1

Surface Plasmon Resonance Imaging Sensors: A Review

Chi Lok Wong1,*

and Malini Olivo1, 2, 3, *

1 Bio-optical Imaging Group, Singapore Bioimaging Consortium, Helios #01-02,

11 Biopolis Way, Singapore 138667. 2 School of Physics, National University of Ireland, Galway, Co. Galway, Ireland.

3 Department of Pharmacy, National University of Singapore, Singapore.

* Corresponding authors

E-mail (Dr. Chi Lok Wong) - dave_wong@sbic.a-star.edu.sg, Tel - (65) 6478 8752, Fax - (65) 6478 9957

E-mail (Prof. Malini Olivo) - malini_olivo@sbic.a-star.edu.sg, Tel – (65) 6478 8752, Fax - (65) 6478 9957

Keywords: Surface Plasmon Resonance Imaging, Review, SPR, non-labeling detection, phase SPR imaging,

spectral SPR imaging.

Abstract

Surface plasmon resonance (SPR) imaging sensors realize label free, real-time, highly sensitive, quantitative, high

throughput biological interaction monitoring and the binding profiles from multi-analytes further provide the

binding kinetic parameters between different bio-molecules. In the past two decades, SPR imaging sensors found

rapid increasing applications in fundamental biological studies, medical diagnostics, drug discovery, food safety,

precision measurement and environmental monitoring. In this paper, we review the recent advances of SPR imaging

sensor technology towards high throughput multi-analyte screening. Finally, we describe our multiplex colorimetric

SPR imaging biosensor based on polarization orientation for high throughput bio-sensing applications.

1. Introduction

Surface-plasmon microscopy was invented by Rothenhäuslar and Knoll in 1988 [1]. Plasmon surface polariton field

was used to image microscopic interfacial structure. Since then, SPR imaging has found rapid increasing research

interests [2-5] and wide applications in drug discovery [6], biomarker screening [7], nucleic acid detection [5], food

safety [8, 9] and environmental monitoring [10] in the past two decades. This paper reviews the recent advances in

SPR imaging sensor technology.

Excitation of surface plasmon

Surface plasmon was observed in 1902 by Wood [11] with the photon excited electrical resonance at small metallic

particles. Plasmonic has gained wide research interests, including surface plasmon enhanced raman scattering

(SERS) [12-18], localized surface plasmon resonance (LSPR) [19], surface plasmon field-enhanced fluorescence

spectroscopy (SPFS) [20], plasmon enhanced near-field scanning optical microscopy (plasmonic NSOM) [21] and

surface plasmon resonance (SPR) [1-5]. In 1968, Otto [22] reported the attenuated total reflection (ATR) coupling

for the excitation of surface plasmon. In the same year, Kretschmann and Raether [23] presented the Kretschmann

configuration ATR coupling, which is the most widely used excitation method in current SPR imaging sensors [1-5].

In ATR coupling, the excitation light passes through a high density medium (glass prism) and it modifies the phase

velocity and wave vector of the excitation light (kin) [24, 25].

pink εθλ

πsin

2⋅= (1)

2

where λ is the wavelength of the excitation light, θ is the incident angle and pε is the dielectric constant of the

prism material. The wave vector of the surface plasmon (ksp) propagating at the metal-dielectric interface is

described by the following equation [24, 25],

sm

smsp

spc

kεε

εεω

+= (2)

where spω is the angular frequency of the surface plasmon, mε is the dielectric constant of the metal film and sε is

the dielectric constant of the dielectric medium.

The surface plasmon resonance (SPR) phenomenon occurs when the energy of the excitation light (kin) matches that

of the surface plasmon wave (ksp). At the resonance condition, part of the energy of the excitation light is transferred

to the energy of surface plasmon. It produces an absorption profile in the reflection spectrum [3, 4, 25], which is

known as the SPR absorption curve.

Working principle of SPR imaging

Intensity SPR imaging

SPR imaging sensors based on intensity [1, 26-31], angular [32, 33], wavelength [34-39], phase [40-45] and

polarization [46-48] interrogations have been reported to date. Intensity SPR imaging has been widely used in

different applications [4, 26-31] and commercialized by GWC Technologies [46]. The operation principle is

explained in Fig. 1a. Monochromatic light is used as the excitation source and the incident angle θ is fixed at the

resonance angle. Due to the energy transfer at the resonance condition, the intensity of the reflection light is

attenuated and it produces an absorption profile in the reflection spectrum. According to Eq (1) and (2), the

refractive index change of the dielectric medium varies the SPR absorption minimum wavelength, which increases

the intensity of the reflection light (Fig. 1a). Intensity SPR imaging therefore provides a two-dimensional (2D)

intensity contrast image of the refractive index distribution of the sensing surface [1, 26-31]. Figure 1b shows a

typical intensity SPR image reported by Corn et al. [108].

