri(ethyleneglycol)-hexadecanoid acid (HSC11 (EG)3 OCH2 COOH) were purchased from Prochimia, Poland. NHydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were obtained from Sigma–Aldrich (USA). Rabbit polyclonal antibodies (Pabs) against E. coli (B65001R) were purchased from Meridian Life Science (USA). The dimensions of the bacterium are about 0.7–0.8 m (diameter) and 2 m ˇ (length) [13]. Heat-killed E. coli HB101P were provided by Dr. Skvor from Charles University, Czech Republic. Buffers: PBM (10 mM phosphate, 2.9 mM KCl, 15 mM Mg2+ , pH 7.4), PBNa (10 mM phosphate, 2.9 mM KCl, 0.75 M NaCl, pH 7.4) and SA (10 mM sodium acetate, pH 5.0) were from Sigma–Aldrich (Czech Republic). The carboxy-modified, biotin-labeled polystyrene beads of 1 m mean size (L8905) were also from Sigma–Aldrich. The beads were obtained as 1% solution in 10 mM PBS buffer. 2.4. Surface functionalization 2.4.1. Surface functionalization for detection of latex beads The gold surface of the SPR chip was functionalized by a mixed alkanethiol self-assembled monolayer (SAM) with carboxyl and hydroxyl terminal groups. Before the functionalization, SPR chips were placed into UV ozone cleaner for 20 min to remove organic contaminants from the gold surface. Then, the chips were rinsed in ethanol, dried by nitrogen stream, rinsed with DI water and again dried by nitrogen. Cleaned gold-coated chips were left dipped in ethanol with 0.7 mM 11-mercapto-1-ethyleneglycolundecanol (HSC11 (EG)OH) and 0.3 mM 16-mercapto-tri(ethyleneglycol)-hexadecanoid acid (HSC11 (EG)3 OCH2 COOH) for more than 16 h at room temperature to allow for SAM formation. Prior to the insertion of the chip into the sensor, the chip was rinsed with ethanol and DI water and dried with a stream of nitrogen. In order to anchor the biotinylated latex beads to the sensor surface, streptavidin was attached to the SAM using NHS/EDC amine coupling chemistry [14]. The carboxyl group of the mixed SAM was activated by flowing freshly mixed 11.5:76.7 mg/ml NHS:EDC in water solution through the assembled sensor flow-cell for approximately 7 min. The sensor surface was then exposed to DI water flow (5 min) followed by SA buffer (5 min) and 50 g/ml solution of streptavidin in SA buffer (20 min). Before the detection, PBNa buffer was flowed through the flow-cell to wash off unbound molecules from the sensor surface. The used flow-rate was 20 l/min. 2.4.2. Surface functionalization for detection of E. coli HB101P The gold surface of the SPR chips was functionalized with mixed alkanethiols using the procedure described in the previous section. Rabbit polyclonal antibodies against E. coli were immobilized to the mixed SAM with NHS/EDC amine coupling chemistry. The carboxyl groups of the mixed SAM were activated by flowing freshly mixed 11.5:76.7 mg/ml NHS:EDC in water solution through the sensor for approximately 8–10 min. Flow-rate was set to 20 l/min during this step. Subsequently, deionized water was flowed for 5 min through the flow-cell followed by SA buffer (5 min) and 100 g/ml of antiE. coli Pabs in SA buffer (10 min). PBNa buffer was used to remove unbound molecules from the sensor surface prior to the detection of bacteria. E. coli HB101P bacteria were obtained in 1 ml of Luria Bertani broth. Diluted sample (1:20 in PBM) was centrifuged for approx-
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imately 10 min at 6000 rpm followed by removal of supernatant from the sample. Sedimented bacteria were then resuspended in PBM and sedimented by centrifugation once more. After removal of supernatant and refilling the sample with PBM, sample was vortexed (1000 rpm) for few seconds to dissolve the bacterial pellet completely. To remove large impurities, samples were left intact for a few minutes to permit large impurities to settle out of the supernatant. Purified samples were then diluted to desired concentrations by addition of appropriate amounts of PBM buffer. Detection of E. coli HB101P was performed using LRSPsupporting chips with 27 nm thick gold film. 3. Results and discussion 3.1. Sensitivity to bulk refractive index changes Sensitivity of the LRSP-based sensor to bulk RI changes was calculated from the response of the sensor to the flow of liquids with different refractive indices along the sensing surface. In this experiment, sensor chip with 25 nm thick gold film was used. A set of NaCl solutions in deionized water with different refractive indices was prepared. Their refractive indices were determined as a function of NaCl concentration using a temperature stabilized multi-wavelength refractometer (DSR-, Schmidt + Haensch, Germany). A typical SPR spectrum measured with LRSP-supporting chip in contact with a pure water sample is shown in Fig. 2. Its resonant dip is almost twice as narrow (full width at half a minimum (FWHM) = 38 nm) as the calculated dip for the cSP-supporting structure (FWHM = 75 nm), which is also shown in Fig. 2 for comparison. Using the refractometric data shown in Fig. 3, the bulk RI sensitivity was determined to be about 59,000 nm/RIU. Using the noise of the baseline, the sensor resolution was calculated to be 5 × 10−8 RIU. 3.2. Detection of latex beads In the first step of detection experiment, the sensor baseline was established in pure PBM. Carboxylate-modified and biotin-labeled polystyrene microparticles (1 m in diameter from Sigma–Aldrich, USA) diluted in PBM buffer were then flowed across the streptavidin-immobilized sensor surface for approximately 10 min. The solution was followed by PBM. The flow-rate was set to
Fig. 2. Comparison of measured and calculated SPR spectra of light reflected with an incidence angle of 61.6◦ to the LRSP-supporting structure (BK7 glass substrate, 1200 nm of Teflon AF layer, 25 nm of gold) in contact with water. Spectrum with cSPconnected SPR dip calculated for structure consisting of BK7 glass substrate coated with 50 nm of gold in contact with water is included for comparison.
