Aluminum Nanostructures for Surface-Plasmon-Resonance-Based

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Aluminum Nanostructures for Surface Plasmon Resonance-Based Sensing Applications Kuang-Li Lee, Meng-Lin You, and Pei-Kuen Wei ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b02325 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on March 4, 2019

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ACS Applied Nano Materials

Aluminum

Nanostructures

for

Surface

Plasmon

Resonance-Based Sensing Applications Kuang-Li Lee, †,* Meng-Lin You,† and Pei-Kuen Wei†,‡,§,** † Research

Center for Applied Sciences, Academia Sinica, 128, section 2, Academia Road, Nangkang, Taipei 11529, Taiwan

‡ Department

of Optoelectronics, National Taiwan Ocean University, Keelung 20224, Taiwan

§Institute

of Biophotonics, National Yang-Ming University, Taipei 11221, Taiwan *[email protected] and **[email protected]

ABSTRACT

Surface plasmon resonance imaging (SPRi) is capable of real-time, label-free, and high-throughput detection for many sensing applications. The conventional prism-based SPRi system is operated with intensity interrogation. As the intensity-based sensors are easily disturbed by the environmental conditions such as light absorption of the analytes and source intensity noise, the intensity variations must be corrected. Besides, the sensitivity of intensity-based SPRi is moderate. To address the issues, we propose a low-cost, portable nanostructure-based SPRi platform, composed of a commercial transmission scanner, aluminum-based nanostructure chips, and self-referencing two-color analysis, for high-throughput sensing applications, such as the thickness of dielectric film, bulk refractive index of solution, and biomolecular interactions. Aluminum nanoslit array and capped nanoslit array are fabricated using nanoimprinting technology and their bulk and thickness sensitivities are studied. The ACS Paragon Plus Environment

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results show that the refractive index and thickness sensitivities are 3260 %/RIU and 16.2 %/nm, respectively. The detectable refractive index resolution and surface thickness are 4.310-5 RIU and 0.053 nm, respectively. The protein-protein interactions between bovine serum albumin (BSA) and anti-BSA proteins are conducted and the minimum concentration of anti-BSA proteins, 10 pg/ml, is detectable. In addition, high-throughput detection of surface thickness is demonstrated and 96 sensing arrays are quickly monitored. Such a portable, low-cost, and high-throughput sensing platform can benefit various multiplex sensing applications.

KEYWORDS: Surface plasmon resonance (SPR), nanostructures, optical sensors, biochips, selfreferencing multicolor analysis

Surface plasmon resonance imaging (SPRi)1 is capable of real-time, label-free, and high-throughput detection for many sensing applications, such as biomarker screening,2 nucleic acid detection,3 drug discovery,4 food safety,5 and environmental monitoring.6 The surface plasmon polaritons are induced on a patterned thin metal through prism-coupling.7 Typical SPR imaging experiments are conducted in a fixed angle format. The single-wavelength polarized light is obliquely incident on the metal film from the prism side, the intensity of the reflected light is then monitored with a charge-coupled device (CCD). This sensing technology is sensitive to the refractive index change near the metal surface and relied on specific binding events between target analytes and surface-immobilized probes. The refractive index resolution of such intensity systems is typically 10-4-10-5 RIU.8-10 With such a technology, multiple surface binding events can be simultaneously monitored at different regions of the metal surface and biomolecular binding affinities can be measured. In addition to intensity-based SPRi, angle-scanning11,12 and wavelength-scanning13 formats are also utilized for SPRi. These formats have higher refractive index resolutions (10-5-10-6 RIU) and wider dynamic ranges (1-1.4 RIU),11 i.e. a linear relationship between the SPR signal and mass of bound material. However, these sensing technologies are timeACS Paragon Plus Environment

