Enhanced PNA Detection Sensitivity based on Polymer-cladded Porous Silicon Waveguide

Transcription

1 Enhanced PNA Detection Sensitivity based on Polymer-cladded Porous Silicon Waveguide Yang Jiao and Sharon M. Weiss* Department of Electrical Engineering and Computer Science, Vanderbilt University, Nashville, TN, USA ABSTRACT In this work, we theoretically and experimentally demonstrate a highly sensitive polymer-cladded porous silicon (PSi) membrane waveguide based on a ~1.55 m thick porous silicon membrane coated on one side with a low loss polymer. The sensor operates in the Kretschmann configuration, which is amenable to microfluidics integration, with a high index cubic zirconium prism. The sensitivity of the sensor is investigated through PNA hybridization in the PSi membrane. We demonstrate that higher angle resonances and a proper ratio of PNA length to PSi pore diameter lead to significantly improved detection sensitivity. A detection sensitivity below 0.1/M is reported for 16mer target PNA. Calculations and complimentary experiments show that careful tuning of the polymer cladding thickness can further improve the detection performance. Keywords: Porous silicon, waveguide, sensitivity, polymer-cladded, PNA, perturbation theory, Kretschmann 1. INTRODUCTION The large available surface area for molecular binding and strong field confinement in porous waveguides has generated much attention recently for biosensing applications. Significantly enhanced detection sensitivity can be achieved using porous waveguide sensors instead of conventional planar waveguide and surface plasmon resonance sensors, especially for small molecule detection. Porous waveguides allow interaction of molecules with a strong localized electric field throughout the volume of the waveguide instead of only on the surface of a sensor. Dielectric and metal-cladded waveguide biosensors in the Kretschmann configuration have been investigated by several groups. For example, a gold cladded porous silica biosensor was reported for the detection of biotin-streptavidin binding [1]. Gold-cladded TiO 2 [2] and porous alumina [3] waveguides have been demonstrated for the detection of aqueous sucrose solution and bovine serum albumin adsorption, respectively. A polymer-cladded porous silicon (PSi) waveguide was shown for the detection of DNA molecules [4]. PSi waveguides in the Otto configuration have also been demonstrated for DNA detection [5], [6]. In this work, we build upon the recent reports of a polymer-cladded porous silicon membrane waveguide [4] and methods [7], [8], [9] to optimize the porous waveguide design, including pore diameter, for the detection of particular target molecules. Among porous materials, porous silicon is an excellent material for biosensing due to the ease of tuning the pore depth, pore morphology, and pore diameter, which can range from micropores (<2nm) to macropores (>50nm), through straightforward tuning of the electrochemical etching parameters [4], [10]. The pore size-dependent sensitivity of detecting variable sized biomolecules was very recently characterized using porous silicon waveguides with optimized designs [11]. Here we show the application of an optimized polymer-cladded PSi waveguide for PNA detection and report on the detection sensitivity. * sharon.weiss@vanderbilt.edu; Phone ; Fax

2 2.1 Mathematical modeling and methods 2. MATERIALS AND METHODS Figure 1 (a) shows the schematic for the PSi membrane waveguide with polymer cladding in an air ambient. The PSi film has a higher refractive index than the polymer and consequently supports waveguide modes when the optical thickness of the film is designed appropriately. Light is coupled into the PSi waveguide using a high index cubic zirconium prism (n= at =1550nm) in the Kretschmann configuration. Transverse electric (TE) polarized light from a 1550 nm diode laser is coupled into the PSi layer through the polymer cladding via an evanescent wave. When the tangential component of the incident wave vector in the prism matches the wave number of a guided mode, the propagating electric field will be confined tightly inside the PSi layer. Waveguide coupling is monitored by measuring the intensity of light exiting the prism. For most angles of incidence, light simply reflects off the prism base, leading to high intensity of light at the detector. A sharp resonance dip is observed in the angle-resolved reflectance spectrum only at angles that satisfy the wave vector matching condition. When molecules are infiltrated and interact with the strong electric field inside the PSi layer, the overall refractive index of the PSi layer changes and leads to a resonance angle shift. Figure 1 (b) shows a cross-sectional SEM image of a ~57% porosity PSi film with ~60 nm pore openings. Since the pore diameters are much smaller than the wavelength of interrogating light, porous silicon can be treated as an effective medium [12]. (a) (b) 100 nm Fig. 1. (a) Schematic diagram of polymer-cladded PSi sensor. This waveguide structure consists of a low index cladding layer and a high index core layer. Multiple waveguide modes are supported in the high index porous core layer. A prism is used to couple the light into the PSi layer via an evanescent wave. (b) Cross-sectional SEM image of a PSi sample with the 57% porosity and ~60 nm pore opening. The well-known transfer matrix method [13] is used to model this multilayer structure. In the transfer matrix method, the incident electromagnetic field in each layer is assumed to be continuous and, at each interface between two mediums, the tangential components of electric and magnetic fields are also assumed to be continuous. First order perturbation theory is used to analyze the detection sensitivity of the polymer-cladded PSi waveguide [14], [15] since the infiltrated

