A two-dimensional (2D) spectral SPR sensor based on a polarization control scheme is reported in this paper. The polarization control configuration converts the phase difference between p- and s- polarization occurring at surface plasmon resonance (SPR) into corresponding color responses in spectral SPR images. A sensor resolution of 2.7 x 10−6 RIU has been demonstrated, which corresponds to more than one order of magnitude resolution improvement (26 times) comparing to existing 2D spectral SPR sensors. Multiplex array detection has also been demonstrated with the spectral SPR imaging sensor. In a 8 x 4 sensor array, 32 samples with different refractive index values were monitored simultaneously. Detection on bovine serum albumin (BSA) antigen-antibody binding further demonstrated the multiplex detection capability of the 2D spectral SPR sensor for bio-molecular interactions. The detection limit is found to be 21ng/ml, which is 36 times better than the detection limit previously reported by phase imaging SPR sensors. In light of the advantages of high sensitivity, 2D multiplex detection and real-time response, the spectral SPR imaging sensor can find promising applications in rapid, high throughput, non-labeling and multiplex detection of protein array for proteomics studies, biomarker screening, disease prognosis, and drug discovery.
©2011 Optical Society of America
Protein array provides the capability of multiplexed detection, which allows large amount of sample spots to be processed in single measurement. It has been widely used in proteomics , disease prognosis , drug discovery  and biomarker screening . The bound antigens on the antibody array are commonly detected by the direct labeling method , and dual antibody sandwich assay [5,6]. Either labeling tags (such as, fluorescence , chemiluminescence  and radioactive isotopes ) or secondary antibodies are required in the bio-assays that can interfere with the original antibody-antigen interactions. In addition, Enzyme-linked immunosorbent assay (ELISA) also requires different second antibody-antigen interaction for each sample in the array.
Surface plasmon resonance imaging (SPRI) is a non-labeling sensing technique for the direct detection of antibody-antigen interaction in an array format [10,11]. It can directly detect the bound protein on the antibody array through the refractive index change occurring on the gold sensor surface . SPR imaging also provides quantitative and kinetic binding information . Common SPR imaging detection technique is based on the intensity interrogation [13,14]. It measures the reflectivity change caused by the refractive index changes on the sensing surface. The major limitation of intensity SPR imaging is the limited sensor resolution  and it can only provide a resolution in 10−5 refractive index unit (RIU).
In recent years, two dimensional (2D) spectral SPR sensors based on the wavelength interrogation have been intensively studied by Jong et al. [15–21] and also Wong and Ho et al. [22–24]. Jong et al. has further applied the imaging techniques for the detection of C-reactive protein  and blood protein . However, their spectral imaging sensors [15,16] mainly rely on the SPR absorption dip determination in the spectrum and scanning throughout the entire sample plane for the reconstruction of the spectral SPR image is required (scanning time of a 2mm diameter spot is approximately 180s). Real-time monitoring of bio-molecular interaction is therefore restricted. In addition, it can only provide a resolution in 7 x 10−5 RIU, which is similar to that of conventional intensity SPR imaging sensors .
In this paper, we present a spectral SPR imaging sensor based on a polarization control scheme. A SPR prism coupler is placed in between two polarizers. The angle between the transmission axes of the two polarizers is 90 degrees and therefore the incident beam cannot be transmitted through the cross polarizers. At (or close to) the excitation wavelength of the surface plasmon wave, a phase difference is introduced between the p- and s- polarization component of the light. The orientation angle of the ellipse is shifted correspondingly and the rotation of the ellipse allows the light interacting with the surface plasmon to be transmitted through the cross polarizers. Since the surface plasmon excitation condition is only matched with particular region of wavelength, particular spectral profile is produced and corresponding color change is produced in the spectral SPR image. A sensor resolution of 2.7 x 10−6 RIU is demonstrated with the spectral SPR imaging sensor and this provides more than one order of magnitude resolution improvement comparing to the existing 2D spectral SPR imaging sensors [15–21] and the most widely used intensity SPR imaging sensors . In addition, entire sensing surface can be imaged in one single spectral SPR image and no time-consuming scanning [15–21] is required. The high throughput array detection ability of the imaging sensor has also been demonstrated in this paper and a 4 x 8 (32 elements) array of refractive index samples are simultaneously monitored with the 2D sensing feature of the imaging sensor. The spectral SPR imaging sensor combines the advantages of real-time response, high sensitivity and 2D multiplex sensing. Thus the sensor can find promising application in multiplex protein array detection for rapid, high throughput and non-labeling detection in proteomics studies, biomarker screening, disease prognosis and drug discovery [1–4].
