Development of plasmonic substrates for biosensing

Development of plasmonic substrates for biosensing

Alexandre G. Brolo*a, Jacqueline Ferreiraa, Marcos Jose Leite Santosa, Carlos Escobedob,David Sintonb, Emerson M. Girottoc, Fatemeh Eftekharid and Reuven Gordond

a Department of Chemistry, P.O. Box 3065 STN CSC, University of Victoria, Victoria, BC, Canada, V8W 3V6;

b Department of Mechanical Engineering, PO Box 3055 STN CSC, Victoria, British Columbia, Canada, V8W 3P6;

c Department of Chemistry, Universidade Estadual de Maringa, Av: Colombo 5790, 87020900 Maringa, Parana, Brazil;

d Department of Electrical and Computer Engineering, P. O. Box 3055 STN CSC, University of Victoria, Victoria, B.C., Canada, V8W 3P6;

*[email protected]; phone 1 250 721-716734; fax 1 250 721-7147.

ABSTRACT

The transmission of normally incident light through arrays of subwavelength holes (nanoholes) in gold thin films is enhanced at the wavelengths that satisfy the surface plasmon resonance (SPR) condition. Our group has been active on the implementation of schemes for the application of this phenomenon for chemical sensing. For instance, we have shown that the interaction between adsorbates with nanoholes modified the SP resonance conditions, leading to a shift in the wavelength of maximum transmission. The output sensitivity of this substrate was found to be 400 nm RIU-1 (refractive index units), which is comparable to other grating-based surface plasmon resonance devices. The array of nanoholes was also integrated into a microfluidic system and the characteristics of the solution flow and detection systems were evaluated. In this work, we will concentrate on improving the efficiency of the nanohole arrays for applications in chemical in chemical sensing. Attempts to improve the sensitivity of the device will be discussed. In-hole sensing is suggested as an alternative to decrease the number of probe molecules, and enhance sensitivity. A biaxial sensing scheme will also be introduced. The biaxial scheme allows for polarization-modulation detection that can account for background fluctuations. A flow-through approach should lead to an optimized transport situation of the analytes to the immobilized species at the surface, which should significantly improve the time and sensitivity of the analysis. Finally, we will discuss the implementation of multiplexing detection using these arrays. Multiplexing detection in zero-order transmission is simpler to implement than the common multiplexing imaging from angle-resolved SPR.

Keywords: Surface plasmon resonance, plasmonics, nanoholes, extraordinary optical transmission, nanooptics, SPR

1. INTRODUCTION

Surface plasmons (SPs) are collective electronic oscillations that can be excited in free-electron metals (Cu, Ag, Au) using visible radiation when the conditions for energy and momentum conservation are satisfied1.The SP dispersion in a planar film is given by:

effx ck (1)

Invited Paper

Biosensing, edited by Manijeh Razeghi, Hooman Mohseni Proc. of SPIE Vol. 7035, 703503, (2008) · 0277-786X/08/$18 · doi: 10.1117/12.793798

Proc. of SPIE Vol. 7035 703503-12008 SPIE Digital Library -- Subscriber Archive Copy

SeS

where kx is the wave vector of the electromagnetic field propagating parallel to the surface, is the angular frequency and c is the speed of light. eff is the effective dielectric constant given by:

md

mdeff (2)

d and m are the real part of the dielectric constants of the adjacent medium and the metal, respectively. It is clear from equations (1) and (2) that the surface plasmon resonance (SPR) conditions are affected by the modification of d. The dielectric properties of the adjacent medium close to the metal surface are highly sensitive to the presence of adsorbates, which constitute the basis for the application of SPR in chemical sensing2.

Moreover, equations (1) and (2) shows that the SP momentum is larger than the parallel component of the

free photon momentum in air, given by c . This means that the SP in a planar film cannot, in principle,

be directly excited in the visible region. In order to excite SPs, it is necessary to provide an extra increase in the kx-value of the light to match the value of the SP k-vector. There are two common methods used to overcome this momentum mismatch. One is to couple the light into SP using a prism. Periodic structures, such as gratings, can also provide the required extra momentum1.

The prism coupling is experimentally realized using the total internal reflection configuration. The most used method is the so-called Kretschmann configuration. According to equations (1) and (2), molecular adsorption modifies the effective value of d, provoking shifts in the SPR conditions. This is the physical basis for the sensitivity of the commercial SPR devices available, which are widely used in bioanalytical applications2.