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consuming. There is a trade-off between the measurement time and detection limit. To further improve the refractive index resolution, complicated optical setups, such as phase SPR imaging14 and polarization contrast-based imaging sensors,15 are proposed and the resolution of 10-7 RIU can be achieved. In addition to the prism coupling method, metallic nanostructures offer an alternative method for SPR excitation16-23 and have been applied to high-throughput sensing applications.24-27 Compared to the prism-based SPRi system, nanostructure-based SPRi platforms have a small detection volume and simple optical setup. The simple system is usually composed of a stable light source with a narrow bandwidth and an intensity imaging two-dimensional CCD. The light is normally incident on the nanostructures and the transmitted light is recorded by the CCD. As the intensity-based sensors are easily disturbed by the environmental conditions such as light absorption of the analytes, source intensity noise, and heat generated in the metallic nanostructures,28 the variations in intensity changes must be corrected. To address the issue, the system with a reference or calibration channel is needed to extract the real signal. The sensing capability for intensity-based SPRi also needs to be improved. To improve the sensitivity of the intensity-based sensors, a biperiodic nanohole array with polarization diversity to have one peak below the source’s wavelength and one above is proposed.29 When the refractive index close to the metal surface changes, it gives rise to an increase in transmission for one polarization and a decrease for the other one, and the difference between the two corresponds to the detection signal, which can achieve self-referencing and double the sensitivity. The refractive index resolution of 6.410-6 RIU is demonstrated. In this study, we proposed a low-cost nanostructure-based SPRi platform, composed of a commercial scanner, aluminum-based SPR chips and self-referencing two-color analysis, for highthroughput sensing applications. The key features of this sensing platform are self-referencing and enhanced sensitivity. Figure 1a shows the optical setup and aluminum-based SPR chips with a narrow resonance dip and peak. The white light generated from light-emitting diode (LED) arrays was incident on the aluminum nanoslits or capped aluminum nanoslits and the transmitted light was recorded by a ACS Paragon Plus Environment

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CCD. Figure 1b and 1c show the proposed concept of two-color analysis for transmission-type SPR sensors with a narrow resonance dip and peak, respectively. Each pixel of the CCD image can be divided into three color bands: red (R), green (G), and blue (B) bands. The periodic nanostructures, aluminum nanoslit arrays and capped aluminum nanoslit arrays, with specific periodicities were utilized to produce a narrow resonance peak or dip in the overlapped region between two bands, such as R and G bands or B and G bands. In Fig. 1a, the narrow resonance dip appeared in the overlapped region between R and G bands, i.e. the dip wavelength was on the right side of the G band and left side, the R band. These nanostructure-based SPR sensors are sensitive to the refractive index change near the metal surface. The adsorbed monolayer or increased bulk refractive index will increase the effective refractive index of surface plasmon waves, which makes the resonance wavelengths of the metallic nanostructures shift to longer wavelengths. When the resonance dip was red-shifted, the transmission intensity for the R band (IR) decreased as the resonance dip was approaching to the R band. On the other hand, the transmission intensity for the G band (IG) increased since the resonance dip left away from the G band. If light source intensity fluctuates, the transmission intensities for the R and G bands will be IR +ΔI and IG+ Δ I, respectively. The normalized intensity difference between two bands can be expressed as follows: I GR 

[( I G  I )  ( I R  I )] (IG  I R ) (I  I R )   G [( I G  I )  ( I R  I )] I G  I R  2I ( I G  I R )

(1)

The (IG+IR) is much larger than 2 Δ I, IGR is equal to (IG-IR)/( IG+IR). It can rule out the common noise, originated from the fluctuation of light source, improve the signal-to-noise ratio and enhance the sensing capability. The similar concept can also be applied to nanostructures with a narrow resonance peak as shown in Fig. 1b. It shows opposite signals for IR and IG and the normalized intensity difference for the resonance peak is expressed as IRG=(IR-IG)/(IR+IG). To prove the concept, aluminum nanoslits and capped nanoslits with different periods were fabricated and the bulk and thickness sensitivities were studied with a commercial scanner and the self-referencing two-color analysis of R and G bands. It is noted that most SPR devices utilize gold or silver as metallic films. However, gold film cannot support ACS Paragon Plus Environment