Spectral SPR imaging

According to Eq (1), the wave vector of the excitation beam is wavelength dependent and it matches with the

propagation constant of the surface plasmon wave at a particular wavelength. In spectral SPR imaging, a

polychromatic excitation source is used. The SPR absorption dip λdip shifts (Fig. 1a) according to the refractive

index change of the dielectric medium and it produces a corresponding color variation in the resultant image. Fig. 1c

shows a spectral SPR image reported by the authors [39] for an array of refractive index samples.

Phase and polarization contrast based SPR imaging

In 1996, S.G. Nelson et al. [47] introduced phase detection for SPR sensing and a steep slope of phase response over

a narrow range of refractive index was reported, which provides three times higher sensor resolution compared to

angular and spectral SPR sensors [47]. As described by the Fresnel model [48], the reflection coefficients (r) of p-

and s- polarizations can be expressed as,

pi

pp errϕ

||= and si

ss errϕ

||= (3)

where φp and φs are the phase of the p- and s- polarization respectively.

3

At surface plasmon resonance, the phase (φp) of the incident light is changed due to the energy transfer between

light and surface plasmon [3, 47]. Fig. 1d shows a typical phase imaging in phase SPR imaging. However, the

oscillation direction of the s- polarization light is perpendicular to the excitation plane of the surface plasmon wave

and it is not affected at surface plasmon excitation. A phase difference (∆φ) is therefore produced between the p-

and s- polarization light. It also causes an orientation to the polarization ellipse of the SPR image, which becomes

the operation principle of the polarization modulated SPR imaging sensors [43-45]. An SPR polarization

contrast image is shown in Fig. 1e.

2. Intensity SPR imaging sensors

Intensity SPR imaging was first demonstrated by E. Yeatman and Ash [49] and Rothenhäuslar and Knoll [1] in late

80’s. In the past two decades, intensive research work on SPR imaging has been conducted by Corn et al. [26-29,

50-62]. In 1997, Jordan and Corn [26] used monochromatic He-Ne laser as excitation source where the expanded

beam was made incident on the gold sensing surface at the resonance angle and the SPR image was subsequently

captured by a monochromatic CCD camera. The system was used to characterize the differential electrostatic

adsorption of proteins and synthetic polypeptides onto photopatterned monolayers of a gold surface. Corn et al. also

applied the system for oligonucleotide arrays [50] and DNA hybridization detection [27, 50]. Later, Nelson and

Corn et al. [28] improved the performance of intensity SPR imaging by utilizing near infrared (NIR) excitation

wavelength. High contrast SPR images have been shown in the NIR wavelength range (800-1152nm). The

utilization of incoherent white source and narrow band-pass filter also eliminates the laser fringes that have been

observed in conventional SPR imaging set-ups [26, 27, 50]. The NIR SPR imaging sensor was further used for

quantitative detection of the hybridization adsorption of RNA and DNA oligonucleotides and the detection limit was

found to be 10nM [29]. In the same year, the system was integrated with a poly(dimethylsiloxane) (PDMS) based

microfluidic channels for the fabrication and detection of 1-D and 2-D DNA hybridization arrays [51]. The NIR

SPR imaging set-up has further been demonstrated for epitope-antibody [52], protein-carbohydrate [53], protein-

protein [54], protein-DNA [54], protein-aptamer [55] interactions detection and protein biomarker screening [56].

Following this, Wark and Corn et al. [56] presented long range surface plasmon (LRSP) imaging. By using a

symmetric dielectric arrangement around the gold thin film, LRSPs possess longer surface propagation length,

higher electric field strength and sharper angular resonance curve than conventional surface plasmon. About 20%

response enhancement has been shown experimentally in DNA hybridization adsorption detection. In recent years,

they enhanced the detection limit of SPR imaging by using metallic nano-particles [57-60]. Gold nano-particles

were adsorbed onto gold diffraction grating at the sensing surface [59, 60] and this detection approach has been

found to improve the sensor sensitivity. Detection of DNA at concentration of 10fM [59] and microRNA [60] have

been demonstrated. Silica-coated gold nano-rod was further used to enhance the sensitivity of SPR imaging for

DNA microarray detection [61]. The sensitivity was found to be 10-100 attomole of polymerase product which is

equivalent to 0.25% of a monolayer [62]. Recently, Halpern and Corn et al. presented a SPR phase imaging system

for ssDNA oligonucleotides detection [61, 62] and further discussion on the work will be provided in the section on

phase SPR imaging sensors.