Another approach for SP excitation is through grating coupling3. In this case, an extra kx component is added to the light parallel momentum due to the grating periodicity. However, the grating sensitivities are generally smaller than the prism coupler arrangement and gratings are seldom used for analytical applications2. Grating-based SPR devices can operate in either reflection or transmission geometries. Our group is recently exploring the excitation of SP modes in transmission gratings consisting of periodic arrays of nanoholes in metallic thin films4, as shown in Figure 1, as chemical sensors.

Figure 1. Scanning electron microscope (SEM) image of a nano-hole array created by focused-ion beam (FIB) milling of a 100 nm gold film on glass. The holes have 200 nm diameters and are spaced by 600 nm. The SEM is imaged at an angle to show side-wall characteristics of the FIB process.

The excitation of SPs in a periodic array of nanoholes leads to the phenomenon of extraordinary optical transmission (EOT). EOT is observed at specific wavelengths that match the Bragg conditions of the structure5. The absolute transmission efficiencies at the peaks are at least twice the amount of light that

Proc. of SPIE Vol. 7035 703503-2

impinge on the holes6. The wavelength for enhanced transmission must satisfy the SP excitation condition. For normal incidence, the wavelength of SP resonance ( SP) can be calculated using:

22),(

ji

pji

eff

SP (3)

Where p is the lattice constant (periodicity) of the array, i and j are integers that define the scattering orders of the array and eff is given by equation (2). The phenomenon of EOT has received a large amount of attention from both theoretical7,8 and experimental groups, due to the potential of these substrates for a wide range of applications. Several groups9-13, including ours14-18, had demonstrated that periodic arrays of nanoholes can be used in bioanalytical applications by exploring either the wavelength shift due to molecular adsorption or the enhanced localized electromagnetic field that yields a significant increase of the spectroscopic response of the analyte. For instance, Figure 2 shows a typical result for a chemical sensing experiment using periodic arrays of nanoholes14,18. In this case, the transmission peak of the SPR observed for a bare array of nanoholes shift to the red when the gold surface is coated with an analyte (a protein in this case). The asterisks in Fig. 2 indicates a minimum before the raise of the SPR peak, which is due to a diffraction phenomenon known as the “Wood’s anomaly”. The dotted line in Fig. 2 indicates the wavelength of a He-Ne laser (632.8 nm), which is typically used in sensing schemes using a single wavelength.

Figure 2: Shift in the transmission resonance of a nano-hole array in a gold film after the adsorption of a self-assembled monolayer. Dashed line shows operating point for single wavelength source.

The periodic arrays of nanoholes has several advantages over other SP-based devices, such as the confinement of analyte in the nanoholes, the large electric field density, the structured (and predicted) plasmonic structure and the collinear geometry of the transmission measurement, which make them ideal for miniaturization, as demonstrated in Figure 314,18.

In this work, we will describe some of the latest advances realized by our group towards the improvement of the sensitivity of these arrays for application in chemical sensing. As mentioned above, the grating based devices are not as sensitive (in terms of sensor output measured) as the commercial SPR systems based on the Kretschmann configuration. We are trying to overcome this problem by decreasing the amount of analyte being detected and by improving the transport conditions to the device. We are also implementing a multiplex scheme for the ultimate goal of simultaneous detection of various biological markers for early cancer diagnostic.

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Figure 3. (top left) Schematic of microfluidic chip for fluid delivery over a chamber incorporating nano-hole arrays on bottom surface. (top right) As fabricated microfluidic chip incorporating nano-hole arrays. (bottom left) Microfluidic control of dyed fluid over nano-hole arrays of same 650 nm periodicity (1-6), and varying periodicity. (bottom right) Shift in peak transmission wavelength observed for sugar solution focused over top two 650 nm arrays.

2. RESULTS AND DISCUSSION

2.1 Improving the analytical sensitivity of arrays of nanoholes

In this section, we will discuss our attempts to improve the analytical sensitivity of the nanohole arrays. The first idea is to try to decrease the number of molecules being detected, without changing too much the output signal of the sensor. This was achieved using an in-hole sensing scheme. The second approach is to improve the S/N of the output signal by implementing a modulated sensing scheme based on a biaxial array.


