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Application of a Plasmonic Chip for Sensitive Biodetection
Keiko Tawa
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DOI: 10.22661/AAPPSBL.2019.29.3.10

Application of a Plasmonic Chip for Sensitive Biodetection



Enhanced fluorescence is a powerful tool for the sensitive detection of analytes. A plasmonic chip is a substrate covered with a thin metal film and has a surface including a periodic pattern with a wavelength-scale pitch. It can make the fluorescence from a fluorescent molecule attached to a plasmonic chip enhance by 100 times and the enhanced fluorescence is based on the excitation of the grating-coupled surface plasmon resonance (GC-SPR) field. In this work, the structure of a plasmonic chip, the mechanism of fluorescence enhancement, and the application of a plasmonic chip to sensitive immunosensor and cell imaging will be introduced.


The principle of propagated surface plasmon resonance (SPR) has been studied for several decades, and SPR was applied to Biacore [1,2], an instrument measuring biomolecular interaction such as antigen-antibody interaction, in the late 1990's. Immediately after the spread of Biacore, studies on near field optics based on localized SPR with metal nanoparticles developed extremely rapidly [3-7] and studies on not only linear optics but also nonlinear optics made intense progress. Following the extensive research on SPR, SPR-field enhanced fluorescence also was studied and the application of enhanced fluorescence to bio-detection has progressed. In this article we focus on the enhanced fluorescence method based not on localized SPR but rather propagated SPR and refer to other studies as for further discussion on localized SPR.

The development of immunosensors, including Biacore, has attracted attention due to the rapid and sensitive detection of analytes by immunosensors. In a propagated-SPR sensor chip [8], analytes can be simply and rapidly measured without labeled-detection antibodies in a sandwich assay; however, the limit of detection (LOD) is not as good as the pico molar (p mol L-1: pM) level. As a popular and sensitive immunosensor with a better LOD, an enzyme-linked immunosorbent assay (ELISA) has been used [9], in which a detection antibody labeled with an enzyme such as horseradish peroxidase (HRP) has been applied. Chromogenic or fluorogenic substances added into the assay chemically react with the enzyme and, after efficient reaction, a number of antigens bound to the chip surface can be quantitatively evaluated by the signal intensity from substances. The sensitive detection, cheap instruments, and the various detection kits available are merits of the ELISA method. However, the sandwich assay of ELISA has many operation steps and takes much time.

Knoll et al. developed surface plasmon field-enhanced fluorescence spectroscopy (SPFS) from the SPR method and applied it to biology in the late 1990's [10,11]. In SPFS, an enhanced electric field based on the SPR field is used as an excitation field for fluorescent dye, so that only fluorescent molecules attached to the substrate surface can be selectively excited and an enhanced fluorescence can be detected. Using SPFS for detecting the fluorescence signal from sensor chips is useful for the immunosensing. However, in general SPFS, the use of a prism is essential for coupling the incident light with a surface plasmon and the optical setup is a little complex. Therefore, general SPFS was modified to surface plasmon- field enhanced fluorescence (SPF) with a plasmonic chip. Using this modification, we improved the detection sensitivity, the rapidity of detection, the ease of operation, and down-sized the instrument. The plasmonic chip is coated with thin metal films such as gold (Au) or silver (Ag) and its surface has a periodic structure with a wavelength-scale pitch. The surface structures measured by atomic force microscopy (AFM) are shown in Fig. 1. Figs. 1 (a) and (b) show the 3D view of the line and space pattern and the hole-array pattern, respectively, and Fig. 1 (c) is a floor plan of the bull's eye pattern. In the bull's eye pattern, a cross section of bull's eye to the center indicates a periodic structure, and all the patterns shown in Figs. 1 (a)-(c) indicate a periodic structure at the cross section of the chip. As for the fluorescence immunosensing method using a plasmonic chip, reagents such as the commercially available antibody, antigen, and buffer solutions in the ELISA kit can be used, and existing fluorescence instruments, such as a spectrometer and microscope, are also available. Therefore, the application of a plasmonic chip to an immunosensor is essentially barrier-free.