Fu and Yager et al. [63, 64] reported a SPR imaging system operating at different wavelengths with tilted

interference filter [63]. The measurement data was averaged from an area of 400 pixels for 100 images and the

detection limit was found to be 3 x 10-5

RIU [64]. Shumaker-Parry and Campbell et al. further demonstrated a high

throughput SPR imaging sensor with 120 sensor sites in a detection limit of 1.8 x 10-5

RIU [65]. Recently, Kihm et

al. [66] applied the intensity SPR imaging to image the near-field fluidic transport properties within 100nm from the

metal surface. Intensity SPR imaging has been commercialized by GWC Technology (USA) and near infrared

imaging technique has been adopted to enhance the sensor response [46].

3. Angular SPR imaging sensors

In angular SPR imaging, the reflection intensity variations for a range of incident angles are scanned. The angular

SPR curves and absorption minimum are then calculated for different refractive index samples or molecular binding

events. Ruemmele et al. [67] reported an automated angle-resolved SPR imaging sensor which provided a wide

dynamic range from 1 – 1.4 RIU based on the variation in excitation angle. Recently, Zhou et al. [68] used a high

4

precision piezoceramic motor to control and scan the intensity SPR images at different incident angles. The system

could distinguish single mismatch in caspase-3 DNA. Since, angle-scanning SPR imaging has also been

commercialized by IBIS Technology B. V. (Netherlands) [69, 70].

4. Phase SPR imaging sensors

Nelson and Yee et al. introduced the phase sensitive SPR detection in 1996 [47], which is proved to provide 3 times

higher resolution compared to conventional detection based on angular and wavelength modulations. In 1998,

Kabashin and Nikitin [71] demonstrated the first SPR imaging sensor based on phase shift measurement. The probe

and reference beam was made to interfere in a Mach-Zehnder interferometer and the phase information over the

sensing surface was captured by a CCD camera. The sensor resolution was found to be 4 x 10-8

RIU for gas

detection. Nikitin et al. [72] later used a birefringent plate to split the p- and s- polarization beams laterally and the

overlapping parts of these two beams were allowed to interfere after passing through an oblique polarizer. These two

beams shared almost the same optical path and the vibration noise during phase detection was suppressed.

Ho and Lam [73] performed fringe shift analysis between the signal and reference SPR interference patterns

captured from a dual channel chamber and the sensor resolution was found to be 10-5

RIU. Su and Chen et al. [74]

then demonstrated a common-path phase-shift interferometry based SPR imaging sensor. A liquid crystal phase

retarder was used for phase modulation and the phase information was unwrapped with a five-step phase shift

reconstruction algorithm [74, 75]. The sensor resolution was found to be 2 x 10-7

RIU in nitrogen and argon gas

detection.

After that, Yu et al reported two designs of phase SPR imaging sensor [76-78]. In the first approach, the p- and s-

polarized beam was separated and they were allowed to interfere after passing through a polarizing prism [76, 77].

The fringe displacements give the phase information and a detection limit of 3 x 10-5

RIU was demonstrated [77].

The second approach utilized an electro-optical crystal to perform time domain phase modulation [78] and the

Stoilov algorithm [79] was used for phase extraction, however the sensor resolution was limited [78].

Recently, Kim and Corn et al. [61] used the polarizer-quartz wedge depolarizer combination to create interference

fringe image of the sensing surface and the interference fringe shifted according to the adsorption of biomolecules. It

has been further combined with nano-particle based detection for signal enhanced [62] and the detection limit in

short single stranded DNA oligonucleotide measurement was found to be 25fM.

In the past decade, Wong and Ho et al. have reported a series of phase SPR imaging sensors [42-45, 80-81]. In 2005,

they invented a multi-pass SPR phase imaging sensor [44]. The SPR sensor head was located in an optical cavity

and the excitation beam was allowed to be incident on the sensor surface for multiple times, which enhanced the

resultant phase shift in the interference image. A phase shift improvement of a factor of 3 was demonstrated and an

US patent has been filed for this technique [44]. It has further been applied for protein-aptamer detection [82, 83].

Conventional phase SPR imaging sensors mainly relied on interference fringe analysis and a large portion of spatial

information has been sacrificed in the phase extraction process [44, 61-62, 71-72, 73, 76-78]. Wong and Ho et al.

presented a two-dimensional (2D) SPR phase imaging sensor [43]. A piezoelectric transducer was used to modulate

the phase of the interference image in the time domain, which allowed every pixel on the SPR image to be converted

into corresponding phase shift value and a 2D SPR phase map could be obtained without the use of expensive

electro-optical modulators. In 2008, they applied a differential phase measurement scheme together with the 2D

phase mapping technique for bio-molecular array detection [45]. At surface plasmon resonance, the phase of the p-

polarized light is altered, while the phase of the s-polarized light remains unchanged [3, 45]. A phase difference is

therefore created between the p- and s- polarized light. In this work, they separated the light interference pattern into

the p- and s- polarized images and the calculation of differential phase eliminated the common optical path noise

and thus enhancing the sensor resolution. A detection limit of 8.8 x 10-7

RIU has been demonstrated [45].