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propagation SPP mode near B/G overlapping band. Silver film is easy to be oxidized and toxic to biosamples. Our recent study30 show aluminum-based nanostructures can form sharp Fano resonances and have high intensity sensitivities in the visible light range. The native aluminum oxide layer can protect the aluminum film from damage and oxidation during sensing processes. The results show the proposed sensing platform can reach a refractive index sensitivity of 3260 %/RIU and thickness sensitivity of 16.2 %/nm. With the system noises, 0.14% and 0.87%, the detectable refractive index resolution and surface thickness were 4.310-5 RIU and 0.053 nm, respectively. To confirm the biological sensing capabilities of the proposed sensing platform, protein-protein interactions between bovine serum albumin (BSA) and anti-BSA proteins were studied and the minimum concentration of anti-BSA proteins, 10 pg/ml, was detectable. In addition, high-throughput detection of dielectric thicknesses with self-referencing multicolor analysis of B and G bands was demonstrated and 96 sensing arrays were simultaneously monitored. These results show that the proposed sensing platform, composed of an inexpensive commercial scanner and Al nanostructures, has a commercial value and can benefit various multiplex sensing applications.

Results and Discussion. Refractive index sensitivity tests with Al nanoslits and multicolor analysis of R, G, and G-R bands. In order to prove the proposed concept, aluminum nanoslits and capped nanoslits with different periods from 420 nm to 500 nm were fabricated and tested. Figure 2a shows the optical image of 96 aluminum nanoslit arrays on a polycarbonate (PC) film. The area of each array is 5×5 mm2. Figure 2b shows the transmission spectra of 430-nm-period aluminum nanoslits in air and different water/glycerin mixtures for normally-incident transverse-magnetic (TM)-polarized light. The polarization of the incident light was perpendicular to the nanoslits so that the gap plasmon resonances in nanoslits and surface plasmon waves on both sides of the periodic aluminum surface can be excited.31 The black line shows the transmission spectrum in an air environment. There were narrow resonance dip, broad resonance peak and narrow resonance peak at wavelengths of 435, 580 and 673 nm, respectively. These ACS Paragon Plus Environment

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features were caused by the gap plasmon resonances in nanoslits and surface plasmon waves on both sides of the periodic aluminum surface. The gap resonances lead to a broadband transmission in the transmission spectrum. The resonance wavelength can be estimated by a Fabry-Perot cavity32 and is expressed as follows:



4h Re( neff ) (2 m  1  2 )

(2)

where neff is the equivalent refractive index in the slit, h is the depth of the nanoslit (cavity length), and

1 and 2 are the phase shifts at the top and bottom interfaces. The gap width is a function of neff, which increases with the decrease of the gap width.32 The resonance wavelength is affected by the gap width and cavity length. On the other hand, a part of energy of the cavity mode was transferred to the surface waves from the edges of the top and bottom interfaces. There are two kinds of surface waves for periodic metallic structures: Bloch wave surface plasmon polaritons (BW-SPPs) and Wood’s anomaly. BW-SPPs occur when the Bragg condition is fulfilled. The condition for one-dimensional arrays can be expressed as follows:16

 m n 2 1/ 2 P SPR (n, i )  {Re[( ) ]} i  m  n2

(3)

where P is the period of the nanostructure, i is the resonance order, n is environmental refractive index, and ɛm is the dielectric constant of the metal. The Wood’s anomaly happens under the condition,

Wood (n, i ) 

P n i

(4)

The measured values are consistent with the theoretical predictions, i.e. 437 (air, n=1, BW-SPPs) and 679 nm (substrate, n=1.58, Wood’s anomaly). In the theoretical calculations, the wavelength dependent permittivity of aluminum was obtained from Rakić.33 When the nanostructures were covered with water, the dip wavelength was moved to 576 nm and red-shifted with the increase of the ACS Paragon Plus Environment