Fig. 1: 3D views of AFM images for a plasmonic chip with (a) a line and space pattern, and (b) a hole-array pattern, and the floor plan of an AFM image with (c) a bull's eye pattern.

A plasmonic chip also can be applied to cell imaging. As sensitive imaging tools, surface-enhanced Raman scattering (SERS), total internal reflection fluorescence (TIRF), and scanning near field optical microscopes (SNOM) are representative examples, but the plasmonic chip shows not only good detection sensitivity but can also be effectively combined with a general microscope and has a simple setup procedure.


In the propagated SPR, the incident light wave can be coupled with the surface plasmon by prism-coupled (PC)-SPR and grating coupled (GC)-SPR methods. The PC-SPR method requires complex optics such as prisms, but in the GC-SPR method, incident light can be directly coupled with surface plasmon without special optics [12,13]. The resonance condition in GC-SPR is


ksp, kphx, and kg are wavenumber vector of a surface plasmon, an incident light component in x direction (propagation direction), and a grating, respectively. kphx corresponds to kph sinθ, in which kph and θ are 2𝜋/λ (λ: wavelength) and the incident angle, i.e., the resonance angle, respectively. Therefore, eq (1) is described as eq (2).


in which εm and εd are complex dielectric constants for metal and dielectric media at an interface, and Λ is the pitch of a plasmonic chip. From above equations, the resonance angle θ is found to be controlled by the pitch Λ.

The mechanism of fluorescence enhanced by GC-SPR includes two processes [14,15]; excitation enhancement and fluorescence enhancement. Utilizing these two processes is important to achieve the most enhanced fluorescence, and in each excitation and emission wavelength, resonance angles are set as satisfying eq. (2), individually. On the other hand, to collect maximum fluorescence the plasmonic chip should be prepared from metal layers and an overlayer of silica with the optimal thickness [16], i.e., the distance from a metal surface needs to be arranged to be optimal for the suppressing fluorescence quench [17], the decay of plasmon field, and the reflection interference effect. From our experimental results, the optimal thickness of the silica layer is considered to be 30-40 nm [18]. Further, the pitch of 400-500 nm is convenient for fluorescence detection and a groove depth of 20-30 nm is found to be appropriate [19, 20]. As a representative metal layer, Au, Ag, and aluminum (Al) are generally used for the SPR sensor. Among these metals, the most enhanced electric field is expected from the Ag layer [15] but chemical stability is poor. The most stable layer is Au but the wavelength range available is narrow at > 550 nm. For Al, the wavelength range available is wide, from UV to near infrared range, but the surface is not stable and the enhancement factor in the visible range is also small compared to Ag and Au. As such, the metal layer should be selected according to the objectives for the metal layer's use.


An incident electric field is enhanced with a plasmonic chip, and the fluorescence from the dye attached to a chip's surface is enhanced by it. As a device based on the application of fluorescence enhancement, sensitive immunosensor and cell imaging systems have been constructed. In an immunosensor for a clinical diagnosis, an analyte (mainly antigen) can be quantitatively detected with its antibody in the sandwich assay. In cell imaging, the same process of scattering a cell is performed on a plasmonic chip instead of in a glass-based dish, so that the enhanced fluorescent image of labeled cells can be obtained.


Fig. 2: Fluorescence intensity measured against the concentration of AFP and fitting the curve of the Langmuir isotherm. The red line corresponds to three standard deviations of mean fluorescence values for nonspecific adsorption.