5

5. Polarization contrast based SPR imaging sensors

Piliarik and Homola et al. [46] reported a SPR imaging sensor based on polarization contrast. The SPR sensing head

was placed between two polarizers with perpendicular orientation. At surface plasmon excitation, a phase shift was

introduced between the p- and s- polarized light and the intensity of the SPR image was increased. A detection limit

of 3 x 10-6

RIU was reported. Later, they improved the sensor resolution by one order of magnitude (2 x 10-7

RIU)

[47] with the subtraction of the dark current signal and intensity fluctuations of the light source. The system has

further been applied for the detection of oligonucleotides [84], protein biomarker in diluted blood plasma [85] and

nucleic acids identification [86].

Patskovsky and Kabashin [87] described a scheme of spatially modulated surface plasmon resonance (SPR)

polarimetry. A birefringt wedge was utilized to produce periodic changes of phase relations between the p- and s-

polarized light and they was allowed to interfere after passing through an analyzer at 45o orientation. The Fourier

Transform method was used for phase extraction.

Recently, Han et al. [88] presented an ellipsometric SPR imaging system and the prism based SPR sensor head was

located between a polarizer and an analyzer. The analyzer was rotated at three different angles and the subsequent

intensity images were used to calculate the phase and ellipsometric parameters. The sensor resolution was found to

be 1.25 x 10-6

RIU.

6. Spectral SPR imaging sensors

Wong and Ho et al. first demonstrated real-time 2D spectral SPR imaging in 2003 [89, 34-37]. P- polarized

polychromatic light source was used as the excitation source. The SPR absorption occurred at particular resonance

wavelength and the resonance wavelength shifted for different refractive index mediums, therefore corresponding

spectral profiles were produced at the spectral SPR image. This imaging approach has been applied for 2D refractive

index mapping in elastohydrodynamic lubricate (EHL) contacts. They also demonstrated the first pixel to pixel color

quantification in spectral SPR image with the Hue extraction algorithm [34-37, 95-97]. It provided full resolution

2D information, which was essential for high throughput array detection. This imaging technique was compared

with the conventional optical interferometry method used in EHL studies [37] and two orders of magnitude (180

times) improvement in accuracy has been shown. In 2006, Yuk et al. also reported a SPR imaging sensor with

wavelength interrogation [90]. The optical fiber probe of a spectrometer was required to scan over the sensing

surface and the SPR absorption minimum values at every scan point were integrated to form a SPR image. The

scanning time for a 2mm spot was 180s and the sensor resolution was limited (7.6 x 10-5

RIU). It has been applied

for protein array [90] and C-reactive protein binding detection [91].

Liu et al. [92] later presented a parallel scan spectral SPR imaging technique. A cylindrical lens was used to focus a

line-shaped light illumination on the sensing surface and the SPR line image was diffracted by a diffraction grating

and then projected on a CCD camera. It was a one-dimensional (1D) scanning technique and the resolution was

limited to 8.1 x 10-5

RIU. Bardin et al. [93] demonstrated a similar optical system with the application of the double

polynomial fit technique [93], the sensor resolution was further improved to 3.5 x 10-7

RIU. However, the imaging

system only provided 1D resolution. Recently, Lee et al. [94] presented a nanohole array based SPR imaging sensor.

White light source was used to excite the surface plasmon resonance at the substrate and the 1D LSPR line image

was processed with an imaging spectrometer and captured with a low-noise CCD camera. The detection limit was

found to be 7.7 x 10-6

RIU.

To date, majority of existing spectral SPR imaging sensors can only provide 1D spatial resolution [92-94] and time-

consuming scanning is required [90-94], while real-time imaging and 2D resolution are two important requirements

in high throughput micro-array detection. In addition, the sensor resolutions are limited in existing spectral imaging

systems [90-92]. In this context, we combine the spectral and phase interrogations and present a new type of

spectral-phase SPR imaging sensor (spectral-phase SPRi) [34, 39]. It provides unique real-time 2D colorimetric SPR

imaging for high throughput micro-array detection and operates with the sensitive phase response, while

complicated and time-consuming phase modulation is avoided.