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glycerin/water mixtures from 1.3290 to 1.3460. The linear correlation between the dip wavelength and the refractive index of outside medium shows that the refractive index sensitivity was 429 nm/RIU (refractive index unit) as shown in Fig. 2b inset. It is close to the theoretical sensitivity (S) which was determined by the periodicity of the nanostructures, i.e. S ~ P nm/RIU. Figure 2c shows a schematic optical setup and Al nanoslit arrays for refractive index sensitivity tests. The refractive indexes of the mixtures were subsequently dropped on the chip and a cover glass was put on the chip. The transmission images for different mixtures were recorded with a commercial scanner (Perfection V800 Photo, Epson) as shown in the right panel of Fig. 2c. Each scanning time for the biochip with an area of 5 cm by 5 cm was 20 secs. The images were then analyzed with multicolor analysis. Figure 2d shows the transmission intensities of the nanoslits for G and R bands (IG and IR) and the normalized intensity difference (IGR) as a function of the refractive index of the mixture. The transmission intensity of IR decreased with the increase of the refractive index as the resonance dip was approaching to the R band; The transmission intensity of IG increased since the resonance dip left away from the G band; The transmission intensity of IGR increased since the changes in refractive index show a decreased intensity for the R band and an increase for the G band. There were linear correlations between the intensity changes and refractive indices as shown in Fig. 2e. The slopes were 657 %/RIU, 332 %/RIU, and 3269 %/RIU for R, G and RG bands, respectively. According to the standard deviations of signals for different mixtures, the average noises were 0.138%, 0.137%, and 0.144%, respectively. Therefore, the detectable refractive index changes were 2.110-4, 4.110-4, and 4.310-5 RIU. Obviously, compared to the resolutions of R and G bands, the resolution of G-R band was improved by factors of 4.8 and 9.5, respectively. Besides, the average signal-to-noise ratio for the R-G band was improved by factors of 5.0 and 11.5 when compared to those of R and G bands. Therefore, the proposed method can achieve self-referencing and enhance the sensing capability of the nanostructures. Such a resolution is comparable to that of the conventional prism-based SPRi (1.8-310-5 RIU).9,10 It was noted that in order to make the narrow resonance dip in an aqueous solution appear in the overlapped region between G and R bands, Al

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nanoslit arrays with periods of 420, 430, 440, and 500 nm were fabricated and tested. We found that the optimal period for the G-R band analysis in aqueous solution was 430 nm. On the other hand, the optimal period for the B-G band analysis in air environment was 500 nm.

Thickness sensitivity tests with 430-nm-period Al nanoslits and multicolor analysis of R, G, and G-R bands. As the surface thickness sensitivity is an important indicator to evaluate the quality of a biosensor, we deposited dielectric thin films on the biochip to mimic the adsorbed molecules. To verify the thickness sensitivity of the biochips in aqueous environment with self-referencing multicolor analysis, Al2O3 films were subsequently deposited on the biochips with atomic layer deposition and the transmission images in a water environment were recorded with a commercial scanner as shown in Fig. 3a. These images were analyzed with self-referencing multicolor analysis of R, G, and G-R bands. Figure 3b shows transmission intensities of Al nanoslits with a period of 430 nm for G and R bands (IG and IR) and the normalized intensity difference (IGR) as a function of the film thickness. As the deposited thin film causes a red-shift of the resonance dip, the resonance dip will approach the R band and the resonance dip will leave away from the G band. Therefore, the transmission intensity will decrease for the R band and increase for the G band. As expected, the transmission intensity of IR decreased; the transmission intensity of IG and transmission intensity of IGR increased with the increased alumina thickness. The linear correlations between the intensity changes and film thicknesses show the thickness sensitivities were 1.07, 0.32, and 16.1 %/nm for R, G, and G-R bands, respectively (see Fig. 3c). According to the standard deviations of signals, the average noises were 0.088%, 0.027%, and 0.87%, respectively. Therefore, the detectable alumina thicknesses were 0.082, 0.084, and 0.054 nm, respectively. The resolution was improved by a factor of 1.6 for G-R band. Such a resolution obtained in water environment is comparable to that of intensity-based capped Al nanoslits with a three mode coupling in air environment (0.04 nm).30 It was noted that the proposed concept can be applied to ACS Paragon Plus Environment

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metallic nanostructures with resonance peaks. The refractive index and thickness sensitivity tests for capped aluminum nanoslits with a resonance peak were shown in Supporting Information (Fig. S1 and S2),. It is notable that though the proposed platform had a similar refractive index resolution with the conventional prism-based SPRi (1.8-310-5 RIU), the resonance wavelengths and the corresponding electromagnetic field decay lengths were quite different. The refractive index resolution considers bulk refractive index changes throughout the entire optical field. However, high surface sensitivity is achieved when a greater proportion of the plasmonic field is occupied by an adsorbate layer.34 The electromagnetic field decay length plays an important role for the surface thickness sensitivity. The surface sensing capability can be improved by reducing the decay length.35 In this work, the resonance wavelength of 430-nm-period Al nanoslits in a water environment was at 576 nm, which was corresponded to a decay length of 612 nm. On the other hand, the resonance wavelength for the prismbased SPRi system was at 854 nm.9,10 The decay length was about 716 nm. Therefore, the proposed platform has a better thickness sensing capability.