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Fig. 3. Sensorgram of the LRSP-based sensor, when aqueous salt solutions with different RI due to different concentrations of NaCl were flowed across the sensor surface.
30 l/min during the whole experiment and the temperature of the chip was kept at 25 ◦ C. Responses of the cSP-based and LRSP-based sensors to binding of latex beads of four different concentrations (0.01%, 0.003%, 0.001%, and 0.0005%) are shown in Figs. 4 and 5, respectively. The responses of the two sensors exposed to the highest concentration of latex beads in PBM (0.01%) were almost the same (10.6 and 10.3 nm for LRSP and cSP-based sensors, respectively). However, for the three lower concentrations of the beads, the sensitivity of the LRSP-based sensor is clearly higher than that of the cSP-based sensor. In particular, the sensor responses were greater about 1.5 times, 2 times and almost 2.5 times for particle concentrations 0.003%, 0.001% and 0.0005%, respectively. Due to the substantially larger sensitivity of LRSP to the bulk RI changes, the effect of a difference between the background RI of buffer and sample is more pronounced than in the case of cSP-based sensor. These bulk RI changes are manifested as step-like responses at the beginning and the end of the detection (see Fig. 5). Detection of latex beads was performed with LRSP-supporting chips with 27 nm thick gold film. These chips exhibited somewhat lower sensitivity to bulk RI changes (32,000 nm/RIU) than chips with 25 nm thick gold film. Binding of high RI beads to the surface of the more sensitive LRSP-
Fig. 4. Response of the cSP-based SPR sensor to the flow of latex beads diluted in PBM in four different concentrations (0.01%, 0.003%, 0.001% and 0.0005%). Arrows indicates where the sensor signal was taken to determine the sensor response.
Fig. 5. Response of the LRSP-based SPR sensor to the flow of latex beads diluted in PBM in four different concentrations (0.01%, 0.003%, 0.001% and 0.0005%). Arrows indicates where the sensor signal was taken to determine the sensor response.
supporting chips with 25 nm thick gold film resulted in the cut-off of the LRSP mode. 3.3. Detection of E. coli HB101P After the sensor baseline was established in PBM, samples containing E. coli HB101P were flowed across the sensor surface with immobilized anti-E. coli Pabs for 10 min. Subsequently, PBM was flowed through the flow-cell and sensor response was determined (see Figs. 6 and 7). The flow-rate was kept at 30 l/min and temperature at 25 ◦ C during all detection steps. Sensorgrams showing responses of the cSP-based and LRSP-based SPR sensors to binding of three different concentrations of E. coli to the sensor surface are given in Figs. 6 and 7, respectively. Similar to the detection of latex beads, greater differences were observed between LRSP and cSP-based sensor responses for lower E. coli concentrations. Samples diluted 1:20 and 1:40 in PBM induced about 3.5-fold (cSP, 2.3 nm; LRSP, 8.5 nm) and 5.5-fold (cSP, 0.97 nm; LRSP, 5.5 nm) greater responses in the case of the LRSP-based sensor. Whereas in the case of a sample diluted 1:200 in PBM, the cSP-based sensor response was below the resolution of the sensor (the sensor
Fig. 6. Response of the cSP-based SPR sensor to the flow of three different concentrations (1:20, 1:40 and 1:200) of heat-killed E. coli HB101P in PBM buffer across the sensor surface. Arrows indicates where the sensor signal was taken to determine the sensor response.