Only fluorescence from dye attached to the chip surface is selectively detected with a plasmonic chip and therefore, it is effective as a sensitive immunosensor. The three elements are required for the development of an excellent immunosensor: 1) a sensitive measurement system, 2) an excellent antibody with good affinity, and 3) an interface that can suppress nonspecific adsorption and that can simply and effectively bind a capture antibody. While satisfying these conditions, a plasmonic chip is used as a good immunosensor for some analytes [21-25], and representative data for an alpha-fetoprotein (AFP) assay [21] and an interleukin-6 (IL-6) assay [22, 23] are introduced here.

In an AFP sandwich assay, a silica layer acting as the surface of a plasmonic chip was modified with (3-aminopropyl)triethoxysilane (APTES) and a capture antibody was bound to an amino group at a surface using a NHS linker [21]. The detection antibody was labeled with Alexa647 using a labeling kit. By the suppressing the non-specific adsorption, in the silver plasmonic chip the limit of detection (LOD) was evaluated as 4pg/mL (55 fM) as shown in Fig. 2. In order to improve the LOD, the stability of optics including incident light intensity and the reproducibility of a plasmonic chip structure should be considered.

In an IL-6 sandwich assay, an SPF image was observed by transmission light illuminated from the rear panel of a plasmonic chip and the fluorescence intensity was analyzed from the image measured for each analyte concentration [22]. The binding process of a capture antibody and preparation of labeled-detection antibody are the same as above description for an AFP assay. After analysis of the SPF image, the LOD was evaluated as 2 pg/mL as shown in Fig.3. This LOD also provides an efficient means to elucidate IL-6 in clinical diagnosis.


Fig. 3: Fluorescence intensity (red solid line) evaluated from fluorescence images plotted against the concentration of IL-6. The blue line indicates the fluorescence intensity without IL-6 (nonspecific adsorption).


Under a fluorescence microscope, a plasmonic chip can provide an enhanced fluorescent image if the resonance angle is included into the illumination angles [26, 27]. In cell imaging, the fluorescent molecules in the membrane side attached to the chip surface are selectively excited by the SPR field and a bright fluorescent image can be obtained. The enhanced fluorescent images of cultured neurons and breast cancer cells are introduced here.

A plasmonic dish was fabricated as a cell-culture dish for in-situ fluorescence imaging applications, in which the cover glass of a glass-bottomed dish was replaced by a plasmonic chip [28, 29]. Neurons were cultured for over two weeks in the plasmonic dish. The fluorescent images of their cells, including dendrites, were simply observed using a conventional upright fluorescence microscope. The fluorescence from neuronal cells growing along the dish surface was enhanced using the surface plasmon resonance field. The fluorescence intensity of the neuron dendrites was found to be enhanced efficiently by an order of magnitude compared with the fluorescence intensity from using a glass-bottomed dish (Fig.4). Even if the culture term is short, the neuron dendrites were clearly observed. Furthermore, in a transmitted-light fluorescence microscope, the surface-selective fluorescent image of a fine dendrite growing along the dish surface was observed with a high spatial resolution (Fig. 5).


Fig. 4: Fluorescence images of neuronal cells cultured on (a) a glass-bottomed dish and on (b) a plasmonic dish. Both images are shown using the same contrast, i.e., they were set to 5500 counts for the maximum - minimum values. The bars correspond to 25 μm.


Fig. 5: Fluorescence images of neuron cells cultured for two weeks on a plasmonic dish observed with: (a) a transmitted-light fluorescence microscope, and (b) an epi-fluorescence microscope. The bar corresponds to a distance of 25 μm.