Bio-interaction measurements with 430-nm-period Al nanoslits and multicolor analysis of G-R band. To further verify the biosensing capability of the proposed sensing platform, we conducted proteinprotein interactions between 66-kDa-sized bovine serum albumin (BSA) and 150-kDa-sized anti-BSA proteins. Figure 4a shows a schematic optical setup and plasmonic biochips for biological experiments (left panel). BSA proteins were first immobilized on the chips and anti-BSA solutions with different concentrations from 10 pg/ml to 1 μg/ml were subsequently injected into the microfluidic channel. The chips were then washing with a 0.01% PBS buffer solution. The images for each step were recorded in the PBS buffer solution as shown in right panel of Fig. 4a. Figure 4b shows transmission intensity changes (ΔIGR/IGR0) of Al nanoslits with a period of 430 nm for different surface conditions. The ACS Paragon Plus Environment

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transmission intensity of the first PBS solution was chosen as a reference. The intensity change increased and then gradually saturated as the concentration increased. To confirm the limit of detection (LOD) of the concentration of anti-BSA proteins, the transmission intensity of the BSA solution was chosen as a reference. The transmission intensity change (ΔIGR/IGR0) as a function of the logarithm of the concentration was shown in Fig. 4c. The signals were 5.9, 10.9, 12.6, 16.8, 19.2, and 23.82 % for 0.00001, 0.0001, 0.001, 0.01, 0.1, and 1 μg/ml anti-BSA solutions, respectively. There was a linear correlation between the signal and the logarithm of the concentration. The calibration curve was described by y=3.3971(log10(x))+23.366, R2=0.98487 and the average noise, extracted from Fig. 4b, was 0.287% (standard deviation of the signal). Based on the current sensitivity and 3 times of noise level (0.861%, 3 times standard deviation of the signal), the LOD of the concentration of anti-BSA solutions can be estimated by a linear regression equation. This yields a theoretical value of 237 fg/mL (1.58 fM). Currently the measured minimum concentration of anti-BSA was 10 pg/ml (66.67 fM), which is lower than those of prism-based SPRi with polarization orientation (8.26 ng/ml (125 pM) BSA)36 and other phase SPR imaging systems (1×10-4 mg/ml (0.67 nM) Immunoglobulin G (IgG)37 and 500 ng/ml (13.6 nM) human chorio gonadotropin (hCG)).38

High-throughput detection of alumina thicknesses with 500-nm-period Al nanoslits and multicolor analysis of B, G, and B-G bands. In order to demonstrate the high-throughput sensing capability of the proposed sensing platform, we conducted multiplex array detection of alumina thicknesses with 96 sensing arrays and selfreferencing multicolor analysis of B and G bands. Figure 5a shows a schematic optical setup and plasmonic biochips with a period of 500 nm for multiplex array detection of alumina thicknesses. Alumina films from 0 nm to 15 nm were subsequently deposited on the 96 sensing arrays. Each area of the sensing array was 5 mm by 5 mm. The images were subsequently recorded in an air environment as shown in the right panel of Fig. 5a. The periodicity, 500 nm, was chosen so that the narrow resonance dip appeared in the overlapped region between B and G bands as shown in Fig. 1a. The resonance dip ACS Paragon Plus Environment

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for 0-nm-thick alumina was at a wavelength of 514.6 nm. It was gradually red-shifted with the increase of the film thickness. The linear fitting curve shows that the thickness sensitivity was 1.19 nm/nm, i.e. a 15-nm-thick alumina film caused a 17.9-nm wavelength shift. The red-shifted dip resulted in a decreased transmission intensity for G band and an increased intensity for B band as shown in Fig. 5c. As there were linear correlations between the intensity change and alumina thickness (see Fig. 5c), it indicated that the dynamic range with intensity interrogation was more than a wavelength range of 18 nm. Figure 5d shows the intensity signal (IBG) against alumina thicknesses for 96 sensing arrays and Fig. 5e indicated their thickness sensitivities. The coefficient of variation of the sensitivities was 17 % and the mean value of detectable thicknesses was 0.52 nm. These results show that 96 sensing arrays can be simultaneously monitored and high-throughput array detection with the low-cost sensing platform can be achieved.