M. Vala et al. / Sensors and Actuators B 139 (2009) 59–63
Fig. 7. Response of the LRSP-based SPR sensor to the flow of three different concentrations (1:20, 1:40, and 1:200) of heat-killed E. coli HB101P in PBM buffer across the sensor surface. Arrows indicates where the sensor signal was taken to determine the sensor response.
response was not higher than three standard deviations of the sensor baseline, which is usually referred to as limit of detection [8]), while the LRSP-based sensor response was about 1.7 nm. 4. Conclusions In this work, we report an SPR sensor based on spectroscopy of LRSP for detection of large analytes such as latex beads and bacteria. Multilayer LRSP-supporting sensing structures based on Teflon AF as a buffer layer and an optical readout system were developed. Performance of the LRSP-based sensor was characterized in a model refractometric experiment. The sensor sensitivity to bulk RI changes was determined to be as high as 59,000 nm/RIU, which is about 8-fold higher than the sensitivity of cSP-based sensors. The LRSP-based sensors were also demonstrated to be capable of more sensitive detection of large analytes than SPR sensors employing cSPs. Two model experiments of large analyte binding – detection of latex beads and detection of E. coli HB101P bacterium – were performed and the LRSP-based sensor exhibited up to 2.5 and 5.5-fold higher response than cSP-based sensors, respectively. Acknowledgements This work was done under support of the Grant Agency of the Czech Republic under contract KAN200670701 and by the US Food and Drug Administration. References [1] B.K. Oh, W. Lee, W.H. Lee, et al., Nano-scale probe fabrication using selfassembly technique and application to detection of Escherichia coli O157:H7, Biotechnology and Bioprocess Engineering 8 (July–August (4)) (2003) 227–232.
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[2] J.W. Waswa, C. Debroy, J. Irudayaraj, Rapid detection of Salmonella enteritidis and Escherichia coli using surface plasmon resonance biosensor, Journal of Food Process Engineering 29 (August (4)) (2006) 373–385. [3] V. Koubová, E. Brynda, L. Karasová, et al., Detection of foodborne pathogens using surface plasmon resonance biosensors, Sensors and Actuators B: Chemical 74 (April 15 (1–3)) (2001) 100–105. [4] G. Bokken, R.J. Corbee, F. van Knapen, et al., Immunochemical detection of Salmonella group B, D and E using an optical surface plasmon resonance biosensor, FEMS Microbiology Letters 222 (May 16 (1)) (2003) 75–82. [5] B.K. Oh, W. Lee, Y.K. Kim, et al., Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi, Journal Of Biotechnology 111 (July 1 (1)) (2004) 1–8. [6] A.D. Taylor, J. Ladd, Q.M. Yu, et al., Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor, Biosensors & Bioelectronics 22 (December (5)) (2006) 752–758. [7] A. Subramanian, J. Irudayaraj, T. Ryan, Mono and dithiol surfaces on surface plasmon resonance biosensors for detection of Staphylococcus aureus, Sensors and Actuators B: Chemical 114 (March (1)) (2006) 192–198. [8] J. Homola, Surface plasmon resonance sensors for detection of chemical and biological species, Chemical Reviews 108 (February (2)) (2008) 462– 493. [9] K. Matsubara, S. Kawata, S. Minami, Multilayer system for a high-precision surface-plasmon resonance sensor, Optics Letters 15 (January 1 (1)) (1990) 75–77. [10] G.G. Nenninger, P. Tobiˇska, J. Homola, et al., Long-range surface plasmons for high-resolution surface plasmon resonance sensors, Sensors and Actuators B: Chemical 74 (April 15 (1–3)) (2001) 145–151. [11] R. Slavík, J. Homola, Ultrahigh resolution long range surface plasmon-based sensor, Sensors and Actuators B: Chemical 123 (April 10 (1)) (2007) 10– 12. [12] H. Raether, Surface-plasmons on smooth and rough surfaces and on gratings, Springer Tracts in Modern Physics 111 (1988) 1–133. [13] O. Pierucci, Dimensions of Escherichia coli at various growth-rates—model for envelope growth, Journal of Bacteriology 135 (2) (1978) 559–574. [14] A.D. Taylor, Q.M. Yu, S.F. Chen, et al., Comparison of E. coli O157:H7 preparation methods used for detection with surface plasmon resonance sensor, Sensors and Actuators B: Chemical 107 (May 27 (1)) (2005) 202–208.