6

The spectral-phase SPR imaging sensor measures the spectral characteristic variation caused by the steep phase

change occurring at surface plasmon excitation. As shown in Fig. 2b, a SPR prism coupler is placed in between two

polarizers with perpendicular transmission axes and the transmission of an incident beam is forbidden. At the

excitation wavelength of surface plasmon, a phase difference is introduced between the p- and s- polarization

component of the light. It rotates the orientation angle of the polarization ellipse (Fig. 2a) and the light interacting

with the surface plasmon is allowed to pass through the crossed polarizers. As the momentum of the surface

plasmon wave only matches with a particular wavelength range, a particular spectral profile is produced, which is

associated with the steep phase response at the surface plasmon excitation. This method enhances the sensitivity of

conventional spectral based SPR sensor through probing the steep phase response at the surface plasmon resonance.

The sensor resolution has been characterized in a refractive index sensing experiment with different concentrations

of salt solutions ranging from 0%-7%, which corresponds to refractive index values in 1.3330-1.3454 RIU. The

spectral-phase SPR images are shown in Fig. 3a-3f. The SPR image for water sample (0%) is red in color and the

green component in the SPR image increases with increasing refractive index values. The corresponding spectra are

shown in Fig. 3g, which indicates clear spectral profile variations for different concentrations of salt solutions.

During image processing, the Hue component in the HSV color space [95-97] is used to quantify the color variations

in the spectral SPR images [36, 37, 95-97]. It enables pixel to pixel information conversion and full resolution 2D

spectral SPR image can be obtained in real-time, which is not available with existing scanning based spectral SPR

imaging sensors [90-94]. The Hue responses of the SPR images (Fig. 3) are extracted and plotted against the

refractive index values in Fig. 4. This gives the response curve of the spectral-phase SPR imaging sensor. In addition,

the measurement standard deviations (S.D.) between five averaged data are given in Table 1. To consider the overall

measurement S.D. value, at 0.032 Hue unit, as the measurement stability of the sensor, the sensor resolution was

found to be 1.6 x 10-6

RIU [3, 4]. The sensor resolutions of existing spectral SPR imaging sensors is reported in [90-

91], [92] and [94] are 7.6 x 10-5

RIU, 8.1 x 10-5

RIU and 7.7 x 10-6

RIU respectively. It clear shows about 1 order of

magnitude improvement in sensor resolution.

( )..DtSMeasuremen

Hueresponse

RIUrangeresolution ×

∆=

(4)

Fig. 5a further shows the spectral-phase SPR image for an array of refractive index samples (1.3333 RIU, 1.3365

RIU and 1.3454 RIU) and the color texture variation has further been quantified to a 2D Hue map. Fig. 5b and 5c are

the spectral SPR images shown in [93] and [91] respectively. Majority of existing spectral SPR imaging sensors [91-

94] rely on the combination of diffraction grating and CCD camera for capturing the whole spectrum. This approach

limits the SPR image to a 1D line format (Fig. 5b) profile is required. Fig. 5c is the SPR image integrated by the

SPR absorption minimum wavelength values. In each pixel of the image, the fiber probe of a spectrometer was used

to record the SPR spectrum and the absorption dip wavelength was determined [90-91]. The scanning time for a

2mm spot was 180s and the spatial resolution is limited by the physical size of the fiber head. Nevertheless, our

spectral SPR image shown in Fig. 5a is a full resolution 2D image, no scanning is required and the response is in

real-time. It is an ideal technique for real-time high throughput protein/DNA microarray imaging detection.

Biosensor array

To demonstrate array based biosensing with the spectral SPR imaging technique, the specific binding between

bovine serum albumin (BSA) antigen and antibody has been measured. Protein array as described in Fig. 6a was

fabricated on the sensing surface. Glucose oxidase and blank sensor spots served as negative controls in the

experiment. The protein array was first kept in PBS buffer for base-line detection. After that, specific BSA antibody

(2.5 µl/ml) was injected to the sensor surface and the binding interactions were allowed to take place for 1 hr.

Finally, the protein array surface was washed with PBS buffer for the removal of non-specific bindings. Fig. 6

shows the spectral SPR images taken at different stages of the binding process. Comparing the SPR image taken

with PBS buffer (Fig. 6b) and after the injection of specific antibodies (Fig. 6f), the color of the specific sites (A1,

A4, B2, B3, C2, C3, D1 and D4) has varied from red to green due to the specific binding between BSA antigens and

antibodies. However, no significant color variation (molecular binding) was seen in the negative control sites (A2,

7

A3, B1, B4, C1, C4, D2, D3) and blank sites (E1-E4). As described in the previous section, the color texture

variations in the SPR images were further quantified with the Hue component as shown in Fig. 7. Fig. 7a and 7b are

the SPR images taken with PBS buffer and Fig. 7c and 7d are the images captured at 1mins and 5mins after the

injection of the specific BSA antibody (2.5 µl/ml) respectively. Clear responses have been shown in the specific

sites of the spectral SPR image within 5 mins of reaction (Fig. 7d). Fig. 7e-7h show that the signals in all specific

sites increased against time, when increased amounts of BSA antibodies bound onto the protein array. The

quantification of the SPR images also provides a series of binding curves for all sensor sites in the protein array and

they are shown in Fig. 8. The sensor resolution for bio-molecules can be calculated from the following [4].