Conclusions. In conclusion, we proposed a low-cost, portable nanostructure-based SPRi platform, composed of a commercial transmission scanner, aluminum-based SPR chips, and self-referencing two-color analysis, for high-throughput sensing applications. To prove the concept of the self-referencing two-color analysis, aluminum nanoslits and capped nanoslits were fabricated and the bulk and thickness sensitivities were studied. The results show that the refractive index and alumina thickness sensitivities are 3260 %/RIU and 16.2 %/nm, respectively. With the system noises, 0.14% and 0.87%, the detectable refractive index resolution and alumina thickness are 4.310-5 RIU and 0.053 nm, respectively. To confirm the biosensing capabilities, protein-protein interactions between BSA and anti-BSA proteins were studied and the minimum concentration of anti-BSA proteins, 10 pg/ml, is detectable. In addition, high-throughput array detection of alumina thicknesses was demonstrated and 96 sensing arrays were simultaneously monitored. As the commercial scanner was capable of scanning an A4-sized area, the number of the sensing array can further increase to a few thousand or more when the current sensing area (5 mm by 5 mm) was reduced.

It was noted that metallic nanostructures include metallic


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nanoparticles and periodic metallic nanostructures, such as nanoslit arrays, nanohole arrays and other periodic nanostructures. The localized surface plasmon resonance (LSPR) was excited through metallic nanoparticles. However, propagating SPR can be excited through a prism (prism-coupling) or metallic gratings (grating-coupling). In this study, aluminum nanoslit arrays play a similar role with metallic gratings in the excitation of propagating SPR on the metal surface. Besides, the nanostructures are not restricted to aluminum nanoslit arrays and capped aluminum nanoslit arrays. Other nanostructures, which can produce a narrow resonance peak or dip in the overlapped region between two bands, can be applied to the proposed concept. Such a portable, user-friendly, low-cost and high-throughput sensing platform can benefit various multiplex sensing applications, such as clinical disease diagnosis, drug screening and protein biomarker discovery.

Experimental Section. Fabrication of metallic nanostructures. 40-nm-thick aluminum nanoslits and capped nanoslits with different periods from 420 nm to 500 nm were fabricated on a polycarbonate (PC) substrate using a home-made hot-embossing nanoimprint machine and thermal evaporator.30,39 Figure 2a shows the optical image of 96 aluminum nanoslit arrays with a period of 470 nm on the PC film and area of each array was 5×5 mm2. The inset shows a scanning electron microscope (SEM) image of Al nanoslits and the silt width was 60 nm.

Optical setup for transmission spectra and images. A home-made optical system was utilized to record transmission spectra of metallic nanostructures. A laser-driven broadband light source (LDLS™) with fiber-coupled output was connected to a fiber cable with a fiber lens for light collimation. To control the polarization of the light, a linear polarizer was added to the optical path. The TM-polarized light was then normally incident on the metallic nanostructures. The transmitted light from the nanostructures was collected by a fiber lens and focused on a fiber cable, connected to a fiber-coupled high performance back-thinned charge-coupled device ACS Paragon Plus Environment

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spectrometer (BWTEK, i-trometerTM). A commercial scanner (Perfection V800 Photo, Epson) was utilized for recording transmission images of metallic nanostructures. In this system, white-light LEDs were used as a light source. The white light was incident on the nanostructures after passing through an A4-sized polarizer, which was fixed on the lid of the scanner and utilized to control the polarization of the incident light. The transmitted light was then collected and recorded by a matrix CCD with micro lens, which has a scanning resolution of 6,400 dpi (Horizontal  Vertical), maximum scanner range of 216 mm  297 mm (Horizontal  Vertical) and 48 bit colour depth. The recorded images by a matrix CCD were analyzed using a Matlab software. A 3030-pixels image area was chosen as an effective detection area for each sensing array. The mean value of the normalized intensity difference between two-color bands, IGR, IRG, and IBG, from 3030-pixels was calculated for each sensing array. It was noted that white light from one-dimensional LED array was utilized as a light source and the transmission image of biochips was recorded with a line scanning approach. As the common LED is utilized, the illumination time of white light is short and the system noise is 0.14%, the heat generated in the metallic nanostructures is not considered here.