Biographies Milan Vala (MS 2006) is a graduate student at Charles University, Prague and carries out his research at the Department of Optical Sensors at the Institute of Photonics and Electronics, Prague (Czech Republic). His research interests are in surface plasmon resonance biosensors. Stacey Etheridge (MS 1997, PhD 2002) is a Biologist in the Office of Regulatory Science in the Center for Food Safety and Applied Nutrition at the US Food and Drug Administration. Her research interests relate to food safety and security and include: detection method development for marine toxins; emerging sources and vectors of marine toxins; and the dynamics of toxic algae and the transfer of toxins to seafood. John A.G. Roach (MS 1970) is a Research Chemist in the Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. His research interests are the use of new mass spectrometry techniques for the analysis of adulterants in foods and the development of practical optical biosensor technology for rapid determination of food safety. Jiˇrí Homola (MS 1988, PHD 1993) is Head of Photonics Division and Chairman of Department of Optical Sensors at the Institute of Photonics and Electronics, Prague (Czech Republic). He also is Affiliate Associate Professor at the University of Washington, Seattle (USA). His research interests are in photonics and biophotonics with emphasis on optical sensors and biosensors. J. Homola is a member of Editorial Board of Sensors and Actuators B (Elsevier), Associate Editor of Journal of Sensors (Hindawi) and Senior Member of IEEE.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Long-range surface plasmons for sensitive detection of bacterial analytes M. Vala a , S. Etheridge b , J.A. Roach b , J. Homola a,∗ a b
Institute of Photonics and Electronics ASCR, Chaberská 57, 18251 Prague, Czech Republic US Food and Drug Administration, College Park, USA
a r t i c l e
i n f o
Article history: Available online 28 August 2008 Keywords: Biosensor Surface plasmon resonance Long-range surface plasmon Bacterium
a b s t r a c t We report a novel prism-coupled surface plasmon resonance (SPR) biosensor based on special surface plasmon (SP) mode referred to as long-range surface plasmon (LRSP). Utilization of LRSP in prism-coupled SPR sensor offers several advantages in comparison with SPR sensors with conventional SPs (cSP) such as extended probe depth of LRSP mode into sensed dielectric and higher sensitivity to the bulk refractive index (RI) changes. The LRSP-supporting multilayer structures and prototype of the sensor setup were prepared. Performance of the LRSP-based sensor was compared to that of the cSP-based sensor in model experiments. LRSP-based sensor was determined to be almost 8 times more sensitive to sample RI changes than the cSP-based sensor. In addition, LRSP-based sensor responses were up to 2.5 and 5.5-fold greater than cSP-based sensor responses for latex beads and the bacterium E. coli HB101P, respectively. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade numerous bacterial pathogens implicated in food safety have been targeted by surface plasmon resonance (SPR) biosensors. These include Escherichia coli O157:H7 [1,2], Salmonella sp. [3–5], Listeria monocytogenes [3,6], Campylobacter jejuni [6], and Staphylococcus aureus [7]. Although SPR biosensor technology has made significant advances both in terms of SPR sensor instrumentation and surface functionalizations [8], further improvements in detection limits for bacterial analytes are highly desired. One of the issues in detection of bacterial analytes is their size, which substantially exceeds the penetration depth of a conventional surface plasmon (cSP), which for gold as a SPR-active medium and a wavelength range 600–1000 nm ranges from 100 to 600 nm [8]. As the sensitivity of SPR sensors decreases with an increasing distance from the surface of metal supporting a SP, only a limited part of the bacterium captured at the surface contributes to response of the SPR sensor. By utilizing a special SP mode referred to as a long-range surface plasmon (LRSP) propagating along a very thin metal layer, the SP probe depth and potentially the sensitivity to large analytes can be increased. The use of LRSPs was first proposed by Matsubara et al. [9]. LRSPs have been demonstrated to propagate along a thin metal film when it is embedded between media with similar refractive indices (RI). To satisfy this condition in an SPR biosensor, a medium (buffer) with a RI close to the sensed medium (typically around 1.33 for
∗ Corresponding author. Tel.: +420 266 773 448; fax: +420 284 680 222. E-mail address: [email protected] (J. Homola). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.08.029
