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A calculation based on surface plasmon coupling condition and Maxwell-Garnett equation was performed for predicting the coupling angle shift and thin film thickness in scanning surface plasmon microscopy (SSPM). The refractive index sensitivity and lateral resolution of an SSPM system was also investigated. The limit of detection of angle shift was 0.01°, the limit of quantification of angle shift was 0.03°, and the sensitivity was around 0.12° shift per nm ZnO film when the film thickness was less than 22.6 nm. Two partially connected Au nano-discs with a center-to-center distance of 1.1 μm could be identified as two peaks. The system was applied to image nanostructure defects and a virus-probe functionalized nanoarray. We expect the potential application in nanobiosensors with further optimization in the future.
© 2015 Optical Society of America
1. Introduction
Surface plasmon microscopy (SPM) is a sensitive technology for imaging the refractive index (RI) perturbations above a metal layer. This high sensitivity has made SPM an useful tool for near-field sensing and imaging [1]. For example, the SPM for imaging additionally deposited metal film down to one-nanometer thickness was reported [2]. However, the decay lengths of surface plasmon propagation limited the lateral resolution of non-scanning SPM [3]. The decay length was 14 to 24 μm for the case with illumination of 805 nm and Au thin film [4]. Besides, the propagation of surface plasmon would cause the distortion and shadow of images. To minimize the effect of the propagation length, Researchers have demonstrated a few technologies to minimize the effect of the propagation length, like changing the excitation wavelengths [5], replacing the metal film material [6], and acquiring images from multi-directions by rotating the target object for imaging analysis [7].
Surface plasmon interference has been proposed for further increasing the lateral resolution of SPM [8].Surface plasmon was excited in many directions by a cylindrically symmetric TM wave by focusing a radially-polarized laser beam and used as a sensing probe [9]. An evanescent Bessel-like standing wave above the metal film could be generated by the longitudinal field when focusing the radially-polarized laser beam [10]. The sensing probe size would be around 300 nm and 200 nm for illumination of 800 nm and 632.8 nm respectively [11, 12]. With the interference of locally excited surface plasmon in scanning surface plasmon microscopy (SSPM), the distortion and shadow of images in non-scanning SPM were improved [13]. This imaging technology has been used to image cell adhesion [14], lipid bilayer composition [15], virus particles [16], and fluorescence nanoparticles [17].
Although SSPM has been proved to be with a sensing probe of diffraction-limited spot size and used in imaging biological samples, the resolving power to two neighbored nanoparticles and the sensitivity to deposited layer thickness were less discussed [18]. For the application of nanobiosensors the sensitivity to a dielectric layer above the metal film, such as aptamers or proteins, and the lateral resolution for imaging nanostructures are important. In this article, the sensitivity of an SSPM system to effective RI changes was characterized with ZnO films of various thicknesses and compared to simulations using Macleod or Maxwell-Garnett theory. The lateral resolution of the SSPM was experimentally verified by imaging Au nano-disc pairs with different separation distances. In addition, the SSPM system was used to image an oligonucleotide-functionalized nanoarray.
2. Materials and methods
2.1 SSPM setup and surface plasmon angle measurement
The schematic diagram of our SSPM is shown in Fig. 1(a). A radial polarization converter (Arcoptix S.A) was used to convert the polarization state of an ultrafast laser beam (Tsunami) of 780 nm to radial polarization (RP). The beam size was expanded to fill the rear aperture of an objective lens (PlanApo 60 × oil objective, NA = 1.4, Olympus). The laser beam was then focused and locally excited surface plasmon resonance (SPR) at a 47 nm Au film. The size of the excited surface plasmon spot was estimated to be around 270 nm [12]. However, the spot size for SPR was expected to be larger than the focal spot because of the side lobes and the propagation of SPR waves. For imaging, raster scanning was performed by a sample stage (Proscan II, PRIOR) for mapping the SPR coupling angles. The scanning step size was 200 nm in a rate of 100 ms/step. The scanning rate was synchronized to the frame rate of a camera (Mightex, frame rate = 10 Hz) for acquiring the back focal plane (BFP) image.