Refractive index sensitivity tests, thickness sensitivity tests and biological sensing experiments To confirm the refractive index sensing capability of the chips with the commercial scanner, the chip was placed on the glass after opening the lid of the scanner. Different water/glycerin solutions were subsequently poured over the nanostructure surface. The refractive indexes of the mixtures ranged from 1.3290 to 1.346, which were confirmed by a commercial refractometer. After putting a cover glass on the chip and closing the lid of the scanner, the transmission images were recorded. Before changing the mixture, the chip was washed with deionized water and blow-dried with a nitrogen gun. To verify the thickness sensitivity of the sensing platform in an aqueous environment, alumina films with different thicknesses were subsequently deposited on the metallic nanostructures and silicon wafers using an atomic layer deposition machine (ALD, Syskey Technology CO., LTD). The thicknesses of alumina


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films deposited on the silicon wafer were then measured with ellipsometry. For deposition of aluminum oxide, the precursors of trimethylaluminum (TMA) and water were used. First, a bare chip was covered with a glass after pouring deionized water on it. The transmission images of the nanostructures were recorded after closing the lid of the scanner. After that, the chip was blow-dried and a 4-nm-thick alumina film was deposited on the nanostructures. The transmission images in a water environment were recorded again. These steps were repeated for alumina films of 4 nm and 9 nm. To demonstrate the biological sensing capability of the platform, protein-protein interactions were conducted using bovine serum albumin (BSA, Sigma-Aldrich) and anti-BSA (Sigma-Aldrich) assay in a 0.01% phosphate buffered saline (PBS) solution. To form amino groups on the surface of aluminum oxide film, the chip were first exposed to a 1% aminopropyltriethoxysilane (APTES) solution for 30 minutes and then baked at 100 oC for 30 minutes. After modification of the amino groups, the chip was modified with a glutaraldehyde bifunctional cross-linker to bind amino groups on proteins. The chip was then integrated to a microfluidic channel. An 80 μL of 100 μg/mL BSA solution was injected into the microfluidic channel with a pipette. After 1 hour immobilization at room-temperature, the chip was washed with the PBS buffer solution to remove the unbound BSA proteins. To immobilize uniform BSA proteins on the chips, the BSA immobilization process was repeated for three times. After that, an 80 μL of 10 pg/mL anti-BSA solution was injected into the structure surface for 1 hour. The chip was then washed with the PBS buffer solution. The binding and washing processes were subsequently repeated for different concentrations of anti-BSA solutions from 10 pg/mL to 1 μg/mL. The transmission images for all steps were recorded and analyzed. It was noted that an aluminum native oxide layer (an oxide protecting layer) was formed on the surface of the Al biochip. However, a higher concentration of the phosphate in the phosphate buffered saline solution would react with the layer and damage the Al biochips. Therefore, we diluted the PBS solution from 1% to 0.01% and conducted the bio-experiments with the 0.01% PBS buffer solution.

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Supporting information

Additional information K.L.L. and P.K.W. conceived and designed the experiments; K.L.L. and M.L.Y. performed the experiments; K.L.L. analyzed the data and prepared the figures; P.K.W. contributed materials/analysis tools; and K.L.L. and P.K.W. co-wrote the paper. Competing financial interests: The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the Ministry of Science and Technology, Taipei, Taiwan, under Contract no. MOST 106-2112-M-001-006-MY3, MOST 106-2627-B-001-001 and Academic Sinica, AS-106TP-3 and AS-KPQ-106-TSPA. Technical support from the nano/micro-fabrication facilities in Academia Sinica is acknowledged.


