SPR-based immunosensor for determining staphylococcal enterotoxin A

Sensors and Actuators B 136 (2009) 8–12

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SPR-based immunosensor for determining staphylococcal enterotoxin A Wen-Chi Tsai ∗ , Ie-Chin Li Graduate Institute of Biotechnology, Chinese Culture University, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Received 12 November 2007 Received in revised form 28 October 2008 Accepted 30 October 2008 Available online 13 November 2008 Keywords: Surface plasmon resonance Mixed self-assembled monolayers Staphylococcal enterotoxin A Immunosensor

a b s t r a c t An immunosensor for determining staphylococcal enterotoxin A (SEA) was developed on the basis of surface plasmon resonance (SPR). The sensor surfaces were constructed from various thiol mixtures of different molar ratios of 16-mercapto-1-hexanol (16-MHA) to 6-mercapto-1-hexanol (6-MCH). The surface functionalized with a 1:20 mixed self-assembled monolayer (SAM) exhibited the best result in terms of SEA detection sensitivity. Through optimization of anti-SEA immobilization, the SPR sensor could detect SEA in phosphate-buffered saline buffer in a linear range from 100 to 1000 ng/ml. Furthermore, studies of recovery and matrix effects were performed to evaluate the suitability of the developed immunosensor for directly analyzing SEA in milk samples. Results indicated that the mixed SAM-based SPR immunosensor has the potential for the specific detection of SEA. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Food borne diseases have a major public health impact. Therefore, methods for rapidly detecting pathogenic microorganisms and toxins in food and water to prevent infection, illness, and economic losses are always desired. Staphylococcal food poisoning is one of the most prevalent causes of gastroenteritis worldwide, which is caused by the ingestion of food that contains pre-formed toxin. The amount of enterotoxin necessary to cause intoxication is small. The emetic dose in a monkey is ∼5–20 ␮g/animal [1]. Staphylococcus aureus makes 12 structurally similar enterotoxins, namely A, B, C1 , C2 , C3 , D, and E as well as the newly discovered G, H, I, J, and K [2]. Usually the intoxication is not lethal. In very rare cases, acute staphylococcal enterotoxicosis can cause death due to complications. Of the different serotypes, staphylococcal enterotoxin A (SEA), a monomeric protein (Mr 27,000), is most commonly associated with food poisoning [3,4]. SEA was selected as a typical protein toxin for this study. Immunoassay techniques, such as radioimmunoassay, fluorescence-labeled antibody assay, and ELISA, are widely used for determining SEA; however, these traditional immunological methods involve time-consuming procedures, hazardous labels, and expensive instruments [5,6]. Development of a sensoror chip-based immunoassay to provide a rapid, easy-to-use, and cost-effective method is needed. Recently, many studies using surface plasmon resonance (SPR)-based biosensors for immune responses have been reported [7]. SPR sensing has been demonstrated to be an exceedingly powerful and quantitative probe of

∗ Corresponding author. Tel.: +886 2 28610511x31821; fax: +886 2 28618266. E-mail address: [email protected] (W.-C. Tsai). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.10.061

interactions of a variety of biopolymers with various ligands. It provides a means for identifying these interactions and quantifying their equilibrium constants and kinetic constants [8–11], and they can be employed in very sensitive, label-free biochemical assays [12–16]. SPR sensors offer several advantages over other detection methods, with which they are commonly compared [17,18]. Two major advantages of SPR detection are that it requires no labeling of the analyte and binding can be monitored in real time. Several SPR-based devices have been proposed in the literature for determining staphylococcal enterotoxins. These include a wavelength interrogation sensor for determining SEB [19], an angle interrogation sensor for SEB [20], and an SPR sensor based on a sandwich assay for SEB [21] and SEA [22]. In our lab, we had developed a QCM-based immunosensor for determining staphylococcus enterotoxin B (SEB) [23]. The biosensing layer was fabricated on PEI-modified gold surface. The determination range for SEB of the method was between 2.5 and 60 ␮g/ml. In the present study, we aim to increase the detection sensitivity for staphylococcal enterotoxins by using an SPR-based immunosensor. As in most of the literature mentioned above, the biosensing layers were fabricated on commercially available prefunctionalized gold surface, such as carboxylated dextran. In this study, we will functionalize the gold surface using a self-assembled monolayer (SAM) technique. It was reported that self-assembled monolayers fabricated using a thiol mixture immobilize proteins better than does a homogeneous SAM [24–27]. This feature can be exploited to immobilize biomolecules in a manner which avoids steric hindrance between these molecules and their binding partners. To obtain an optimal surface for the immobilization of anti-SEA, various mixed SAMs prepared with carboxylic- and hydroxyl-terminated thiols were examined. The performances of the mixed SAM surfaces were evaluated in terms of effects of the degree of antibody immobilization