Fig. 1 Radially-polarized illumination was used to locally excite surface plasmon. (a) The schematic diagram of our SSPM. (b) The image of the BFP was acquired for the coupling angle calculation. (c) is the effective refractive index of the target sample including the deposited material and medium. (LP: linear polarizer, RPC: radial polarization converter, RP: radial polarization, BS: beam splitter, L: lens, OBJ: objective, BFP: back focal plane, BFPI: back focal plane image)
The spatial distribution in BFP was the angular distribution of the reflected light from front focal plane of the objective lens [Fig. 1(b)] [10]. A dark ring in BFP, also called SPR ring, was the reflectivity dip caused by SPR coupling. The SPR coupling angle could be calculated from the radius of the ring by Eq. (1) [19]:
denotes the SPR angle, denotes the maximum illumination angle under the numerical aperture of the objective, denotes the radius of the SPR ring, and is the maximum radius of the back pupil image in BFP. For calculating the coupling angle in SSPM, numerical aperture of objective (NA) and environmental RI (n0) between the objective and the metal film is used to replace the maximum angle in Eq. (1):At the SPR angle, the evanescent wave, which penetrates through the metal film, couples with the surface plasmon [Fig. 1(c)]. Moreover, the propagation constant of the penetrated evanescent wave βEW and the surface plasmon wave βSP would be equal while the resonance coupling occurs:
∆β is the influence of the metal film and the glass layer with finite thicknesses, βSP is the surface plasmon propagation constant influenced by ∆β, and βSP0 is the propagation constant in absence of the influence. The propagation constant in Eq. (3) can be rewritten as the RI times the wavenumber in vacuum, as follows [20]: refers to the effective RI of surface plasmon, refers to the effective RI of surface plasmon without the ∆β, is the complex permittivity of the metal and refers to the effective permittivity of sample above the metal film [20]. In addition, the decay length () of the intensity of the penetrated evanescent wave can be calculated as [21]where refers to the effective RI of sample above the metal film. Equation (2) was used to calculate the SPR coupling angle in our experiment. Equation (4) and (5) were used to simulate and predict the SPR coupling angle in Sec. 3.1.2.2 Nanostructure Fabrication
Three fabrication methods were used in producing different nanostructures on SPR chips, i.e. thin film deposition, Dip-Pen Nanolithography (DPN), and microsphere lithography. Glass slides were cleaned with methanol, water, and detergent sequentially in ultrasonic water bath. After the substrates were dried by blowing with N2, Ti or ZnO was deposited as an adhesion layer on the slides. Au film of 47 nm was coated above the adhesion layer with electron beam evaporation.
2.2.1 Thin film deposition
Zinc oxide films were stacked on the prepared SPR chip by sputtering. The mask was shifted 5 mm toward the same direction after each sputtering (see Fig. 2). The deposition rate for ZnO film was 2.05 nm/min under 35 Watt input power and 10−5 Torr of atmospheric pressure. ZnO layer with gradually increased thickness was built after a few round of coating process. The thickness of ZnO films were measured from the cross section images captured by scanning electron microscope (SEM).
Fig. 2 The fabrication methods used in this article are illustrated. (a) Thin film was sputtered sequentially to stack ZnO layers with variant thicknesses. (b) MHA ink was written on Au film with DPN for producing the Au nanoarray. (c) Microsphere lithography was performed for making a large area nanoarray. (Unit: nm)
2.2.2 Dip-pen nanolithography
Au nano-disc pairs and nano-disc nanoarray were produced by DPN. Tip of AFM probes were dipped into 16-mercapto-hexadecanoic acid and then drew mask patterns on Au film [22]. The chip was then immersed to etching solution which included 0.076 g thiourea, 0.2686 g ferric nitrite, and 18 μl 1-octanol in 50 ml water. The pH value was adjusted to 1.85 by adding HCl. After 15 minutes etching, the chip was washed in ultrasonic water bath. An extra 30 nm Au film was deposited on the chip for matching SPR condition. The final disc size after etching was on average 637.7 nm in diameter and 20 nm in height. The diameters of nano-discs and distances between nanostructures were measured by SEM. The intervals were from 0.8 to 3 μm for nano-disc pairs and 3 μm for nanoarray.
2.2.3 Microsphere lithography
A hexagonal nanogold array for oligonucleotide modification was made by microsphere lithography. Microsphere lithography is an efficient and cost-effective method to produce large area nanostructures. SPR chip was immersed into 1 mM 6-mercapto-1-hexanol (MCH) in 99% ethanol for 2 hours. A microsphere-detergent mixture was dropped on the MCH modified surface and dried in room temperature. The mixture was composed of 3.5 μl microsphere solution (5 μm polystyrene sphere in 3%, SIGMA-ALDRICH) and 100 μl 0.125% Triton-100. A 10 nm thick Au film was then deposited on the chip, while the hexagonal microsphere structure formed on chip performed as a mask. The spheres were lifted off by ultrasonic water bath. The shape of deposited Au structure was defined by the projection of the interstices between particles [23]. The diameter of each nano-dot was about 1.13 μm, and the interspace between structures was 2.885 μm in average.
2.3 Nucleotide functionalized nanoarray
The hexagonal nanogold array in Sec. 2.2.3 was functionalized with an oligonucleotide probe for hepatitis C virus (HCV). The probe was pre-conjugated with thiol group at the 5′ end for binding to the Au structure. A drop of 20 pmol oligonucleotide probe was dropped onto the nanoarray and incubated overnight. The chip was then washed and dried before imaging.