(5)

It is because the injection of 2.5 µl/ml BSA antibody produces an averaged overall response of 39.13 (Hue unit) and

the overall measurement S.D. is 0.13 (Hue unit), the bio-sensing resolution was found to be 8.26 ng/ml (125pM).

This value is 12.1 times and 93.2 times better than the sensor resolution reported in [98] and [45] respectively for

IgG and BSA antibody-antigen binding detections with phase SPR imaging.

7. Diffraction grating based SPR imaging sensors

Diffraction grating coupler is not as widely used as the prism coupler in SPR imaging for the excitation of surface

plasmon [2-5]. The working principle of grating based SPR sensing is described in Fig. 9a. Excitation light is

incident onto a gold/silver coated diffraction grating and the surface plasmon propagating at the metal-dielectric

interact will be excited when the momentum of the diffraction light matches with that of the surface plasmon wave,

which is described in equation (6) [4],

��

�� sin �

��

�� �� (6)

where Λ is the period of the diffraction grating, m is an integer and ksp is the wave vector of the surface plasmon

wave.

Diffraction grating based SPR sensing was first demonstrated in 1987 by Cullen et al. [99]. A gold coated grating

was used for the excitation of surface plasmon at the metal-dielectric interface. The reflection intensity was plotted

as a function of incident angle and the shift of the angular SPR dip corresponded to the bio-molecular binding.

Grating coupler was then demonstrated for imaging detection in 2001 by Brockman and Fernandez [100]. In their

design, monochromatic light at 860nm was illuminated on a gold-coated diffraction grating and 400 sensing

channels were allowed to operate in parallel. This approach was commercialized by HTS Biosystems in 2005 [101]

and the technique was further acquired by Biacore International AB in the same year.

Homola et al. reported another design of high throughput SPR sensor with an array of minimized diffraction grating

spots (216 elements) [102]. A 635nm laser diode was used as the monochromatic excitation source. It was focused

on each row of diffraction grating with scanning optics and the reflected angular spectrums were captured with a

CCD detector. The detection limit was found to be 5 x 10-6

RIU. The system was further applied for multiplexed

protein-analyte scanning [103] and the detection limit was improved to 5 x 10-7

RIU with reduced sensing sites

(120). They then developed a portable device with a gold coated grating sensor chip [104]. In the optical

configuration, 10 angular spectrums captured from 10 sensing channels were projected on the CCD detector with a

cylindrical lens and the resonance angle shifts indicated the refractive index changes or bio-molecular bindings.

Micro-fluidic sample delivery system, heat insulation and cooling system are integrated in the device. The refractive

index resolution was found to be 6 x 10-7

RIU. Detection of the hybridization of oligonucleotide probes have been

demonstrated and the detection limit was found to be 1nM.

StabilitytMeasuremenresponseSensor

moleculebioofionConcentratLimitDetection ×

−=

8

Singh et al. reported the use of gold coated commercial compact disk (CD) as grating substrate for SPR imaging

measurement [105]. Array of functionalized monolayer layers were spotted on different regions of the CD-grating

and measurements on protein bindings (bovine serum albumin) have been performed with the system. Lynes et al.

also developed a grating based imaging system, which relies on angle scanning of SPR dip [106]. Recently, the

system was applied for CD4+ T cells detection which found applications in disease diagnostics [108].

8. Conclusion

Since the first invention of surface plasmon microscopy by Rothenhäuslar and Knoll in 1988 [1], SPR imaging has

found rapid increasing research interests [2-5] and wide applications in drug discovery [6], biomarker screening [7],

nucleic acid detection [5], food safety [8, 9] and environmental monitoring [10] in the past two decades. SPR

imaging sensors based on intensity [1, 23-28], angular [29, 30], wavelength [31-36], phase [37-42] and polarization

[43-45] modulations have been reported to date. However, intensity based SPR imaging sensors are the most widely

used method [4, 23-28]. Among different detection approaches, phase SPR imaging [37-42, 47, 61-62, 71-78] are so

far the most sensitive technique, however the practical usage has been limited by the sensitivity to background noise.