2. Theory and experimental set-up
2.1 Surface plasmon resonance
At surface plasmon resonance, the energy of the incident photons is transformed into surface plasmons and the resonance condition is described by the equations ,
Since refractive index equals the square root of (i.e. ), the refractive index change in the dielectric medium alters the characteristic of surface plasmon wave and SPR resonance condition.
2.2 Phase difference between p- and s- polarization
S. G. Nelson et al.  reported that a rapid phase change is produced to the p- polarization component during the surface plasmon excitation. However, the s- polarization component contains no resonant feature, because the oscillation direction is perpendicular to the excitation plane of the surface plasmon wave [12,13].27,29,30],
Refractive index variations in the dielectric medium alter the value of, (). In p- polarization, the refractive index change alters the Fresnel coefficient rp 23 and therefore the complex reflection coefficient rp is modified. According to Eq. (5), a corresponding phase response is produced. However, no phase shift is produced to the s- polarization, because the oscillation direction is perpendicular to the excitation plane of the surface plasmon wave [12,13,27]. A phase difference is therefore produced between the p- and s- polarizations ,
2.3 Elliptical Polarization
Considering an elliptically polarized light and the two orthogonal optical disturbances (p- and s- components) can be represented as ,
The elliptically polarized lightcan be further described by the following equation of ellipse 31] such thatEq. (11), a phase difference between the p- and s- polarizations is produced at surface plasmon excitation,Eq. (16) and Eq. (17), the equation of the elliptically polarized lightand the angle with the (,) coordinate system are also modified.Fig. 1(a) , the SPR prism coupler is placed in between two polarizers. The angle between the transmission axes of these two polarizers is 90 degree. Thus the broad band incident beam can’t be transmitted through the cross polarizers. At (or close to) the excitation wavelength of the surface plasmon wave, a phase difference is introduced between the p- and s- polarization component of the light. As described by Eq. (19), the orientation angle of the ellipse is shifted correspondingly (as shown in Fig. 1(b)) and the rotation of the ellipse allows the light interacting with the surface plasmon to be transmitted through the cross polarizers. At wavelength out of the excitation spectral range, the interaction with the surface plasmon wave is weak and the ellipse is almost parallel to the original orientation angle of the incident beam, therefore no significant transmitting light is observed in the wavelength region longer or shorter than the excitation wavelength. The modeling work reported by Homola et al.  shows that different spectral responses are produced for different phase differences . . between the p- and s- polarization.
In this paper, the spectral SPR imaging relies on the color change caused by the spectral response variation, which corresponds to the differential phase change between p- and s- polarization. Figure 1(a) shows the experimental set-up of the sensor system. Halogen illuminator is used as a broad band light source. The beam is collimated and expanded with a 10x objective lens and a bi-concave lens. After passing through the first polarizer (input polarizer), the polarized beam enters a gold sensing layer coated on glass prism. A PDMS based micro-fluidic flow cell is attached on the sensor surface for feeding in samples with different refractive index values. Then, the beam passes through a quarter wave plate and the second polarizer (output polarizer). The angle of rotation of the input and output polarizer is chosen to be perpendicular to each other. Finally, the resultant SPR images are captured by a color CCD camera and they are analyzed using our internally developed software.
3. Results and discussions
To estimate the performance of the spectral SPR imaging sensor, measurements on different concentrations of salt solution samples ranged from 0%, 1%, 2%, 3%, 4% and 7%, which corresponding to refractive index values from 1.3333 – 1.3454 RIU  have been performed.
Figure 2 shows the Spectral SPR images for different concentrations of salt solutions. As shown in Fig. 2(a), the Spectral SPR image of water sample (0%) is major in deep red color. When the concentration of salt solution increases from 0% to 7%, the SPR image changes from deep red to green in color (Fig. 2(f)). The color changes in SPR images refer to the spectral characteristics changes caused by the different differential phase shifts (between p- and s- polarization), which is produced by the variation of surface plasmon resonance conditions for different refractive index dielectric samples.