W.-C. Tsai, I.-C. Li / Sensors and Actuators B 136 (2009) 8–12

and the subsequent immune response sensitivity. The feasibility of detecting and quantifying SEA in a buffer, as well as in a morecomplex solution, i.e., milk, was demonstrated. The specificity of the immunosensor was also studied. 2. Experimental procedures 2.1. Materials SEA, SEB, affinity-purified rabbit anti-SEA immunoglobulin G (IgG), glycine, 1-ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC), and N-hydroxy-succinimide (NHS) were purchased from Sigma Chemical (St. Louis, MO, USA). 16-Mercaptohexadecanoic acid (16-MHA), and 6-mercapto-1-hexanol (6-MCH) were supplied by Aldrich (Milwaukee, WI, USA). Other chemicals used were of analytical grade. 2.2. Apparatus The SPR measurements were conducted using a single-channel AUTOLAB ESPR (Eco Chemie, Utrecht, The Netherlands). It works with a laser diode fixed at a wavelength of 670 nm, using a vibrating mirror to modulate the angle of incidence of the p-polarized light beam on the SPR substrate. The instrument is equipped with a cuvette. A gold sensor disk (17 mm in diameter) mounted on a hemi-cylindrical lens through index-matching oil forms the base of the cuvette. A syringe pump and a peristaltic pump perform all liquid handling. Each pump has its own defined task. The syringe pump is used for sample mixing in the cuvette and for sample dispensing. This experimental arrangement maintains a homogeneous solution and hydrodynamic conditions. The peristaltic pump is used for draining the cuvette, with the waste going into a waste flask. The SPR angle shift (millidegree change; ) was measured at a non-flow liquid condition, i.e., with the circulating pump paused. The degree of substance immobilization is given by the change in millidegrees. Generally, one millidegree is roughly equivalent to a change in concentration of about 1 pg/mm2 on the sensor surface. 2.3. Preparation of mixed SAMs on gold surfaces [25,26] Prior to use, bare gold disks were exposed to a freshly prepared piranha solution (a 3:1 mixture of H2 SO4 and H2 O2 ) for 5 min and then rinsed with absolute ethanol and deionized water. The cleaned bare gold disk was then mounted onto the SPR cuvette.

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Surface modification based on a SAM and carbodiimide chemistry was employed in the study. Gold-coated disks were modified by thiols with carboxyl (16-MHA) and hydroxyl (6-MCH) terminal groups. One millimolarity of mixed thiols consisting of different ratios of 16-MHA to 6-MCH and homogeneous 16-MHA were prepared in absolute ethanol. The mixed thiol solution at 100 ␮l was applied to a cleaned bare gold disk and mixed for 1 h. After 1 h of SAM deposition, the gold disks were thoroughly rinsed with ethanol and distilled water for 5 min to physically remove any unadsorbed thiols. The four different SAMs are hereby designated SAM1 (homogeneous16-MHA), SAM2 (1:3 ratio of 16-MHA to 6MCH), SAM3 (1:10 ratio of 16-MHA to 6-MCH), and SAM4 (1:20 ratio of 16-MHA to 6-MCH). These thiol-coated gold disks were used in the following SPR experiments. 2.4. Antibody immobilization and SEA measurement All experiments were carried out at 35 ◦ C unless otherwise stated. The terminal carboxylic groups of the mixed SAMs were activated with a freshly prepared 1:1 mixture of EDC (0.4 M) and NHS (0.1 M) in distilled water for 7 min. After activation, 1 mg/ml (unless otherwise stated) of anti-SEA was applied and allowed to react for 15 min. Following antibody immobilization, the surface was deactivated by 0.2 M glycine for 10 min to block remaining NHS-ester groups and minimize nonspecific effects. Subsequently, the SEA solution diluted to various concentrations in PBS buffer or milk was applied over the antibody-bound surface. After 1 h of reaction, PBS buffer was flushed over the surface to remove any loosely associated SEA. 3. Results and discussion 3.1. Typical sensorgram A typical sensorgram for the stepwise sensor fabrication and detection is shown in Fig. 1. The sensorgram exhibits the response of a thiol-coated SPR sensor to successive EDC/NHS activation, anti-SEA immobilization (1 mg/ml), glycine deactivation, and SEA binding (1 ␮g/ml). To eliminate the background interference, the SPR angle shift between each two neighboring baselines of the PBS buffer was calculated as the net response. A large SPR angle shift was observed with EDC/NHS activation which suggests a bulk refractive index change. Upon rinsing with PBS, the SPR angle shift nearly returned to the original baseline. The reaction with EDC/NHS allows