In recent years, SPR imaging sensors based on polarization contrast [37-42] and wavelength [31-36] interrogations

have been reported to provide high sensitivity and high stability detection. In this context, we combine the spectral

interrogation of phase and polarization detection and demonstrate the use of a multiplex spectral SPR imaging

biosensor array based on polarization orientation. It enhances the sensitivity of conventional spectral based SPR

sensor through probing the steep differential phase response at the surface plasmon resonance between the p- and s-

polarization. One order of magnitude improvement in sensor resolution has been demonstrated with this imaging

sensor compared to existing spectral SPR imaging sensors [90-92] [94] and phase SPR imaging sensors reported for

high throughput bio-molecular detection [99, 42]. Our spectral SPR imaging sensor also provides real-time 2D

resolution imaging, while only 1D resolution is enabled with existing spectral SPR imaging techniques [92-94].

Such imaging sensors can find promising applications in clinical disease diagnosis, protein biomarker and drug

screening.

Acknowledgment

This project is supported by Singapore Bio-Imaging Consortium (SBIC). Acknowledgment also goes to Li-Tin Ho

for the contribution in experimental design and ideas.

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16

List of captions

Table 1 The measurement standard deviation of different concentration salt solutions.

“Reprinted with permission from (Chi Lok Wong et al., Optics Express v19 (20), pp. 18965-18978, (2011)[39]).

Copyright (2011) OSA.”

Fig. 1

a) Reflection intensity a function of wavelength at fixed incident angle for samples with different refractive index

values.

“Reprinted with permission from (Jiří Homola et al., Methods, v37(1), pp. 26-36 (2005) [2]). Copyright (2005)

Elsevier.”

b) Intensity SPR image obtained for the specific adsorption ofβ2m (50nM).

“Reprinted with permission from (Hye Jin Lee et al., Anal. Chem., v78, pp. 6504-6510 (2006) [107]). Copyright

(2006) American Chemical Society.”

c) Spectral SPR image for an array of different refractive index samples

(the scale bar represents 1mm in length)

“Reprinted with permission from (Chi Lok Wong et al., Optics Express v19 (20), pp. 18965-18978, (2011)[39]).

Copyright (2011) OSA.”

d) Phase SPR image of MUAM monolayer and bare gold surface. The intensity distributions refer to the

interference fringe pattern.

“Reprinted with permission from (Aaron R. Halpern et al., Anal. Chem., v83, pp. 2801-2806 (2011) [61]).

Copyright (2011) American Chemical Society.”

e) SPR image with polarization contrast. The vertical bands correspond to referencing areas. “Reprinted with

permission from (Marek Piliarik et al., Sensors and Actuators, v134, pp. 353-35(2008) [47]). Copyright (2008)

Elsevier.”

Fig. 2 a) Ellipse of the elliptically polarized light E'

b) Schematic diagram of the spectral SPR imaging sensor based on polarization orientation.

“Reprinted with permission from (Chi Lok Wong and Malini Olivo et al., Biosensors and Bioelectronics v47, pp.

545-552, (2013)[34]). Copyright (2013) Elsevier.”

Fig. 3

Spectral SPR images for different concentrations of salt solutions

a) Water (0%)

b) 1% salt solution

c) 2% slat solution

d) 3% salt solution

e) 4% salt solution

f) 7% salt solution

g) Spectrums measured for different concentrations of salt solutions ranged from

0%-7% (Normalized with s- polarization)

“ Reprinted with permission from (Chi Lok Wong et al., Optics Express v19 (20),

pp. 18965-18978, (2011)[39]). Copyright (2011) OSA.”

Fig. 4 The response curve of the spectral SPR imaging sensor in the refractive index range between 1.3333-1.3454

RIU. The error bar is obtained from the standard deviation (S.D.) between 5 averaged measurement data.

“Reprinted with permission from (Chi Lok Wong et al., Optics Express v19 (20), pp. 18965-18978, (2011)[39]).

Copyright (2011) OSA.”

17

Fig. 5

a) 2D spectral SPR image of an array of refractive index samples (water (0%), salt

solution (2%) and salt solution (7%)). Applying the Hue extraction, the color distribution is quantified. 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). (the scale bar represents 1mm in length)

“ Reprinted with permission from (Chi Lok Wong et al., Optics Express v19 (20), pp. 18965-18978, (2011)[39]).

Copyright (2011) OSA.”

b) 1D spectral SPR image of a biochip reported in [93]

“Reprinted with permission from (Fabrice Bardin et al., Biosensors and Bioelectronics, v24(7), pp. 2100-2105

(2009). [93]). Copyright (2009) Elsevier.”

c) SPR image integrated by the SPR absorption minimum wavelength values. In each pixel of the image, the fiber

hand of a spectrometer was used to record the SPR spectrum and the absorption dip wavelength was determined

[90-91]. The scanning time for a 2mm spot was 180s and the spatial resolution is limited by the physical size of

the fiber hand. The first SPR image was Au, and the final SPR image was anti-CRP on the Au/DTSP/CRP

surface.