In addition, a portable spectrometer (Ocean Optics USB2000) was used to show the spectral characteristic profiles for different concentrations salt solutions. The spectrums of different salt solution samples are shown in Fig. 3 . For water (0%) sensing, the spectrum shows that the signal in the red color spectral band (after 600nm) is much higher than that in the green spectral band, therefore the resultant SPR image color is dominant red in color (as shown in Fig. 2(a)). The results also show that the signal from the red spectral band decrease for increasing concentration of salt solutions, while the signals from the green spectral band remains unchanged. These results correlate well with the experimental finding obtained in Fig. 2 that the SPR images change from red to green in color for increasing concentration of salt solutions.
In the analysis of the spectral SPR images, we treat all colors as linear combinations of three primary colors, namely red (R), green (G) and blue (B). An established RGB imaging model has already been defined by the CIE (Commission International de I’Eclarage) to contain three standard primary components (R, G and B refer to monochromatic spectral energies at wavelengths 700nm, 546.1nm and 435.8nm respectively) so that the color of a pixel can be represented by a linear combination of these primary components . Besides, several other color models have also emerged to provide equivalent representation for different engineering applications. The XYZ, CMYK, YIQ and HSV models are most commonly used color coding systems [34,35]. Nonetheless, only the Hue (H) component in HSV coding directly refers to the dominant wavelength of a color [34,35]. In the present case, since the spectral SPR images are produced by the wavelength dependence spectral characteristic variations, the use of Hue value for analyzing the imaging is a more appropriate approach.
In our previous works [22–24], the quantification of the color changes in spectral SPR images with the HSV color coding system  has been demonstrated. The HSV color space model was first introduced by Smith in 1978  and it was originally developed for the digital control of color television monitors. There are three dimensions in the HSV color space - Hue (H), saturation (S) and value (V). The Hue (H) component in HSV coding directly refers to the dominant wavelength of a color [34,35]. In addition, Smith also introduced a RGB to HSV algorithm  to transform colors from RGB colorcube model to HSV hexcone model (the detail algorithm is given in the appendix).
To quantify the color changes in the spectral SPR images, the Hue (H) profiles of the spectral SPR images shown in Fig. 2(a)–2(f) are extracted and plotted against the refractive index changes. It gives the response curve of the spectral SPR imaging sensor. Figure 4 shows the sensor response for different concentration salt solutions ranged from 0%-7% and it can be seen that the sensor response (Hue values) increases with increasing refractive index values (RIU).
The error bar in Fig. 4 is obtained from the standard deviation (S.D.) between five averaged measurement data. The S.D. values for different concentration samples are listed in Table 1 . The overall measurement S.D. of the system is calculated from the average S.D. values among 0%-7% measurement S.D. and this value is used as the Measurement S.D. in the calculation of the sensor resolution.As reported in [13,37], sensor resolution can be calculated by the relationship shown in Eq. (20) and the sensor resolution of the spectral SPR imaging sensor is found to be 2.7 x 10−6 RIU. It provides one order of magnitude resolution improvement (26 times) over the existing 2D spectral SPR sensors [15–21].15–21]. However, color CCD camera is used in our sensor configuration for the real-time capturing of spectral SPR images. The real-time 2D sensing feature not only enables the detection of fast bio-molecular interactions, but also allows high-throughput detection of a range of reaction sites in an array format.
The 2D sensing feature of the spectral SPR imaging sensor has been demonstrated in the results shown in Fig. 5 . A 8 x 4 array of salt solution spots were spotted in the gold sensing surface and a color CCD camera was used to capture the responses from all the 32 elements in one single spectral SPR image. Figure 5(a) shows the spectral SPR image taken for three different concentrations (0%, 2% and 7%) of salt solution spots. Different color responses have been demonstrated for different concentration solutions. Deep red, deep green and green color responses are shown for 0%, 2% and 7% salt solution spots respectively. The spectral response in the SPR image was further quantified through Hue (H) profile extraction and it is shown in Fig. 5(b). The responses of 0%, 2% and 7% salt solution spots are 25.7, 69.1 and 83.4 (hue unit) (average value from all sensing sites).
Multiplex detection of antigen-antibody binding interactions has been carried with the 2D spectral SPR sensor. Bovine serum albumin (BSA) antigen and corresponding antibody (anti-BSA) were used as the testing sample in the experiment. As shown in Fig. 6(a) , four sensor sites of the protein array were immobilized with BSA antigen (1mg/ml). In addition, a non-specific protein sample (glucose oxidase, 1mg/ml) was immobilized in another four sensor sites and they served as the negative control of the experiment (Fig. 6(a)).