3 molecules will only slightly change the overall refractive index of the PSi layer. Compared to the original refractive index of the PSi layer, the refractive index change due to the molecule infiltration is much smaller. This small refractive index change n will generate a corresponding change of the waveguide effective index N. The effective index, N, can be related to the resonance angle inside the prism via the function N=n p sin where n p is the refractive index of the prism. Since sensitivity is defined as the ratio of the shift of resonance angle to the change of environmental index in the case of angular interrogation, the sensitivity of the waveguide can be calculated by [8] sensitivity d dn PSi E x 2 dx Ex 2 dx n PSi N 1 n p cos where the n p and n PSi are the refractive index of the prism and PSi respectively, and is the resonance angle in the prism. The first term of the right-hand side of Eq (1) is the power confinement factor, which is defined as the ratio of power confined in the porous silicon layer to the total power distributed throughout the entire waveguide structure. The waveguide detection sensitivity is directly proportional to the power confinement factor and the incident angle. It has been shown that the 1/cos term in Eq (1) is the dominant term and the highest sensitivities are achieved at the largest resonance angles [8]. The resonant coupling angle can be tuned by proper design of the PSi film porosity and thickness. 2.2 Waveguide fabrication The single layer PSi membrane waveguide structure was fabricated on highly doped n-type (0.01 -cm) silicon wafers by straightforward electrochemical etching method. An electrolyte containing 5.5% hydrofluoric acid (HF) in deionized water (25 ml 50% aqueous HF ml deionized water) was used to etch the PSi samples. By applying different continuous current densities and etching time, PSi samples with different porosities and corresponding pore diameters were formed on the silicon substrate. In this work, 30 ma/cm 2 current density was applied for 72 s to form PSi films with approximately 57% porosity, 1.55 m thickness (50 nm), and ~60 nm pore diameters. The PSi films were removed from the silicon substrate by applying a series of 160 ma/cm 2 current pulses for 4 s with 50% duty cycle. The lifted-off PSi films were then thermally oxidized at 500C for 5 min in an Omegalux LMF-3550 oven, after insertion at 300C [8]; a 0.8 nm thin oxide layer was formed on the pore walls of the porous silicon membrane. The polymer-cladding layer was fabricated by dropping approximately 0.15% formvar polymer in ethylene dichloride (Ernest F. Fullam, Inc) onto the cubic zirconium prism with a refractive index of (Metricon). After the evaporation of ethylene dichloride, an approximately 700 nm thick polymer layer with a refractive index of ~1.49 at 1550 nm was formed. The PSi membrane was placed on top of the polymer solution before the solvent dried out to ensure that there was no air gap between the polymer layer and PSi layer. A uniform and robust adhesion between polymer layer and PSi layer has been demonstrated using this method [8]. (1) 2.3 Porous silicon waveguide surface functionalization In order to detect particular target biomolecules, functionalization steps need to be carried out to link organic molecules to the inorganic silicon pore walls. Selective detection of a particular target biomolecule is achieved by proper choice of a complimentary probe molecule that is immobilized in the pores. In our experiment, the PSi sample was firstly exposed to 4% 3-aminopropyltriethoxysilane (3-APTES) in a humid environment for 20 min, and baked at 100C for 10 min. A resulting 0.8 nm uniform silane layer was formed on the pore walls [16]. The cross-linker Sulfo-SMCC in 50% water and 50% ethanol was attached to the silane monolayer to provide the necessary surface chemistry for probe DNA attachment. Next, 200 M of 16mer probe DNA in HEPES buffer was mixed 1:1 by volume with TCEP in water and ethanol [7]to obtain 100 M probe DNA solution, and this 100 M 16mer probe DNA solution was infiltrated into the PSi sample. For the sensing experiments, complimentary, uncharged 16mer PNA molecules were exposed and hybridized to the negatively charged probe DNA molecules in a humid environment for one hour. Here we used the PNA molecules to avoid the possible corrosion of the PSi pore walls caused by hybridizing negatively charged DNA molecules [17].