biosensing applications) has to be used on the opposite side of the metal film. The use of LRSPs was shown to significantly improve both the sensor sensitivity and resolution. Nenninger et al. demonstrated an SPR sensor based on spectroscopy of LRSPs capable of resolving refractive index (RI) changes as small as 3 × 10−7 RIU (RIU: refractive index units) [10]. Subsequently, Slavík and Homola demonstrated an even better RI resolution of 3 × 10−8 RIU [11]. In this contribution, we advance the use of LRSPs for the detection of large analytes. An optical multilayer structure supporting LRSPs and an optical system for spectroscopy of LRSP have been developed. In order to evaluate the potential of LRSPbased sensors for detection of large analytes, detection of dielectric microparticles (latex beads) and the bacterium E. coli HB101P was evaluated. 2. Experimental 2.1. Preparation of SPR sensing chips 2.1.1. LRSP-supporting sensor chip Polished BK7 glass substrates (from Schott, USA) were first cleaned by acetone and dried in a clean air stream. Then, the substrates were placed into a UV ozone cleaner (UVO cleaner 42, Jelight Company, USA) for 20 min, rinsed with pure ethanol, dried in a dry nitrogen gas stream, rinsed with deionized (DI) water and again dried with nitrogen. In order to improve the adhesion of buffer layer material (Teflon AF) to the glass substrate, substrates were immersed in a boiling 2% solution of silane primer (1H,1H,2H,2Hperfluorodecyltriethoxysilane, Lancaster Synthesis, USA) in 20:1 ethanol:DI water. After 5 min, substrates were removed from the
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solution, rinsed with ethanol and dried with a stream of nitrogen. A solution of 6% Teflon AF fluropolymer (Teflon AF 1601S40, DuPont, USA) dissolved in fluorinated solvent FC-40 (3M, USA) was spincoated onto silane-pretreated substrates. Substrates were spun for 120 s at 600 rpm to obtain homogeneous layer thickness. Coated substrates were left for more than 1 h at a room temperature and then baked for 1 h at 160 ◦ C. The thicknesses of prepared Teflon AF layers were between 1150 and 1200 nm and were measured by profilometer (Tencor Alpha Step, USA). SPR-active gold films (with thicknesses ranging from 25 to 27 nm) were prepared by thermal evaporation in a PFEIFFER PLS 570 deposition facility at a vacuum greater than 10−7 mbar. About 1.5 nm of titanium was deposited prior to the deposition of the gold film to improve the gold adhesion to the Teflon AF surface. Substrate temperature during both deposition steps was kept at 150 ◦ C. Thicknesses of the deposited gold and titanium layers were controlled by a quartz crystal oscillator.
total reflection (ATR), see Fig. 1. Temperature stabilization of both the sensing chip and liquid samples flowed through the flow-cell was available in both types of SPR sensors.
2.1.2. cSP-supporting sensor chip Sensor chips supporting conventional SPs consisted of BK7 glass substrate coated with 1.5 nm of titanium layer and 50 nm of gold layer. Procedures for substrate cleaning and deposition of titanium and gold layers were similar to those described in the previous section for LRSP chips.
2.2.2. LRSP-based sensor Due to the high angular sensitivity of the resonant position of LRSP, a high degree of collimation of the incident optical beam is needed to avoid degradation of the SPR dip. For this reason, a broadband superluminescent diode (SLD) coupled to a single mode (SM) optical fiber (EXS8505-1411, Exalos, Switzerland) was chosen as a light source. Light emitted from the SM fiber was collimated by a diffraction-limited collimator (OZ Optics, Canada) prior to total reflection on the LRSP-supporting chip optically matched with the same ATR coupling prism as in the case of cSP-based sensor. Reflected light was coupled into two output optical fibers (M25L02, Thorlabs, USA) using GRIN lenses (GRINTECH, Germany) and deliv-
2.2. SPR sensor instrumentation In this work, we used two in-house-built SPR sensors based on spectral interrogation of surface plasmons. SPR conditions were investigated in Kretschmann configuration [12] of the attenuated
2.2.1. cSP-based sensor Light from a broadband halogen lamp was transmitted by a multimode optical fiber (M28L02, Thorlabs, USA) and then collimated by means of a home-made collimator consisting of two cylindrical lenses with 20 and 50 mm effective focal lengths (CLB1515-20P and CLB-2020-50P, Sigma Koki, Japan) and coupled in the ATR coupling prism (BK7 glass, custom-made) in optical contact with the SPR chip. Light reflected from four distinct sensing spots where cSPs were excited was coupled into four output optical fibers (M25L02, Thorlabs, USA) using graded-index (GRIN) focusing lenses (GRINTECH, Germany) and delivered into a 4-channel spectrometer (model S2000 with 0.37 nm/pixel, Ocean Optics, USA).
Fig. 1. Scheme of the sensor setup and layered sensing structures supporting (a) LRSP and (b) cSP.