2.1.1 In-hole sensing

The main advantage of the in-hole sensing is illustrated in Figure 4, which depict a side view of a nanohole. In all sensing experiments reported so far, the gold surface is fully exposed to the analyte. This means that adsorption of the analyte will happen inside the hole and in the space between holes (Fig. 4a). However, by blocking the gold surface between the holes, it is possible to decrease the amount of molecules being probed. For a typical array used in our group, as the one presented in Fig. 1, this would translate in about 10 times less molecules being probed.

Figure 4. (a) Schematic representation of a typical situation in nanohole sensing experiments. The red ellipses represent the analyte. In this case, we observe signal from species adsorbed inside the hole and on the top surface. (b) Structure proposed in this work. The top gold surface is blocked by an inert layer and molecular adsorption only takes place inside the holes.

The question remains what would be the effect of the in-hole sensing on the signal output of the sensor? In order to address this point, the relative importance of the surface plasmons on the surface around the holes, and the surface plasmon contribution from the modes within the hole was calculated using FDTD methods. The results of the calculations are presented in Figure 5.

a ba b


wavelen gil {nm)

S....S * S

• * .S....E

Figure 5. FDTD simulation of transmission spectrum through a nano-hole array. The square array has a 400 nm periodicity and the holes in the array are cylindrical with 50 nm radius. The array is made in a 100 nm thick gold film, supported by a glass substrate, with a 5 nm Cr layer between the gold and glass (as typically used to aid adhesion). A 5 nm thick surface layer with refractive index step of 0.1 is added to the top surface (gray-solid), to the inner surface of the hole (black-dashed), and to both the top surface and to inside the hole (gray-dashed).

It is clear from Fig. 5 that the molecules inside the nanohole carry the major contribution to the SPR shift observed from this substrate. Therefore, a significant improvement in the analytical sensitivity should be possible by blocking the top surface and sensing only the molecules inside the hole. We have realized this experimentally, by using a sol-gel procedure to grow an ultra thin SiOx layer (50 nm thick) on top of a gold film and then milling the holes through the layer and the gold. The resulting structure now corresponds to the schematic representation of Fig. 4b, with the SiOx being represented by the “blocking layer” (represented in blue in Fig. 4b). The SEM of the resulting structure and the white light spectra when exposed to solutions with different refractive index is shown in Figure 6. Notice that the observed shift now corresponds mainly to the interaction of the solution with the walls inside the hole.

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Figure 6. (left) arrays of nanoholes on a gold surface covered by SiOx. The only gold surface with direct access to the solution is inside the holes. (right) transmission spectra through the array on the left in contact with solutions of different refractive indexes.


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2.1.2 Biaxial Arrays

Multiplexing of the arrays of nanoholes as chemical sensors would be facilitated by an intensity detection scheme in contrast to the wavelength detection demonstrated so far in this work. In this case, variations in intensity at a single wavelength are indicative of molecular adsorption. He-Ne and diode lasers are the common light source for this type of schemes. The light transmitted through the structure can then be detected by a photodiode or by a CCD camera. One drawback of this detection scheme is fluctuations caused by optical scattering due to the solution and other impurities. A modulation procedure would be ideal to eliminate this type of noise and increase the S/N ratio. We propose a biaxial array, with different periodicities in the x and y directions, as defined in Figure 7, as a possible sensing element that would allow polarization modulation and the elimination of unwanted noise. The different periodicities in both directions would allow the excitation of SP resonances at different wavelengths by controlling the polarization of the incident light (the SP is excited at the same direction of the electric field polarization).

Figure 7 (left) shows a typical biaxial array of nanoholes and the transmission spectra obtained at two incident polarizations, defined at the figure. Different SP resonances are accessed for both polarizations. As demonstrated earlier, the transmission spectra shifted when an organic molecule is adsorbed at the surface. The two resonances are well separated, allowing the monitoring of intensity variations at a particular wavelength. Following the initial demonstration presented in Fig. 6, we are now working on the implementation of a polarization modulation scheme and on the quantification of the S/N improvement achieved by this approach.

2.2 Improving the transport characteristics of the analyte using a flow-through system

The in-hole sensing demonstrated in section 2.1.1 already showed a significant improvement on the analytical characteristics of the arrays of nanoholes. In this section, we will describe our attempts to also improve the transport characteristics of the in-hole sensing scheme. The nanoholes do not provide only a chemical and optical platform for sensing, but they are also nanochannels. The nanometric dimensions of the holes offers an opportunity for increase transport of the analytes in solution to the capturing species attached to the gold wall inside the holes, if a flow-through approach is implemented. Preliminary fluid dynamics calculations were performed and are presented in Figure 8. The total adsorption of the bio-analyte is predicted and it can be controlled through the flow rate. An schematic representation of the proposed flow-through device is also shown in Figure 8.