4 3. EXPERIMENTAL RESULTS AND SENSITIVITY ANALYSIS 3.1 Experimental measurements Fig. 2 demonstrates the experimentally measured changes of resonance angles of the polymer-cladded PSi waveguide due to the pore surface functionalization, including the infiltration of 3-APTES, Sulfo-SMCC, 100 M probe DNA and 10 M complimentary PNA. By matching the experimentally measured resonances with simulation results, the refractive index of the PSi film is calculated to be ~2.05, which corresponds to the porosity of ~57% based on the Maxwell-Garnett effective medium theory [12]. From Fig. 2, it is clear that in the limited Metricon prism coupler measurement range (32-68) this PSi waveguide supports 3 rd, 2 nd, and 1 st order modes, which are initially located at ~38, ~51, and ~62, respectively, as shown by the black solid line in this figure. Each functionalization step was detected as the resonance angle shifts to the higher values. The magnitude of the resonance shift directly correlates to the size and number of molecules attached. 1.0 Reflectance (a.u.) rd 2 nd Oxidized Silanized SMCC Probe DNA PNA 1 st Internal Angle (deg.) Fig. 2. Total reflectance spectra of a 1.55 m thick, 57% porosity PSi waveguide after (from left to right) oxidation, and infiltration of 3-APTES, Sulfo-SMCC, 100 M probe DNA and 10 M target PNA. The resonance dips on the left, middle and right part of this figure are 3 rd, 2 nd and 1 st order modes, respectively. The resonance dips at higher angle (lower order mode) show larger shifts for all infiltrated molecules. 3.2 Sensitivity analysis of PNA detection In order to achieve higher detection sensitivity, both the ratio of PNA length to the pore diameter and the initial resonance angle must be considered. We recently showed that different DNA lengths give different infiltration efficiencies for different pore sizes [11]. Based on the results of that study, in this work we choose to use a PSi film with ~60 nm pore diameters to allow easy infiltration of 16mer PNA (3.25 nm length) into pores functionalized with 3- APTES (0.8 nm), Sulfo-SMCC (1.9 nm), and 16mer single strand DNA (3.52 nm). Based on the theory described in section 2.1 and [8], lower order waveguide modes are supported at higher resonance angles, which leads to higher detection sensitivity. From Fig. 2, we can observe that the shifts for 3 rd, 2 nd, and 1 st order modes after the infiltration of 0.8 nm silane molecules are 1.1, 1.25, and 1.43 respectively. Here we assume that the pore walls are approximately 90% uniformly covered by the small silane molecules [11]. Similarly the resonance angle shifts from 16mer probe DNA to 16mer complimentary PNA for 3 rd, 2 nd, and 1 st order modes are 0.46, 0.67, and 0.72 respectively, which shows the similar trend of increased sensitivity for higher angle resonances, as predicted. We note that the 16mer complimentary PNA shows a smaller resonance shift compared to the resonance shift from oxidization to silanization although the length of PNA molecules is much larger than the size of 3-APTES molecules. This smaller magnitude of resonance shift can be attributed to the much lower surface coverage of the larger 16mer probe PNA, which was reported to be ~15% [11].