3. Results
The change of surrounding medium above the Au film would disturb the surface plasmon polariton. This disturbance caused the changing of diameter of SPR rings. While the SSPM sensing probe passed through the location with different effective RI, like nanostructures or deposited films, the expansion and contraction of SRP ring was observed.
3.1 Effective RI sensitivity
The trend of SPR angle shift and the sensitivity of SSPM to effective RI change were investigated experimentally and theoretically. ZnO layers with different thicknesses were used as the target sensing material with different effective RI. The resonance conditions were firstly simulated by Essential Macleod V8.7.21 (Thin Film Center Inc.). The simulation results in Fig. 3(a) show that the coupling angle shift was nonlinear proportional to the thickness of ZnO film. Further calculations were performed to understand the relationship between the thin film thickness and the coupling angle shift. To perform the calculation, the permittivity of Au () and a series of given effective RI () was used. The effective permittivity of the sensing material was obtained from , while the imaginary permittivity was assume to be extremely low and could be ignored. The value of the effective RI was contributed by the surrounding air and the ZnO layer above the Au film [Fig. 1(c)]. Predicted SPR coupling angle of the thin film structures was then calculated from Eq. (5) (Sec. 2.1). It should be noted that the influence of Au and glass with finite thickness was neglected in this calculation.
Fig. 3 (a) The SPR angle shifted with the thickness of the ZnO layer (performed with Macleod. (b) The trend of coupling angle shifts predicted with Macleod, MG model, and the equation published by Jung et al. [24] (c) BFP images with the SPR angles of sample with different ZnO thickness. Region in 10 um2 size was scanned for each sample and calculated the average angle and standard deviation.
The ZnO film thickness was estimated by the equation shown by Jung [24]:
in which was the RI of the sensing material at a z position away from the metal surface (z = 0). For a single thin film with thickness , the equation was reduced to:where and referred to the RI of ZnO (1.96) and air (1.0005) respectively. In the case of an ultrathin film deposition, the above equation could be further simplified to [21]:The calculated film thickness and coupling angle was plotted in Fig. 3(b). However, this equation was used in the condition with small effective RI shift and without the shape deformation of the reflectivity dip in SRP curve [21, 24].Meanwhile, another effective medium theory, Maxwell-Garnett model (MG), was also utilized to simulate the film thickness [25]:
where, denoted the permittivity of ZnO, denoted the permittivity of air, and denoted the volume fraction of ZnO. In the MG model, ZnO was assumed to be homogeneously distributed in the sensing volume. A series of ZnO thicknesses was divided by the penetration depth [Eq. (3)] to yield the volume fractions:The effective thickness was then shown with the correspondent coupling angle in Fig. 3(b). Although MG theory was not refined for the two layer structure including ZnO film and air, the trends of the results strongly agreed with the Macleod simulation results when ZnO film was thin.Furthermore, a ZnO deposited SPR chip was imaged by SSPM. The BFP images and SPR angle maps are shown in Fig. 3(c). A 10 um2 area was scanned for each ZnO thickness. The SPR angle of the bare Au film was 43.01° on average with a standard deviation (SD) of 0.003°. The limit of detection was around 0.01° (3SD) [26], and the limit of quantification was 0.03° (10SD) [27]. The radius of SPR ring increased with the thickness of deposited ZnO, and the width of the refractivity dip increased as well. The sensitivity of SSPM was around 0.12° shift per nm ZnO when the thickness was less than 22.6 nm. Here we assumed that the curve in the range less than 22.6 nm was linear. The r-square value was 0.9924 while the y-intercept of the fitting curve was set to 43.01°.
The simulation and experimental results have shown similar trends of SPR angle shift [Fig. 3(b)]. While the SPR condition was satisfied, both of the results indicated that the coupling angle was nonlinearly proportional to the thickness of ZnO film. That is, the slope increased with the thickness of ZnO in a proper range of thickness. The results implied that the angle shift of SSPM increases with the thickness of the ZnO film. However, the full width at half maximum (FWHM) of the reflectivity dip should be considered in calculating intrinsic sensitivity [28]. Higher FWHM would cause larger standard deviations of the fitting angle and deteriorate the limit of detection. The standard deviation of the coupling angles was 10−3 for film thickness less than 22.6 nm and 10−2 for thickness more than 32.8 nm [Fig. 3(c)]. The increased standard deviations were mainly caused by the fitting error of the coupling angle when the reflectivity dips in SPR ring become broader. In addition, the shift of y intercept in Fig. 3(b) might be caused by the ∆β which was ignored in the calculation.