“Reprinted with permission from (Jong Seol Yuk et al., Sensors and Actuators B: Chemical, v119, pp. 673-675

(2006) [91]). Copyright (2006) Elsevier.”

Fig. 6

a) The protein array consists of 3 different array elements: bovine serum albumin

(BSA) (red spots, positive sample), glucose oxidase (GOx) (black spots, negative sample) and blank sites (white

spots, background control)

Spectral SPR images for specific BSA antigen-antibody binding detection (the scale bar represents 1mm in length)

b) at the beginning of the experiment, the protein array was kept in PBS buffer. c) (46mins) 15 minutes after the

injection of anti-BSA. d) (90mins) 1 hour after the injection of BSA antibody. e) (106mins) 15 minutes after the

PBS buffer washing process.

“Reprinted with permission from (Chi Lok Wong and Malini Olivo et al., Biosensors and Bioelectronics v47, pp.

545-552, (2013)[34]). Copyright (2013) Elsevier.”

Fig. 7 2D hue profiles extracted from the spectral SPR images

a) At the beginning of the experiment, the array was kept in PBS buffer.

b) (30mins) the array was kept in PBS buffer.

c) (32mins) 1 minute after the injection of anti-BSA (2.5µl/ml in PBS).

d) (36mins) 5 minutes after the injection of anti-BSA. The average response in the specific BSA antigen sites is

increased from 0.19 (hue unit) to 13.95 (hue unit), which is equivalent to 66.21% increase. However, less than 3.05

% increases are recorded in the non-specific GOx sites.

e) (46mins) 15 minutes after the injection of anti-BSA.

f) (61mins) 30 minutes after the injection of anti-BSA.

g) (76 mins) 44 minutes after the injection of anti-BSA.

h) (90mins) 59 minutes after the injection of anti-BSA.

i) (105mins) 15 minutes after array surface washing with PBS buffer. Less than 1% signal decrease is recorded in

the specific sites (Figure 7i). It reveals the specificity of the binding interactions. Comparing the 2D Hue profiles

shown in Figure 7i and Figure 7a, 162.1 % increase is found in the specific BSA antigen sites, while only 4.45%

increase is indicated in the non-specific GOx sites.

(the scale bar represents 1mm in length)

“Reprinted with permission from (Chi Lok Wong and Malini Olivo et al., Biosensors and Bioelectronics v47, pp.

545-552, (2013)[34]). Copyright (2013) Elsevier.”

18

Fig. 8

Binding curves for all sensor sites in the array. Figure 8a, 8d, 8f, 8g, 8j, 8k, 8m and 8p reveal the binding curves of

the specific BSA antigen sites, and the binding curves of the non-specific GOx sites are illustrated in Figure 8b, 8c,

8e, 8h, 8i, 8l, 8n and 8o. Specific bindings are clearly shown in the specific BSA antigen sites (A1, A4, B2, B3, C2,

C3, D1, D4), while less than 5% variations are recorded in the non-specific GOx sites (A2, A3, B1, B4, C1, C4, D2,

D3).. At the equilibrium stage, the average response recorded in the specific BSA antigen sites is 24.8 times higher

than that of the non-specific GOx sites. In addition, it is 6.2 times higher than that of the blank control sites (Figure

8q-8t, E1-E4).

“Reprinted with permission from (Chi Lok Wong and Malini Olivo et al., Biosensors and Bioelectronics v47, pp.

545-552, (2013)[34]). Copyright (2013) Elsevier.”

Fig. 9

a) Working principle of diffraction grating based angular SPR imaging sensor

b) Imaging of an array of SPR angular spectra from a row of diffraction gratings [103].

“Reprinted with permission from (Jakub Dostálek et al., Sensors and Actuators B v107, pp. 154-161, (2005) [102]).

Copyright (2005) Elsevier.”

19

Table 1

0% (water) 1% 2% 3% 4% 7%

Refractive Index (RIU)

1.3333 1.3347 1.3365 1.3383 1.3400 1.3454

Measurement Standard Deviation

(Hue unit 0-255) 0.030 0.023 0.073 0.023 0.024 0.017

Fig. 1

(a)

(b)

20

(c)

Fig. 1

(d)

21

Fig. 1

(e)

22

Fig. 2

23

Fig. 3

(a)

(c)

(e)

(b)

(d)

(f)

24

25

Fig. 3

(g)

26

Fig. 4

(a)

Fig. 5

(a)

(b)

27

Fig. 5

(c)

SPR absorption minimum wavelength

(nm)

28

SPR absorption minimum wavelength

Fig. 6

(a)

(b)

(d)

(b)

(d)

(c)

(e)

(c)

(e)

29

30

Fig. 7

31

Fig. 8

(b)

Fig. 9

(a)

32