5 In order to calculate the complimentary PNA detection sensitivity, different concentrations of 16mer PNA solutions were infiltrated into the porous silicon samples to hybridize with the immobilized DNA molecules. Fig. 3 shows the result of the experimentally measured resonance angle shifts of the 2 nd order mode due to the infiltration of 5, 10, and 50 M 16mer complimentary PNA solution. The PNA detection sensitivity is defined as the resonance angle shift per micromolar concentration of PNA (degree/m), and higher concentration of PNA solution will correspond to an increased PNA coverage. Therefore the slope of the fitted curve in the linear region indicates the PNA detection sensitivity is ~0.1/M. The resonance angle shift saturates for PNA concentrations greater than 10 M. The maximum resonance shift for the 2 nd mode is 0.67, corresponding to a surface coverage of 15%. The surface coverage of PNA is limited by the low density of DNA probes on the pore walls Resonance Shift (degrees) mer 16-mer PNA Concentration PNA concentration (M) Fig. 3. Experimentally measured resonance shifts for PSi membrane waveguides infiltrated with different concentrations of 16mer complimentary PNA (black square). The slope of the fitted curve in the linear region gives a PNA detection sensitivity of ~0.1/M. The resonance shift saturates at 0.67 for concentrations above 10M when the available immobilized probe DNA molecules are all hybridized. 3.3 Optimization of the cladding thickness Optimal sensor performance requires a narrow and deep reflectance resonance in addition to a large degree/m shift. Therefore, after adjusting the porosity, pore opening and thickness of the PSi core layer based on the methods described previously, the optimum cladding thickness was also investigated. If the cladding layer is too thin, light will be coupled efficiently back to the prism, causing the reflectance resonance dip to become shallow and broad. If the cladding layer is too thick, a significant fraction of the field will decay in the cladding layer before reaching the PSi waveguide, leading to a shallow and small resonance dip. From the pole expansion method stated in [18], [19], we know that the optimum cladding thickness is related to the resonance angle; different order modes have different optimum cladding thickness. Therefore, for a single PSi sample, the cladding thickness can only be optimized based on one single mode. Fig. 4 shows the comparison results for 2 nd order resonance dips of the 57% porosity, 1.55 m thick porous silicon membrane waveguides with different concentrations of formvar polymer solution. Since the thickness of the formed polymer layer is directly related to the concentration of the formvar polymer solution, the cladding thickness can be tuned by diluting the formvar polymer solution to different concentrations. We note that the ~5 degree deviation in resonance angle between the different samples is likely due to the non-uniformity of the different PSi samples. A negligible shift results from the different cladding thicknesses over the given range. From the width and the minimum of those resonance dips, it is clear that the formvar polymer solution with the 0.139% concentration (red dashed line) gives

6 the optimized cladding thickness for 2 nd order mode. With the 0.178% concentration polymer solution (green dot-dashed line), a shallower and broader dip was obtained due to under coupling of the PSi structure, while with the smaller concentrations of 0.096% (black solid line) and 0.114% (blue dotted line), the resonance dips become broader and shallower due to overcoupling. 1.0 Reflectance (a.u.) % 0.114% 0.139% 0.178% Resonance Angles (degrees) Fig. 4 Comparison of experimentally measured 2 nd order resonances of the proposed PSi membrane waveguides with different concentrations of polymer solution. The polymer-cladding thickness can be tuned by adjusting the concentration of the formvar polymer solution. 4. CONCLUSION The increased available surface area for biomolecule attachment and the strongly confined field inside porous waveguides enable improved detection sensitivity of small molecules. We used a polymer-cladded PSi membrane waveguide to demonstrate a highly increased detection sensitivity of 16mer complimentary PNA molecules. The detection sensitivity directly corresponds to the mode order and corresponding position of the waveguide resonance angle. Lower order waveguide modes with higher resonance angles give larger responses upon infiltration of molecules. Using a polymer cladded 1.55 m thick 57% porosity PSi waveguide with 60 nm pores, we showed that the angular shift of the 1 st order mode was approximately 50% larger than that of the 3 rd order mode upon infiltration of 10M PNA. With optimized waveguide parameters and cladding thickness, detection sensitivities below 0.1 /M are achievable. 5. ACKNOWLWDGMENTS This work was supported in part by the Army Research Office (W911NF ) and the National Science Foundation (ECCS ). The authors gratefully acknowledge Judson Ryckman and Jenifer Lawrie for technical assistance and useful discussion. Scanning electron microscopy imaging was performed at the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE). REFERENCES [1] K. Awazu, C. Rochstuhl, M. Fujimaki, N. Fukuda, J. Tominaga, T. Komatsubara, T. Ikeda and Y. Ohki, High sensitivity sensors of perforated waveguides, Opt. Express 15, (2007).

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