In breast cancer cell observations [30, 31], the epithelial cell adhesion molecule (EpCAM) and the epidermal growth factor receptor (EGFR) were observed in the Michigan cancer foundation-7 (MCF-7) and MDA-MB-231 using a plasmonic chip, i.e., the surface plasmon field-enhanced fluorescence. Expression level of EpCAM in MCF-7 cells is generally known to be more than that in MDA-MB-231. In our first study of breast cancer cells, EpCAM was labelled with an allophycocyanin-labeled anti-EpCAM antibody (APC-EpCAM), and the brighter fluorescent images of MCF-7 and MDA-MB-231 cells were obtained on the plasmonic chip with 480-nm pitch compared with those on the glass slide [30]. In the second study, these membrane proteins, EpCAM and EGFR, that acted as the surface markers used to differentiate breast cancer cells were then detected with the dye Alexa 488 - labeled anti-EGFR antibody (488-EGFR) and APC-EpCAM, respectively [31]. For the MDA-MB231 cells, 3-fold and 7-fold fluorescence enhancement in 488-EGFR were observed on the bull's eye-type plasmonic chip with 480-nm pitch and the 400-nm pitch (Fig. 6 (c)), respectively (compared with the fluorescence intensities on a glass slide). On the other hand, 9-fold fluorescence intensity in APC-EpCAM was obtained on a 400-nm pitch plasmonic chip (Fig. 6(b) and (d)). So, dual-color fluorescence of 488-EGFR and APC-EpCAM in MDA-MB231 was clearly observed on the plasmonic chip with a 400-nm pitch as shown in Fig. 6. Fluorescence enhancement with a plasmonic chip depends on the wavelength. The surface plasmon coupling at the 400-nm pitch contributed to the enhancement of the excitation field for APC-EpCAM and to the collection of the surface plasmon-coupled emission (SPCE) for 488-EGFR effectively under the microscope. Therefore, the 400-nm pitch contributed to the dual-color fluorescence enhancement for these wavelength ranges.


Fig. 6: Fluorescent images of 488-EGFR (a,c), and of APC-EpCAM (b,d) in MDA-MB-231 cells. The upper and lower images correspond to those on the glass slide and the bull's eye-plasmonic chip with 400-nm pitch, respectively. The 488-EGFR and APC-EpCAM images shown in (a,c) or (b, d) were adjusted to the same scales between minimum and maximum brightness. Bar corresponds to 10 µm.


The structure and mechanism of a plasmonic chip in providing enhanced fluorescence is explained and the application of plasmonic chips to immunosensor and cell imaging is introduced. The plasmonic chip is an excellent and simple tool for the sensitive detection of analytes, but sensor interface suppressing non-specific interaction and densely binding capture antibodies are required for more effective biodetection. With the improvement of the sensor interface, further sensitive detection can be implemented. On the other hand, in the fluorescent images of cells, the fluorescent molecules existing in the membrane side attached to the chip surface, i.e., the fluorescent molecules located within 200-nm distance from the surface, are selectively excited by SPR field and a bright fluorescent image can be obtained. Distribution of EpCAM and EGFR into cells is clearly observed on the plasmonic chip with an appropriate pitch; this distribution cannot be observed on a glass slide. The enhancement of fluorescence using a plasmonic chip is useful to detect small signals, and further applications of plasmonic chips are expected to be discovered in other fields.

Acknowledgements: I thank Prof. Dr. J. Nishii, Prof. Dr. XQ. Cui, Dr. K. Kintaka, Dr. S. Yamamura, Prof. Dr. C. Hosokawa, and Dr. T. Kaya, for their respective collaborations and discussions. I also thank C. Yasui, F. Kondo, M. Tsuneyasu, and S. Izumi for performing experiments and analyzing data. I thank Toyo Gosei for providing the UV-curable resin PAK-02-A. This work was supported by JSPS KAKENHI Grant Numbers 19049016 on Priority Area "Strong Photons-Molecules Coupling Fields (No. 470)", JP15H0110 in Scientific Research on Innovation Areas "Photosynergetics", 25286032 in Scientific Research (B), and JP16H02092 in Scientific Research (A).


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Keiko Tawa is a professor at the School of Science and Technology, Kwansei Gakuin University, Japan. She received her PhD in engineering from Kyoto University, Japan in 1995. Her research interests are spectroscopy and plasmonics, specifically in the field of nano-biophysics including sensor and optical imaging of cells and proteins.

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