Enhancing performance of a miniaturized surface plasmon resonance sensor in the reflectance detection mode using a waveguide-coupled bimetallic chip
© Lee et al.; licensee Springer. 2013
Received: 13 May 2013
Accepted: 26 July 2013
Published: 6 August 2013
The characteristics of a waveguide-coupled bimetallic (WcBiM) chip in a miniaturized surface plasmon resonance (SPR) sensor and its detection capability for a low molecular weight biomolecule were investigated. The configuration of the WcBiM chip was gold (Au)/waveguide (ZnS-SiO2)/silver (Ag). In the intensity measurement mode, the sensitivity could be improved by reducing the full width at half maximum (FWHM) of the reflectance curve. The FWHM of the WcBiM chip is narrower than that of the Au chip, which suggests that the slope of the reflectance curve for the WcBiM chip is steeper. In order to generate enhanced resolution, the reflectance should be monitored at the specific angle where the slope is the steepest in the reflectance curve. For the detection of biotin that is a low molecular weight biomolecule, streptavidin was formed on the SPR sensor chip surface. The response of the SPR to biotin at various concentrations was then acquired. The sensitivities of the WcBiM chip and the Au chip were 0.0052%/(ng/ml) and 0.0021%/(ng/ml), respectively. The limit of detection of the biotin concentration for both the WcBiM and Au chips was calculated. The values were 2.87 ng/ml for the WcBiM chip and 16.63 ng/ml for the Au chip. Enhancement of the sensitivity in the intensity detection mode was achieved using the WcBiM chip compared with the Au chip. Therefore, sufficient sensitivity for the detection of a disease-related biomarker is attainable with the WcBiM chip in the intensity measurement mode using a miniaturized SPR sensor.
With continuous research and advancement over the last several decades, a surface plasmon resonance (SPR) sensor has been developed as a promising technology for biomolecular interaction analysis (e.g., antigen-antibody reaction, DNA) due to its merits of real-time monitoring and higher sensitivity compared with any other sensor system [1–3]. In addition, an SPR sensor does not require any chemical procedures such as fluorescence. Thus, this sensor has been studied for the detection of disease-related biomarkers, which requires immediate detection and simple operation [4, 5]. The SPR sensor is based on variations in permittivity, such as the refractive index on a metal surface, and is very sensitive to subtle changes. When a small amount of the target analyte binds with the bioreceptors immobilized on the metal surface, the reflectance curve, acquired by monitoring the reflected light intensity on changing the incident angle of the light source, shifts depending on the changed refractive index of the bound target biomolecule. Based on these principles, various diseases can be diagnosed by detecting disease-related biomarkers [6, 7].
The SPR-based sensor relies on the extraordinary optical properties of noble metals such as gold (Au), silver (Ag), aluminum (Al), and copper (Cu) . Among these metals, Au has been commonly used as an SPR sensor chip since it has merits of great stability, durability, and outstanding biocompatibility [8–10]. Although a single Au layer leads to stable performance, the commercialized Au-based sensor chip has a sensitivity limitation when it comes to the detection of biomolecules with very low molecular weight or trace level concentration . The detection ability of biomolecules at trace level concentration or very low molecular weight plays an important role in the instrument for the early diagnosis of diseases. The SPR sensor utilizes the evanescent field, which measures changes in the refractive index in proximity to the metal surface . Compared to Au, Ag enhanced an evanescent field better, resulting in a sharper SPR reflectance curve [13, 14]. However, Ag is easily oxidized when exposed to an air or liquid environment due to its high oxygen affinity [13, 15]. As a remedy for the shortcomings of the Au and Ag sensor chips, the Ag-Au bimetallic SPR chip has been proposed to exploit their advantages [9, 16]. Commonly, the thin Au film is coated over the surface of the Ag film due to the chemical stability of the Au metal . In addition, the waveguide layer has been adopted to obtain a sharper reflectance curve and moderate decay length . As materials for the waveguide layer, Si3N4, SiO2, and ZnO  have been extensively studied.
There are four different SPR detection modes, which indicate angular interrogation, intensity measurement, phase interrogation, and wavelength measurement [19, 21]. Among these approaches, the angular and intensity detection schemes have been widely used as the SPR measurement mode. Angular interrogation  detects the SPR angle change by monitoring the SPR reflectance dip shift. This offers highly sensitive performance by measuring extremely small angle changes of the SPR using the Au chip with a broad SPR reflectance curve. The intensity measurement  monitors the intensity of the reflected light at a fixed angle where the maximum slope of the SPR reflectance curve is located. This method is very effective in the case of an SPR reflectance curve with a narrower full width at half maximum (FWHM), leading to great reflectance variation at this fixed angle [24, 25].
In the present work, we experimentally investigated the characteristics of a waveguide-coupled bimetallic (WcBiM) chip in the intensity measurement mode using the miniaturized SPR sensor system, and extended the study to the system sensitivity for the detection of biotin with very low molecular weight (MW 341.38) at a low concentration level. The noble metal materials applied to the WcBiM chip were Ag as the inner metal layer and Au as the outer metal layer. Moreover, ZnS-SiO2 was used as a waveguide layer due to the high force of adhesion between the two metals. It is easy and robust to integrate this waveguide layer with electrical and optical systems . The characteristics of the WcBiM chip in the intensity measurement were investigated by evaluating the FWHM and slope of the SPR reflectance curve. The comparison analysis of streptavidin-biotin interaction was carried out using a miniaturized SPR sensor in the intensity measurement with both the WcBiM and Au chips.