Initially, the protein array was kept at phosphate buffered saline (PBS) buffer solution. Then, anti-BSA (antibody, 0.01mg/ml) was injected to the protein array surface. The subsequent antigen-antibody binding interactions at all sensor sites were imaged by the 2D spectral SPR sensor in parallel. Figure 6(b) shows the spectral SPR image of the protein array, when it was kept at the buffer solution. As shown in the image, red color responses were observed in both specific and non-specific sensor sites. Figure 6(c) further shows the SPR image captured at 41 minutes after the injection of BSA antibody. As shown in the result, the spectral responses of all the specific sites (BSA antigen) were changed from red to green color, which were corresponding to the binding interactions between the BSA antigen and BSA antibody. However, no significant change of spectral response was observed at the non-specific sites (glucose oxidase).
In order to quantify the spectral response changes in the SPR image, Hue extraction has been performed in every sensor site. The average Hue value of the BSA antigen sites is 13.96 ± 0.11 (Hue unit) when the protein array was kept at buffer solution and the Hue value increases to 66.24 ± 0.21 (Hue unit) after the injection of BSA antibody. The injection of 0.01 mg/ml BSA antibody therefore results in a sensor response of 52.28 (Hue unit). In addition, the standard deviation between 10 measurements is 0.11 (Hue unit), when the BSA antigen sites were placed in buffer solution. It gives the measurement stability of the system. Finally, the detection limit of the 2D spectral SPR sensor is calculated with Eq. (21). It is found to be 21ng/ml.38] and Wong et al. (7.7 × 10−4 mg/ml)  with phase imaging SPR sensors, the 2D spectral SPR sensor has demonstrated 36 times improvement in detection limit. The experimental results also demonstrate the capability of the 2D spectral SPR sensor for multiplex bio-molecular binding interactions imaging.
A two-dimensional (2D) spectral SPR sensor based on a polarization control scheme has been demonstrated. The polarization control configuration converts the phase shift between p- and s- polarization occurring at surface plasmon resonance (SPR) into corresponding color responses in spectral SPR images. A sensor resolution of 2.7 x 10−6 RIU has been demonstrated, which corresponds to one order of magnitude resolution improvement (26 times) comparing to the existing 2D spectral SPR imaging sensors [15–21]. The multiplex array detection ability of the imaging sensor has further been demonstrated. In a 4 x 8 sensor array, 32 samples with different refractive index values were simultaneously monitored with the 2D spectral SPR sensor. Detection on bovine serum albumin (BSA) antigen-antibody binding further demonstrated the multiplex detection capability of the 2D spectral SPR sensor for bio-molecular interactions. The detection limit is found to be 21ng/ml, which is 36 times better than the detection limit previously reported by phase imaging SPR sensors [11,38]. The polarization control scheme also provides a simplified way for probing the phase difference between p- and s- polarization, which offers real time sensor response and the information on the entire 2D sensor surface is provided (i.e. pixel to pixel conversion), because no information in the time domain and spatial domain is scarified in the process of phase modulation and phase extraction [10,39–41]. In light of the advantages of real-time response, high sensitivity and multiplex sensing feature of the spectral SPR imaging sensor, it can find promising applications in rapid, high throughput and non-labeling detection in protein array detection for proteomics studies, biomarker screening, disease prognosis, and drug discovery.
Appendix: RGB to HSV algorithm 
The equivalent H, S and V are on the range [0, 1] and r, g and b values (corresponding to the R, G and B values respectively) are on the range [0, 1],
- 1) Let V = max (r, g, b); (i.e. choosing the maximum component - either r, g or b)
- 2) Let X = min (r, g, b); (i.e. choosing the minimum component)
- 3) Let S = (V - X) / V,
- 4) Let k = (V – r) / (V - X);
m = (V – g) / (V - X);
n = (V – b) / (V - X);
- 5) Conditions
If r = V and g = X, h = (5 + n);
If r = V and g ≠ X, h = (1 – m);
If g = V and b = X, h = (1 + k);
If g = V and b ≠ X, h = (3 – n);
If g ≠ V and r = X, h = (3 + m);
If g ≠ V and r ≠ X, h = (5 – k);
- 6) hue = h / 6
This project is supported by Singapore Bio-Imaging Consortium under grant RP C-015 /2007. The author (Chi Lok Wong) would like to give thanks to the grace of Jesus Christ throughout every experiment. The prayer from his mother, Vanessa Ho, is also acknowledged.
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