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ered to a 2-channel spectrometer (model S2000 with 0.12 nm/pixel, Ocean Optics, USA). 2.3. Materials 11-mercapto-1-ethyleneglycolundecanol (HSC11 (EG)OH) and 16-mercapto-tri(ethyleneglycol)-hexadecanoid acid (HSC11 (EG)3 OCH2 COOH) were purchased from Prochimia, Poland. NHydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were obtained from Sigma–Aldrich (USA). Rabbit polyclonal antibodies (Pabs) against E. coli (B65001R) were purchased from Meridian Life Science (USA). The dimensions of the bacterium are about 0.7–0.8 m (diameter) and 2 m ˇ (length) [13]. Heat-killed E. coli HB101P were provided by Dr. Skvor from Charles University, Czech Republic. Buffers: PBM (10 mM phosphate, 2.9 mM KCl, 15 mM Mg2+ , pH 7.4), PBNa (10 mM phosphate, 2.9 mM KCl, 0.75 M NaCl, pH 7.4) and SA (10 mM sodium acetate, pH 5.0) were from Sigma–Aldrich (Czech Republic). The carboxy-modified, biotin-labeled polystyrene beads of 1 m mean size (L8905) were also from Sigma–Aldrich. The beads were obtained as 1% solution in 10 mM PBS buffer. 2.4. Surface functionalization 2.4.1. Surface functionalization for detection of latex beads The gold surface of the SPR chip was functionalized by a mixed alkanethiol self-assembled monolayer (SAM) with carboxyl and hydroxyl terminal groups. Before the functionalization, SPR chips were placed into UV ozone cleaner for 20 min to remove organic contaminants from the gold surface. Then, the chips were rinsed in ethanol, dried by nitrogen stream, rinsed with DI water and again dried by nitrogen. Cleaned gold-coated chips were left dipped in ethanol with 0.7 mM 11-mercapto-1-ethyleneglycolundecanol (HSC11 (EG)OH) and 0.3 mM 16-mercapto-tri(ethyleneglycol)-hexadecanoid acid (HSC11 (EG)3 OCH2 COOH) for more than 16 h at room temperature to allow for SAM formation. Prior to the insertion of the chip into the sensor, the chip was rinsed with ethanol and DI water and dried with a stream of nitrogen. In order to anchor the biotinylated latex beads to the sensor surface, streptavidin was attached to the SAM using NHS/EDC amine coupling chemistry [14]. The carboxyl group of the mixed SAM was activated by flowing freshly mixed 11.5:76.7 mg/ml NHS:EDC in water solution through the assembled sensor flow-cell for approximately 7 min. The sensor surface was then exposed to DI water flow (5 min) followed by SA buffer (5 min) and 50 g/ml solution of streptavidin in SA buffer (20 min). Before the detection, PBNa buffer was flowed through the flow-cell to wash off unbound molecules from the sensor surface. The used flow-rate was 20 l/min. 2.4.2. Surface functionalization for detection of E. coli HB101P The gold surface of the SPR chips was functionalized with mixed alkanethiols using the procedure described in the previous section. Rabbit polyclonal antibodies against E. coli were immobilized to the mixed SAM with NHS/EDC amine coupling chemistry. The carboxyl groups of the mixed SAM were activated by flowing freshly mixed 11.5:76.7 mg/ml NHS:EDC in water solution through the sensor for approximately 8–10 min. Flow-rate was set to 20 l/min during this step. Subsequently, deionized water was flowed for 5 min through the flow-cell followed by SA buffer (5 min) and 100 g/ml of antiE. coli Pabs in SA buffer (10 min). PBNa buffer was used to remove unbound molecules from the sensor surface prior to the detection of bacteria. E. coli HB101P bacteria were obtained in 1 ml of Luria Bertani broth. Diluted sample (1:20 in PBM) was centrifuged for approx-
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imately 10 min at 6000 rpm followed by removal of supernatant from the sample. Sedimented bacteria were then resuspended in PBM and sedimented by centrifugation once more. After removal of supernatant and refilling the sample with PBM, sample was vortexed (1000 rpm) for few seconds to dissolve the bacterial pellet completely. To remove large impurities, samples were left intact for a few minutes to permit large impurities to settle out of the supernatant. Purified samples were then diluted to desired concentrations by addition of appropriate amounts of PBM buffer. Detection of E. coli HB101P was performed using LRSPsupporting chips with 27 nm thick gold film. 3. Results and discussion 3.1. Sensitivity to bulk refractive index changes Sensitivity of the LRSP-based sensor to bulk RI changes was calculated from the response of the sensor to the flow of liquids with different refractive indices along the sensing surface. In this experiment, sensor chip with 25 nm thick gold film was used. A set of NaCl solutions in deionized water with different refractive indices was prepared. Their refractive indices were determined as a function of NaCl concentration using a temperature stabilized multi-wavelength refractometer (DSR-, Schmidt + Haensch, Germany). A typical SPR spectrum measured with LRSP-supporting chip in contact with a pure water sample is shown in Fig. 2. Its resonant dip is almost twice as narrow (full width at half a minimum (FWHM) = 38 nm) as the calculated dip for the cSP-supporting structure (FWHM = 75 nm), which is also shown in Fig. 2 for comparison. Using the refractometric data shown in Fig. 3, the bulk RI sensitivity was determined to be about 59,000 nm/RIU. Using the noise of the baseline, the sensor resolution was calculated to be 5 × 10−8 RIU. 3.2. Detection of latex beads In the first step of detection experiment, the sensor baseline was established in pure PBM. Carboxylate-modified and biotin-labeled polystyrene microparticles (1 m in diameter from Sigma–Aldrich, USA) diluted in PBM buffer were then flowed across the streptavidin-immobilized sensor surface for approximately 10 min. The solution was followed by PBM. The flow-rate was set to
Fig. 2. Comparison of measured and calculated SPR spectra of light reflected with an incidence angle of 61.6◦ to the LRSP-supporting structure (BK7 glass substrate, 1200 nm of Teflon AF layer, 25 nm of gold) in contact with water. Spectrum with cSPconnected SPR dip calculated for structure consisting of BK7 glass substrate coated with 50 nm of gold in contact with water is included for comparison.