Fig. 1. SPR sensorgram of covalent immobilization of an anti-staphylococcal enterotoxin A (SEA) antibody on a mixed self-assembled monolayer (SAM) of thiols. (1) PBS baseline, (2) activation of the surface with EDC/NHS, (3) PBS wash, (4) binding of anti-SEA, (5) PBS wash, (6) deactivation of the surface using glycine, (7) PBS wash, (8) PBS baseline, (9) blank, (10) baseline, (11) binding of SEA, and (12) PBS wash.

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Table 1 Surface plasmon resonance (SPR) angle response of the antibody and antigen binding on different mixed thiol-treated surfaces. Mixed thiols (16-MHA: 6MCH)

Immobilized material

 (millidegrees)a

CV (%)

Molar binding ratiob

Anti-SEAc SEAd

267.7 ± 82 39 ± 8.2

30.6 21.0

0.80

Anti-SEA SEA

323.7 ± 100.1 47 ± 2.7

30.9 5.7

0.81

Anti-SEA SEA

314.3 ± 56.6 62.3 ± 19.1

18.0 30.7

1.10

Anti-SEA SEA

331.3 ± 66.4 85.7 ± 2.5

20.0 2.9

1.44

SAM1 (1:0)

SAM2 (1:3)

SAM3 (1:10)

SAM4 (1:20)

a b c d

Average values of three measurements. Refer to Su et al. [31]. Concentration of antibody (1 mg/ml). Concentration of antigen (1 ␮g/ml).

the formation of highly reactive O-acylisourea intermediates, which promote the formation of amide bonds between the carboxylic acids of 16-MHA and the amino groups of anti-SEA. An irreversible SPR response was observed during the subsequent anti-SEA injection. The binding of anti-SEA causes about a 313-millidegree angle shift, and confirmed successful immobilization of the antibody. A glycine solution was subsequently applied to block the remaining unreacted NHS-ester groups and minimize the non-specific binding of SEA. The glycine deactivation process was also observed to be a bulk refractive index change. After antibody immobilization and glycine blocking, the SEA solution was applied to the sensor surface. As shown in Fig. 1, the immunointeraction led to about a 91-millidegree angle shift. 3.2. Functionalization of the SPR sensor disks with various mixed SAMs The SAM technique represents a powerful and attractive strategy which provides well-defined and controlled structures of monomolecular interfaces of biological elements on a variety of substances. This technique was employed to functionalize the sensor chip in this study. In an SPR-based biosensor, the intensity of the response signal is related to the amount of immobilized ligand on the sensor surface. To obtain a high surface density of the ligand, it is important that the terminal groups of the SAMs are more accessible to the protein. In a mixed SAM format, the steric hindrance between the immobilized biomolecules and their binding partners is avoided. Therefore, to obtain an optimal surface for immobilization of the anti-SEA antibody, we examined four surfaces constructed by mixing different molar ratios of thiols. 16-MHA was used to anchor the antibodies (linkers), while 6-MCH was used to form a stable non-fouling background. The sensitivities of the immunoresponses of the four different SAMs were compared by measuring changes in the SPR angle shift caused by the interaction between the anti-SEA antibody (1 mg/m) and SEA (1 ␮g/ml). As Table 1 shows, mixed thiol surfaces (SAM2, SAM3, and SAM4) immobilized protein more effectively than did the homogeneous SAM1. This suggests that the mixed SAM, consisting of two thiols with different chain lengths, created a suitable environment for antibody immobilization. As far as the sensitivity for detecting SEA is concerned, anti-SEA immobilized on SAM4 exhibited the highest molar binding ratio. On the contrary, SAM1, consisting of the linker only, possessed the most severe steric hindrance and thus impeded effective SEA binding. Therefore, the lowest binding ratio was observed with SAM1.

Table 2 Surface plasmon resonance (SPR) angle response caused by anti-staphylococcal enterotoxin A (SEA) immobilization and SEA binding performed at 25 and 37 ◦ C. Temperature (◦ C)

Anti-SEAa immobilization  (millidegrees)

CV (%)

SEAb binding  (millidegrees)

CV (%)

25 35

324 ± 42.3 322.7 ± 33.9

13.1 10.5

16.3 ± 7.1 72.7 ± 8.1

43.6 11.1

a b

Concentration of anti-SEA = 1 mg/ml. Concentration of SEA = 1.0 ␮g/ml.