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Figure 1. Concepts of multicolor analysis for transmission-type SPR sensors. (a) Schematic illustrations optical setup and nanostructures with a narrow resonance dip and peak. Schematic illustrations depict the concepts of multicolor analysis for transmission-type SPR sensors with (b) a narrow resonance dip and (c) a narrow resonance peak. The white-light LED was recorded by a CCD. Each pixel of the CCD image can be divided into three color bands: red (R), green (G), and blue (B) bands. The nanostructures were designed to make its narrow resonance peak or dip appear in the overlapped region between two bands, such as R and G bands or B and G bands. For a nanostructure with (1) a resonance dip ((2) a resonance peak), when it was red-shifted due to the adsorbed monolayer or increased bulk refractive index changes, the transmission intensity for the R band (IR) decreased (increased) and the transmission intensity for the G band (IG) increased (decreased) as shown in the right panel of a (b). The normalized intensity difference between two bands (IGR=(IG-IR)/(IG+IR) or IRG=(IR-IG)/(IR+IG) ) can rule out the common noise, originated from the fluctuation of light source, and enhanced the sensing capability.


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Figure 2. Refractive index sensitivity tests with Al nanoslits and multicolor analysis of R, G, and G-R bands. (a) Optical image of Al nanoslits arrays. The area of each array was 5 mm by 5 mm. The inset shows an SEM image of Al nanoslits and the silt width was 60 nm. (b) Transmission spectra of Al nanoslits with a period of 430 nm for different water/glycerin mixtures. The inset shows the dip wavelength against the refractive index of the mixture. (c) Schematic optical setup and plasmonic biochips for refractive index sensitivity tests (left panel). Transmission images of Al nanoslits for different refractive index mixtures were shown in the right panel. (d) Transmission intensities of the nanostructures for G and R bands (IG and IR) and the normalized intensity difference (IGR) as a function of the refractive index of the mixture. (e) Intensity changes as a function of the refractive index for G, R, and G-R bands.


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Figure 3. Thickness sensitivity tests with 430-nm-period Al nanoslits and multicolor analysis of R, G, and G-R bands. (a) Schematic optical setup and plasmonic biochips for thickness sensitivity tests (left panel). Alumina films from 0 nm to 9 nm were subsequently deposited on the biochips and the images were recorded in a water environment (right panel). (b) Transmission intensities of Al nanoslits with a period of 430 nm for G and R bands (IG and IR) and the normalized intensity difference (IGR) as a function of the film thickness. (c) Intensity changes as a function of the film thickness for G, R, and G-R bands.


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Figure 4. Bio-interaction measurements with 430-nm-period Al nanoslits and multicolor analysis of G-R band. (a) Schematic optical setup and plasmonic biochips with a microfluidic channel for biological experiments (left panel). The materials from top to bottom were a PMMA film with 2 holes, doublesided tape with a channel and biochip. The microfluidic channel was formed by assembling these three parts. BSA proteins were first immobilized on the chips and anti-BSA solutions with different concentrations from 10 pg/ml to 1 μg/ml were subsequently injected into the microfluidic channel with a pipette. The chips were washing with a 0.01% PBS buffer solution. The images for each step were recorded in aqueous environment (right panel). (b) Transmission intensity changes (ΔIGR/IGR0) of Al nanoslits with a period of 430 nm for different surface conditions. The transmission intensity of the first PBS solution was chosen as a reference. (c) Transmission intensity change (ΔIGR/IGR0) as a function of the logarithm of the concentration of the anti-BSA solution. The transmission intensity of the BSA solution was chosen as a reference. There was a linear correlation between the intensity change and the logarithm of the concentration of the anti-BSA solution. The calibration curve was described by y=3.3971(log10(x))+23.366, R2=0.98487 and the average noise was 0.287%


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Figure 5. High-throughput detection of alumina thicknesses with 500-nm-period Al nanoslits and multicolor analysis of B, G, and B-G bands. (a) Schematic optical setup and plasmonic biochips for multiplex array detection of alumina thicknesses (left panel). Alumina films from 0 nm to 15 nm were subsequently deposited on the biochips with 96 sensing arrays. Each area of the sensing array was 5 mm by 5 mm. The images were subsequently recorded in an air environment (right panel). (b) Transmission spectra of Al nanoslits with a period of 500 nm for different alumina thicknesses. (c) Intensity changes as a function of the film thickness for B and G bands. (d) Intensity signal (IBG) against alumina thickness. (e) Thickness sensitivities of 96 sensing arrays for the B-G band.

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Aluminum Nanostructures for Surface-Plasmon-Resonance-Based

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