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Fig. 3. Sensorgram of the LRSP-based sensor, when aqueous salt solutions with different RI due to different concentrations of NaCl were flowed across the sensor surface.
30 l/min during the whole experiment and the temperature of the chip was kept at 25 ◦ C. Responses of the cSP-based and LRSP-based sensors to binding of latex beads of four different concentrations (0.01%, 0.003%, 0.001%, and 0.0005%) are shown in Figs. 4 and 5, respectively. The responses of the two sensors exposed to the highest concentration of latex beads in PBM (0.01%) were almost the same (10.6 and 10.3 nm for LRSP and cSP-based sensors, respectively). However, for the three lower concentrations of the beads, the sensitivity of the LRSP-based sensor is clearly higher than that of the cSP-based sensor. In particular, the sensor responses were greater about 1.5 times, 2 times and almost 2.5 times for particle concentrations 0.003%, 0.001% and 0.0005%, respectively. Due to the substantially larger sensitivity of LRSP to the bulk RI changes, the effect of a difference between the background RI of buffer and sample is more pronounced than in the case of cSP-based sensor. These bulk RI changes are manifested as step-like responses at the beginning and the end of the detection (see Fig. 5). Detection of latex beads was performed with LRSP-supporting chips with 27 nm thick gold film. These chips exhibited somewhat lower sensitivity to bulk RI changes (32,000 nm/RIU) than chips with 25 nm thick gold film. Binding of high RI beads to the surface of the more sensitive LRSP-
Fig. 4. Response of the cSP-based SPR sensor to the flow of latex beads diluted in PBM in four different concentrations (0.01%, 0.003%, 0.001% and 0.0005%). Arrows indicates where the sensor signal was taken to determine the sensor response.
Fig. 5. Response of the LRSP-based SPR sensor to the flow of latex beads diluted in PBM in four different concentrations (0.01%, 0.003%, 0.001% and 0.0005%). Arrows indicates where the sensor signal was taken to determine the sensor response.
supporting chips with 25 nm thick gold film resulted in the cut-off of the LRSP mode. 3.3. Detection of E. coli HB101P After the sensor baseline was established in PBM, samples containing E. coli HB101P were flowed across the sensor surface with immobilized anti-E. coli Pabs for 10 min. Subsequently, PBM was flowed through the flow-cell and sensor response was determined (see Figs. 6 and 7). The flow-rate was kept at 30 l/min and temperature at 25 ◦ C during all detection steps. Sensorgrams showing responses of the cSP-based and LRSP-based SPR sensors to binding of three different concentrations of E. coli to the sensor surface are given in Figs. 6 and 7, respectively. Similar to the detection of latex beads, greater differences were observed between LRSP and cSP-based sensor responses for lower E. coli concentrations. Samples diluted 1:20 and 1:40 in PBM induced about 3.5-fold (cSP, 2.3 nm; LRSP, 8.5 nm) and 5.5-fold (cSP, 0.97 nm; LRSP, 5.5 nm) greater responses in the case of the LRSP-based sensor. Whereas in the case of a sample diluted 1:200 in PBM, the cSP-based sensor response was below the resolution of the sensor (the sensor
Fig. 6. Response of the cSP-based SPR sensor to the flow of three different concentrations (1:20, 1:40 and 1:200) of heat-killed E. coli HB101P in PBM buffer across the sensor surface. Arrows indicates where the sensor signal was taken to determine the sensor response.
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Fig. 7. Response of the LRSP-based SPR sensor to the flow of three different concentrations (1:20, 1:40, and 1:200) of heat-killed E. coli HB101P in PBM buffer across the sensor surface. Arrows indicates where the sensor signal was taken to determine the sensor response.