+

•

Figure 8: (left) Fluid dynamics modeling results showing biomarker transport to active sites at the surface of a nanohole: a.) nanohole-cross-section, and b.) in-plane, concentration profiles for low average in-hole velocity case (1 m/s ); c.) in-plane concentration profile for the high average in-hole velocity case (1 cm/s). Reaction rates at the surface were set to 106 M-1s-1, characteristic of antibody-antigen reactions. The diffusivity was set to D = 4x10-11 m2s-1 based on the hydrodynamic radii of large proteins such as lysozymes and glycoproteins. Red to blue colour contours indicate analyte concentration with initial inlet concentration as red. (right) Schematic of the integrated flow-through nanohole concept.

We have recently been able to fabricate the flow-through structures, by milling the holes on a gold film deposited on a silicon nitride membrane. The optical and mechanical characteristics of the substrate are currently being tested.

2.3 Multiplexing

Among the main advantages of the arrays of nanoholes as SPR sensing is the fact that the normal transmission simplified the optical setup and facilitates multiplexing. In fact, multiplexing using arrays of nanoholes have already been reported by several groups19,20. We are also developing this aspect, but with a specific focus of implementing a device for multiple detection of specific targets for ovarian cancer. Ovarian cancer is among the hardest to be diagnostic, and the proposed device would allow a fast blood sample screening.

Figure 9 shows the SEM and the optical images of a testing device fabricated by our group. The device consists of two columns with three rows containing four arrays of different periodicities. Four arrays per row is used to provide redundant measurements to validate the methodology. The arrays were illuminated by an uniform He-Ne laser that probes the whole area of interested and were imaged using a CCD camera. The laser wavelength is close to the SPR resonance of the arrays in pure water, and the addition of adsorbates shift the SPR, leading to a change in the intensity of the laser light transmitted. A typical result is illustrated in Figure 10, where a typical intensity profile of one of the arrays investigated. The profile’s intensity changes when the nanonoles in phosphate buffer solution (PBS) is modified by a monolayer of cysteamine – biotin species. The biotin group is well known to capture the protein straptavidin with high affinity. Addition of straptavidin leads to further changes in the intensity, indicating the interaction of the protein with the surface. The results presented in Figure 10 for one array is reproduced by the others, indicating that the adsorption events were detected by all arrays. We are now implementing schemes for


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multiple detection of distinct analytes and are working towards the determination of the sensitivity of these devices to ovarian cancer antibodies.

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425455

Figure 9: (left) columns or arrays of nanoholes integrated in microfluidics channels for imaging. (right) SEM pictures of one of the rows containing four arrays.

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5

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15

20

25

30

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40

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Figure 10: Intensity profile over one of the arrays of nanoholes (see inset). The different profiles correspond to an affinity test experiment where the surface is first modified with a biotin linker. Then, a solution containing the protein straptavidin is introduced. The binding between the biotin and the straptavidin leads to a change in the intensity for this array.


3. CONCLUSIONS

We have discussed some of the projects that are currently being developed in our group aiming at the improvement of the sensing capabilities of arrays of nanoholes on gold films. We have shown that in-hole sensing can detect less molecules without significant affecting the sensor output. This important effect, allied to the improved diffusion of analytes to the gold wall during flow-through, indicate that the utilization of the nanoholes as channels might provide a breakthrough device in terms of sensitivity, efficiency and analysis speed. The S/N ratio, which might suffer for unwanted variations from the microfluidic environment, may also be improved by using a polarization modulation scheme. We demonstrated that biaxial arrays (with different periodicities in both directions) allow this polarization modulation. Finally, we also show that the multiplex detection of simultaneous adsorption events are also possible with micro-arrays of arrays of nanoholes. The results presented here indicate the potential of arrays of nanoholes as chemical sensors and explores the advantages of these structures over the commercial technologies for future implementation.

4. ACKNOWLEDGEMENT

We gratefully acknowledge funding support for this work through an NSERC Strategic Grant with the BC Cancer Agency, and Micralyne Inc. Infrastructure funding was provided by CFI and BCKDF.

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Development of plasmonic substrates for biosensing