3.3. Optimization of anti-SEA immobilization and SEA detection Optimal conditions for anti-SEA immobilization were investigated by varying the immobilization temperature (Table 2) and anti-SEA concentration (Fig. 2). Initially, the assay was performed at room temperature (about 25 ◦ C) without temperature control. However a fluctuation in the baseline (data not shown) was observed. This could be ascribed to the fact that the refractive index is sensitive to temperature [28]. In addition, as shown in Table 2, the sensitivity of SEA detection was greater at 35 ◦ C than at 25 ◦ C. Therefore, all the following SPR measurements were performed at a temperature of 35 ◦ C, which was controlled by a water bath. The concentration-dependent anti-SEA immobilization signals and consequent SEA binding signals at 1 ␮g/ml are shown in Fig. 2. Within the examined anti-SEA concentration range (0.1–2.0 mg/ml), the immobilization signal initially sharply increased and then gradually leveled off. As far as the subsequent

Fig. 2. Concentration-dependent anti-staphylococcal enterotoxin A (SEA) binding signals and subsequent SEA binding signals at 1 ␮g/ml.

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Table 3 Cross-reactivity of the staphylococcal enterotoxin A (SEA) immunosensor to various concentrations of SEB. SEB concentration (␮g/ml)

 (millidegrees)

Cross-reactivity (%)

0.2 1.0

– 10 ± 3.6

– 11.8

tion limit; however this might make real-time monitoring more complicated. 3.5. Selectivity Because of the relatedness of SEA to other staphylococcal enterotoxins, the selectivity of the SEA immunosensor was examined. Since SEA and SEB are the most often occurring enterotoxins, interference from SEB was evaluated. Table 3 shows the cross-reactivity resulting from various concentrations of SEB. As Table 3 shows, at a low concentration of SEB (0.2 ␮g/ml), cross-reactivity was not detectable. At a high concentration (1.0 ␮g/ml), the interference was about 11.8%. This could be ascribed to the fact that there is 31% homology at the predicated amino acid level between SEA and SEB [4]. 3.6. Detection of SEA in milk samples

3.4. Calibration curve obtained in PBS buffer

The food processing industry is interested in detecting SEA in milk and food samples. These environments are much more complex than simple buffers. To investigate the performance of the SPR biosensor in a complex medium, milk was selected as a representative sample. Milk samples spiked with various concentrations of SEA standard solutions were directly used in the assay. The standard curve of SEA obtained from the milk samples is shown in Fig. 4. Compared to Fig. 3B, a clear matrix effect was observed. This was primarily due to the refractive index of the milk differing from that of the buffer and partly due to the non-specific adsorption of molecules from the milk sample onto the sensor surface. Within the explored SEA concentration range (0.1–1.0 ␮g/g), there was a linear relationship with a correlation coefficient of 0.98. The result showed a good working range comparable to the one obtained in buffer, and thus it is possible to measure the analyte directly in milk without dilution pretreatment. The recovery was evaluated by spiking blank milk samples with known amounts of SEA. Based on the calibration curve obtained in the milk samples, it was possible to calculate the recovery of the analyte. As shown in Table 4, the calculated recoveries varied

The sensorgram of the developed SPR sensor with various concentrations of SEA in PBS buffer is shown in Fig. 3A. When the SEA solution was applied to the sensor surface, a rapid increase in the signal occurred. This increase is associated with the specific binding of SEA to anti-SEA molecules on the sensor surface. Specific sensor responses were determined to be 20.3 ± 9.1, 27.3 ± 2.5, 40.0 ± 10.4, 64.3 ± 6.4, and 85.7 ± 2.5 millidegrees for SEA concentrations of 0.1, 0.2, 0.5, 0.75, and 1.0 ␮g/ml respectively. The SPR signals from Fig. 3A were used to construct the calibration curve for determining SEA (Fig. 3B). The sensor response appeared to be linear between 100 and 1000 ng/ml with a correlation coefficient of 0.98. These experimental results indicated that the SPR sensing device was capable of directly detecting SEA at a concentration of as low as 100 ng/ml. It was reported that a SPR biosensor based on a sandwich method could detect SEA with a sensitivity of 10–100 ng/ml depending on the material tested [22]. In other sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml [19] and 2.5 ng/ml [21] for SEB respectively. As the data showed, the sandwich method is advantageous for its lower detec-

Fig. 4. Calibration curve obtained with milk samples. Milk samples were spiked with various concentrations of staphylococcal enterotoxin A (SEA) and applied to the sensor surface.