response was not higher than three standard deviations of the sensor baseline, which is usually referred to as limit of detection [8]), while the LRSP-based sensor response was about 1.7 nm. 4. Conclusions In this work, we report an SPR sensor based on spectroscopy of LRSP for detection of large analytes such as latex beads and bacteria. Multilayer LRSP-supporting sensing structures based on Teflon AF as a buffer layer and an optical readout system were developed. Performance of the LRSP-based sensor was characterized in a model refractometric experiment. The sensor sensitivity to bulk RI changes was determined to be as high as 59,000 nm/RIU, which is about 8-fold higher than the sensitivity of cSP-based sensors. The LRSP-based sensors were also demonstrated to be capable of more sensitive detection of large analytes than SPR sensors employing cSPs. Two model experiments of large analyte binding – detection of latex beads and detection of E. coli HB101P bacterium – were performed and the LRSP-based sensor exhibited up to 2.5 and 5.5-fold higher response than cSP-based sensors, respectively. Acknowledgements This work was done under support of the Grant Agency of the Czech Republic under contract KAN200670701 and by the US Food and Drug Administration. References [1] B.K. Oh, W. Lee, W.H. Lee, et al., Nano-scale probe fabrication using selfassembly technique and application to detection of Escherichia coli O157:H7, Biotechnology and Bioprocess Engineering 8 (July–August (4)) (2003) 227–232.
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[2] J.W. Waswa, C. Debroy, J. Irudayaraj, Rapid detection of Salmonella enteritidis and Escherichia coli using surface plasmon resonance biosensor, Journal of Food Process Engineering 29 (August (4)) (2006) 373–385. [3] V. Koubová, E. Brynda, L. Karasová, et al., Detection of foodborne pathogens using surface plasmon resonance biosensors, Sensors and Actuators B: Chemical 74 (April 15 (1–3)) (2001) 100–105. [4] G. Bokken, R.J. Corbee, F. van Knapen, et al., Immunochemical detection of Salmonella group B, D and E using an optical surface plasmon resonance biosensor, FEMS Microbiology Letters 222 (May 16 (1)) (2003) 75–82. [5] B.K. Oh, W. Lee, Y.K. Kim, et al., Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi, Journal Of Biotechnology 111 (July 1 (1)) (2004) 1–8. [6] A.D. Taylor, J. Ladd, Q.M. Yu, et al., Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor, Biosensors & Bioelectronics 22 (December (5)) (2006) 752–758. [7] A. Subramanian, J. Irudayaraj, T. Ryan, Mono and dithiol surfaces on surface plasmon resonance biosensors for detection of Staphylococcus aureus, Sensors and Actuators B: Chemical 114 (March (1)) (2006) 192–198. [8] J. Homola, Surface plasmon resonance sensors for detection of chemical and biological species, Chemical Reviews 108 (February (2)) (2008) 462– 493. [9] K. Matsubara, S. Kawata, S. Minami, Multilayer system for a high-precision surface-plasmon resonance sensor, Optics Letters 15 (January 1 (1)) (1990) 75–77. [10] G.G. Nenninger, P. Tobiˇska, J. Homola, et al., Long-range surface plasmons for high-resolution surface plasmon resonance sensors, Sensors and Actuators B: Chemical 74 (April 15 (1–3)) (2001) 145–151. [11] R. Slavík, J. Homola, Ultrahigh resolution long range surface plasmon-based sensor, Sensors and Actuators B: Chemical 123 (April 10 (1)) (2007) 10– 12. [12] H. Raether, Surface-plasmons on smooth and rough surfaces and on gratings, Springer Tracts in Modern Physics 111 (1988) 1–133. [13] O. Pierucci, Dimensions of Escherichia coli at various growth-rates—model for envelope growth, Journal of Bacteriology 135 (2) (1978) 559–574. [14] A.D. Taylor, Q.M. Yu, S.F. Chen, et al., Comparison of E. coli O157:H7 preparation methods used for detection with surface plasmon resonance sensor, Sensors and Actuators B: Chemical 107 (May 27 (1)) (2005) 202–208.
Biographies Milan Vala (MS 2006) is a graduate student at Charles University, Prague and carries out his research at the Department of Optical Sensors at the Institute of Photonics and Electronics, Prague (Czech Republic). His research interests are in surface plasmon resonance biosensors. Stacey Etheridge (MS 1997, PhD 2002) is a Biologist in the Office of Regulatory Science in the Center for Food Safety and Applied Nutrition at the US Food and Drug Administration. Her research interests relate to food safety and security and include: detection method development for marine toxins; emerging sources and vectors of marine toxins; and the dynamics of toxic algae and the transfer of toxins to seafood. John A.G. Roach (MS 1970) is a Research Chemist in the Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. His research interests are the use of new mass spectrometry techniques for the analysis of adulterants in foods and the development of practical optical biosensor technology for rapid determination of food safety. Jiˇrí Homola (MS 1988, PHD 1993) is Head of Photonics Division and Chairman of Department of Optical Sensors at the Institute of Photonics and Electronics, Prague (Czech Republic). He also is Affiliate Associate Professor at the University of Washington, Seattle (USA). His research interests are in photonics and biophotonics with emphasis on optical sensors and biosensors. J. Homola is a member of Editorial Board of Sensors and Actuators B (Elsevier), Associate Editor of Journal of Sensors (Hindawi) and Senior Member of IEEE.