Surface plasmon resonance sensor system
Waveguide-coupled bimetallic chip
The configuration of the WcBiM SPR chip is shown in Figure 1b. This was prepared by the deposition of gold (Au), waveguide (ZnS-SiO2), and silver (Ag) onto the glass substrate using an RF magnetron. The thickness of each layer was Au (31 nm)/ZnS-SiO2 (190 nm)/Ag (25 nm), which was optimized using a commercial optical thin film software (SCI Film Wizard™, Carlsbad, CA, USA). ZnS-SiO2 was adopted as a waveguide because it exhibits a good adhesion property between Ag and Au. For verification of the performance of the WcBiM chip, it was compared with the commercialized Au chip (K-MAC, Daejeon, Korea). The Au chip consists of Au (50 nm)/Cr (2 nm) on a glass substrate. Experimental setup is represented in Figure 1c, and both WcBiM and Au chips are shown in the inset of Figure 1c.
Materials and detection of biotin
Streptavidin (Sigma-Aldrich, St. Louis, MO, USA) was immobilized on the sensor chip modified by a self-assembled monolayer (SAM; K-MAC, Daejeon, Korea) containing N-hydroxysuccinimide and ethyl(dimethylaminopropyl) carbodiimide so that the amine group would react easily. The WcBiM SPR chip was dipped in 1 mM SAM solution in ethanol (2.5 ml) overnight. The streptavidin molecules were covalently immobilized onto the sensor chip by injection of the streptavidin solution into the sensor system. Next, the biotin (Sigma-Aldrich, St. Louis, MO, USA) was made to flow into the SPR sensor system in order of concentration at 50, 100, 150, and 200 ng/ml. All proteins were diluted in the phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO, USA) solution.
Results and discussion
SPR parameters for WcBiM configurations when the refractive index is changed from 1.335 to 1.35
Reflectance at n= 1.335
Reflectance at n= 1.35
(Rn= 1.35 − Rn= 1.335)
(Δ R/Δ θ)
Au(31 nm)/WG/Ag(25 nm)
Au(25 nm)/WG/Ag(25 nm)
Au(31 nm)/WG/Ag(20 nm)
Au(31 nm)/WG/Ag(35 nm)
Au(35 nm)/WG/Ag(25 nm)
The limit of detection (LOD) of this SPR sensor system was obtained using Equation 1 . The standard deviation (SD) of the signal was recorded over 100 s in the stable state. The SDs of the blank measures for the WcBiM chip and the Au chip were 0.0026% and 0.0046%, respectively. Moreover, the SPR responses of 50 ng/ml biotin for both sensor chips were 0.1360% and 0.0415%, respectively. Therefore, the LOD of the concentration (concentrationLOD) was calculated from Equation 1; the respective values were 2.87 ng/ml for the WcBiM chip and 16.63 ng/ml for the Au chip. Thus, the WcBiM chip can detect biomolecules at a very low level of concentration. From these results, if the SPR reflectance curve has a narrower FWHM and the detection mode is based on the intensity measurement, it is expected that the sensitivity of the sensor system can be enhanced compared with the conventional device. In particular, for the early diagnosis of diseases through the detection of a disease-related biomarker with very low molecular weight or trace level concentration, the SPR sensor in the reflectance detection mode using the WcBiM chip will be very useful tool for medical applications.
The performance of a simplified SPR sensor with a WcBiM chip was investigated. Since the SPR sensor was simple and miniaturized, the incident angle of the beam was fixed. Thus, the reflectance curves for the designated incident angle were obtained by successive numerical fitting of the intensity profiles from 2D-CMOS for both WcBiM and Au chips. The FWHM of the Au chip was about twice as large as that of the WcBiM chip, which implied that the slope of the WcBiM reflectance curve was steeper. In order to achieve better performance, the reflectance was monitored at the specific pixel of the 2D-CMOS corresponding to the angle where the slope is the steepest in the reflectance curve. The slope was obtained by differentiating the reflectance curve with respect to the incident angle. The steepest slopes for the WcBiM chip and the Au chip were −237.52%/° and −115.92%/°, respectively. The WcBiM chip's slope was about twice as steep as that of the Au chip. For the detection of a disease-related biomarker, it is necessary for biomolecules with very low molecular weight such as biotin to be detected. For the verification of the detection capability of the WcBiM chip, an experiment of the streptavidin-biotin interaction was carried out. Streptavidin at a concentration of 50 μg/ml formed on the SPR sensor chip surface, and the response of the SPR to the biotin with various concentrations of 50, 100, 150, and 200 ng/ml was acquired in triplicate. The sensitivities of the WcBiM chip and the Au chip were 0.0052%/(ng/ml) and 0.0021%/(ng/ml), respectively. In addition, the concentrationLOD of this SPR sensor system was calculated. The results were 2.87 ng/ml for the WcBiM chip and 16.63 ng/ml for the Au chip. Thus, for the detection of a disease-related biomarker, an SPR sensor in the reflectance detection mode using the WcBiM chip would be very useful in the medical field.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2010028).
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