Fig. 3. (A) An overlay plot of sensorgrams showing the sensitivity of detecting of staphylococcal enterotoxin A (SEA). Responses of SEA varying from 0.1 to 1.0 ␮g/ml against the immobilized antibody on self-assembled monolayer 4 (SAM4) with a 1:20 ratio of 16-MHA: 6MCH. (B) Calibration curve obtained with PBS buffer.

SEA binding sensitivity is concerned, the binding signals increased with concentration and reached a maximum at 1 mg/ml. Therefore, 1 mg/ml of anti-SEA was considered to be the optimal concentration for surface preparation and was used throughout the study.

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Table 4 Recovery of spiked staphylococcal enterotoxin A (SEA) in milk samples. SEA added (␮g/g)

 (millidegrees)

SEA detected (␮g/ml)

Recovery (%)

0.5 0.75 1.0

178.0 ± 24.3 224.7 ± 19.4 265.3 ± 45.2

0.36 0.63 0.87

72 84 87

[10]

[11]

[12]

from 72% to 87% over the concentration range examined. Although at low concentrations of SEA the recovery was not very satisfactory, the experiment demonstrated the feasibility of applying the immunosensor in a complex sample.

[13]

[14] [15]

4. Conclusions [16]

The effects of different mixed SAMs on the degree of anti-SEA antibody immobilization and the immunoresponse sensitivity were studied. It was found that the SAM with a molar ratio of 1:20 (16MHA: 6-MHA) exhibited the best sensitivity for SEA determination. This could be ascribed to a decrease in steric hindrance, and the concomitant enhanced detection sensitivity. The immunosensor exhibited a working range of 100–1000 ng/ml of SEA in both buffer and a milk sample. It was reported that in an outbreak of gastroenteritis in United States due to chocolate milk containing SEA, the total amount of SEA varied from 94 to 184 ng with the average being 144 ng [29]. In another outbreak of staphylococcal food poisoning occurred in Japan, the amount of SEA in powdered skim milk was approx. 3.7 ng/g [30]. As compared to the reported concentration level of contamination, the method proposed here seems to be not very sufficient. To increase the sensitivity further, we are working on the control over the orientation of the immobilized antibody molecules. In the meantime, it is suggested that for low contaminated samples, pretreatment such as concentration may be needed to make the proposed method applicable. The cross-reactivity of the assay with SEB was satisfactory. Recovery of SEA from spiked milk samples ranged from 72% to 87% with an average value of 81%. This suggested that the immunosensor fabricated in this study can be applied to detect SEA in complicated mixtures such as milk. In addition, the reported SPR sensor can provide a generic platform which can be tailored to detect various food-borne pathogens and agents for food analysis and testing. References [1] M.S. Bergdoll, The staphylococcal enterotoxins, in: R.I. Mateles, G.N. Wogan (Eds.), Biochemistry of Some Foodborne Microbial Toxins, MIT Press, Cambridge, 1967, pp. 1–25. [2] E. Antunes, E.A. Camargo, I.A. Desouza, C.F. Franco-Penteado, C.S.P. Lima, M.N. Muscara, G.D. Nucci, S.A. Teixeira, Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats, Toxicol. Appl. Pharmacol. 217 (2006) 107–113. [3] D.L. Archer, F.E. Young, Contemporary issues: diseases with a food vector, Clin. Microbiol. Rev. 1 (1988) 377–398. [4] N. Balaban, A. Rasooly, Staphylococcal enterotoxins, Int. J. Food Microbiol. 61 (2000) 1–10. [5] A.R. Bhatti, Y.M. Siddiqui, V.V. Micusan, Highly sensitive fluorogenic enzymelinked immunosorbent assay: detection of staphylococcal enterotoxin, B. J. Microbiol. Meth. 19 (1994) 179–187. [6] C.E. Park, M. Akhtar, M.K. Rayman, Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit (TECRA), Appl. Environ. Microbiol. 59 (1993) 2210–2213. [7] W.M. Mullett, E.P.C. Lai, J.M. Yeung, Surface plasmon resonance-base immunoassays, Methods 22 (2000) 77–91. [8] M.A. Cooper, Biosensor profiling of molecular interactions in pharmacology, Curr. Opin. Pharmacol. 3 (2003) 557–562. [9] A.A. Lathrop, Z.W. Jaradat, T. Haley, A.K. Bhunia, Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9

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Biographies Wen-Chi Tsai is an associate professor at the Chinese Culture University, Taiwan. She received her PhD in biotechnology from Imperial College of London in 1994. Her current research interests are QCM-based biosensors and SPR-based biosensors. Ie-Chin Li received her MSc in biotechnology from Chinese Culture University in 2007. She works for a biotechnology company currently and her interest is in immunosensors.