3.2 Lateral spatial resolution of SSPM
Nano-disc pairs with gradually decreased interspaces were imaged by SSPM. And then, the intensity profiles of SPR angle maps were analyzed to estimate the resolution of SSPM [Figs. 4(a) and 4(b)]. The distance between the nano-disc pairs in the SPR angle profiles was compared to the corresponding center-to-center distance in SEM images [Fig. 4(a)]. The disc-pair with center-to-center distance of 1.6 μm (interspace: 0.936 μm) was identified as 1.6 μm by SSPM. Additionally, the partially connected discs with a center-to-center distance of 1.1 μm in, Fig. 4(a) were identified as two peaks with a distance of 0.8 μm. Figure 5 compared the distances in SEM and SSPM images of a series of nano-disc pairs.
Fig. 4 (a) Nano-disc pairs with gradually decreased center-to-center distances were imaged by SSPM and SEM. (b) The distances between peaks of the intensity profiles of the SSPM image in (a) were measured.
3.3 Quality inspection of nanostructure on SPR chip
The SSPM was used to scan a large nano-disc array. The result revealed the high sensitive and high resolution of SSPM image (Fig. 6). The nano-disc array of 20 nm height was clearly presented in the SPR angle map. Comparing to the SEM image, the averaged disc diameter was 637.7 ± 180 nm in SEM image and 555 ± 59 nm in SSPM image. Also, the incompletely etched Au residues were also clearly presented in the SSPM image. Besides, the scratch on chip was also shown in SSPM image by changing the coupling condition and caused the fitting error of SPR ring [Fig. 6(c)]. The results showed the high sensitivity of SSPM to the RI variation on surface and demonstrated the capability of SSPM to examine the quality of nanostructures on SPR chip.
Fig. 6 A nano-disc array produced by DPN was imaged by SEM (a) and SSPM (b). The SSPM image clearly showed the nanoarray structure and some defects on the SPR chip (c). No.1 is the image of residual Au after etching, and No. 2 is the image of a scratch on the surface.
3.4 Mapping oligonucleotide functionalized nanoarray
SSPM was utilized to map SPR angle images of a hexagonal nanogold array before and after single-strand DNA (ssDNA) functionalization. Figure 7 is the SPR angle maps of the nanostructure before and after oligonucleotide modification. The SPR angles were slightly larger after ssDNA functionalization as shown in the pixel counting histogram [Fig. 8(a)]. In the histogram region for nanostructure, a slight right shift was observed. We further simulated the SPR angle shift caused by ssDNA functionalization based on Maxwell-Garnett model and the equation published by Jung et al. [Fig. 8(b)]. The refractive index of ssDNA was assumed to be 1.46 [29]. Since the average distance between a nucleotide base pair is 1.08 nm [30], the thickness (height) of the ssDNA lying on a gold film was estimated to be a half of 1.08 nm. In the simulation, the gold surface was assumed to be completely covered by a monolayer of flat-lying ssDNA. The shifts of angle predicted by the MG theory and the equation of Jung were 0.037°/nm and 0.02°/nm respectively, which means that the shift was less than 0.02 o. According to the result, ssDNA modification might be detected, but would be hard to quantify.
Fig. 7 The SSPM image before and after single-strand DNA modification. (HCV probe sequence: 5′-thiol-TATGGCTCTCCCGGGAGGGGTTGCCATGGCGTTAGTATGAGT-3′)
Fig. 8 (a) The histogram of SPR angle showed a slight right shift after ssDNA modification. (b) The simulation results for ssDNA modification based on Jung et al. and MG model.
4. Discussion
Although conventional SPR has been widely used in biosensing, to the best of our knowledge, this is the first time that objective-based SSPM was used in a nanobiosensor array. However, we noted that the performance of the SSPM system could be further improved. First, the fitting error of coupling angle could be reduced with image analyzing, improved fitting algorithms [17], or designed chip configuration. Second, the image quality could be improved. Waiting for the settling time of the stepper motor before image acquisition could minimize vibrations of the stage due to scanning. Scanning with a smaller step size and higher precision could also improve the smoothness of images. Third, the lateral resolution could be further improved by minimizing the effect of transverse fields at the focal point with an annular aperture [31, 32]. We expect that SSPM could be helpful in developing SPR nanobiosensors for stacked biomaterials on SPR chips in the future.
To conclude this work, we investigated the refractive index sensitivity of a SSPM system and introduced a simple method based on Maxwell-Garnett theory for predicting the SPR angle shift. Further, we investigated the lateral resolution of the SSPM system by using nano-disc pairs. Finally, using SSPM for detecting functionalized oligonucleotides on a high-density nanoarray was demonstrated, which shows the potential to be applied to micro-/nano-biosensing chips in the future.
Acknowledgments
We would like to thank the Ministry of Science and Technology of Taiwan (Project Nos.102-2218-E-002-014-MY3 and 103-2917-I-002-168) for financial support and the Center for Emerging Material and Advanced Devices of National Taiwan University for facility and equipment support.
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