Ag Nanocomposites on Paper Substrate

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Nanoplasmonic Alloy of Au/Ag Nanocomposites on Paper Substrate for Biosensing Applications Moonseong Park, Charles Soon Hong Hwang, and Ki-Hun Jeong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16182 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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ACS Applied Materials & Interfaces

Nanoplasmonic Alloy of Au/Ag Nanocomposites on Paper Substrate for Biosensing Applications Moonseong Park, Charles S. H. Hwang, Ki-Hun Jeong* Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291-Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. *E-mail : [email protected] KEYWORDS Plasmonic alloy, Paper substrate, Surface-enhanced Raman spectroscopy, Metal-enhanced fluorescence, Localized surface plasmon resonance

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ABSTRACT :

Plasmonic alloy has attracted much interest in tailoring localized surface plasmon resonance (LSPR) for recent biosensing techniques. In particular, paper-based plasmonic substrates allow capillary-driven lateral flow as well as three-dimensional metal nanostructures and therefore they become actively transferred to LSPR-based biosensing such as surface-enhanced Raman spectroscopy (SERS) or metal-enhanced fluorescence (MEF). However, employing plasmonic alloy nanoislands on heat-sensitive substrate is still challenging, which significantly inhibits broad-range tailoring of the plasmon resonance wavelength (PRW) for superior sensitivity. Here we report paper-based plasmonic substrate with plasmonic alloy of Au/Ag nanocomposites for highly sensitive MEF and SERS biosensing applications. The nanofabrication procedures include concurrent deposition of Au and Ag below 100 degrees Celsius without any damage on cellulose fibers. The Au/Ag nanocomposites feature nanoplasmonic alloy with single plasmon peak as well as broad-range tunability of PRW by composition control. This paper-based plasmonic alloy substrate enables about two-fold enhancement of fluorescence signals and selective MEF after paper chromatography. The experimental results clearly demonstrate extraordinary enhancement in SERS signals for picomolar detection of folic acid as a cancer biomarker. This new method provides huge opportunities for fabricating plasmonic alloy on heat-sensitive substrate and biosensing applications.


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INTRODUCTION : Plasmonic alloy of different noble metals provides high opportunities for tuning plasmon resonance.1-5 Localized surface plasmon resonance (LSPR), i.e. a collective oscillation of free electron cloud on the metal-dielectric interface, creates an intense electromagnetic field within the extremely confined area, so-called plasmonic hotspots.6,7 This unique behaviour

significantly

enhances

both

electromagnetic

field

near

plasmonic

nanostructures and optical signals from small molecules8 and therefore it provides highly sensitive biosensing techniques such as surface-enhanced Raman spectroscopy9,10 (SERS) and metal-enhanced fluorescence11 (MEF). SERS provides highly sensitive label-free fingerprint of small molecules from their Raman modes12-16 and MEF also allows highly sensitive fluorescence detection of fluorescent molecules in small amounts.17 For last decade, many previous works on LSPR-based biosensing techniques have extensively focused on securing superior sensitivity in terms of LSPR such as alignment of target molecules within plasmonic hotspots,18-20 matching plasmon resonance wavelength (PRW) to excitation laser wavelength,21-23 or multiple plasmonic hotspot generation by nanogap-rich metal nanostructures.24-26 In particular, plasmonic alloys have extensively contributed to tailoring of PRW to secure the superior sensitivity in biosensing.8 However, the previous works still struggle with introducing plasmonic alloys in low temperature for heat-sensitive substrate such as paper substrate or polymer substrates, which significantly restricts tailoring of PRW in broad range for small molecule detection with superior sensitivity.4,5,27 Paper substrate provides cellulose micro-/nanofiber matrices, which allow capillary-driven lateral flow through the hygroscopic micro-/nanopores.28-31 The cellulose fiber matrices as both microfluidic channel and three-dimensional backbone for metal


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nanoislands have been utilized as and microfluidic paper-based analytical devices32 and SERS dipsticks.33 Moreover, this unique configuration of paper has further enabled clinical applications such as plasmonic Schirmer strip for SERS diagnosis of gouty arthritis using human tears34 and lateral flow sensor for nucleic acid detection in the blood.35 In particular, multiple hotspot generation has been recently achieved by introducing the cellulose fiber matrices as three-dimensional hierarchical backbone for metal nanostructures for plasmonic biosensing techniques such as MEF and SERS.36 Some facile methods for plasmon resonance tuning have been achieved by controlling metal nanostructures on a paper substrate, however, they still suffer from the limited tuning range of a single metal.32,36 As a result, paper-based nanoplasmonic substrates are still under development for broad-range tuning of PRW for highly sensitive plasmonic biosensing.32,36 Here we report plasmonic alloy of Au/Ag nanocomposites on hierarchical cellulose micro-/nanofiber matrices for paper-based plasmonic substrate for highly sensitive biosensing. The Au/Ag nanocomposites exhibit one single extinction peak at PRW and they serve as a nanoplasmonic alloy, whereas the corresponding individual metal nanostructures clearly have two different extinction peaks (Fig. 1a). Moreover, the plasmonic alloy allows high tunability of PRW in broad range between the PRW of Au and Ag. The concurrent thermal evaporation of Au and Ag apparently employs the plasmonic alloy on the Whatman chromatography paper substrate as well as provides composition control under the precise regulation of the film thickness and the deposition rate (Fig. 1b and see the Experimental Section). This approach enables not only the lowtemperature wafer-level fabrication of plasmonic alloy in Volmer-Weber mode37 but also


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the tailoring of the PRW in broad range, which is clearly shown in plasmonic color change. Note that this fabrication procedures include only low-temperature process below 100 degrees Celsius to avoid any damage on cellulose fibers or burning of paper substrate. The scanning electron microscope (SEM) image clearly shows the densely packed Au/Ag nanocomposites on the top surface of cellulose micro-/nanofibers. This particular configuration allows both the capillary-driven lateral flow for paper chromatography and the intense localization of electromagnetic field within threedimensional plasmonic hotspots for highly sensitive plasmonic biosensing techniques such as MEF and SERS.

Figure 1. (a) The plasmonic alloy of Au/Ag nanocomposites. The discrete Au nanoislands and Ag nanoislands have two distinct extinction peaks from Au and Ag. On the contrary, Au/Ag nanocomposites act as plasmonic alloy with one single extinction peak, i.e., plasmon resonance wavelength. The extinction peak depends on the composition of each metal. (b) The plasmonic alloy fabrication includes concurrent thermal evaporation of Ag/Au nanocomposites on the top surface of cellulose fiber matrices. The delicately controlled deposition provides both efficient regulation of each metal composition and tailoring plasmon resonance in broad range. The SEM image on the right side clearly shows nanogap-rich plasmonic alloy nanoislands on cellulose


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fibers, which secure generation of intensely localized electromagnetic field within volumetric plasmonic hotspots for SERS and MEF.

RESULTS AND DISCUSSION The plasmonic alloy on paper substrate shows plasmonic color change from pale orange to steel blue (Fig. 2a). The photographic images apparently show the wide tailoring of PRW with plasmonic color change. Each paper-based plasmonic alloy substrate was fabricated in wafer-level, and then cut to the dimension of 5 mm X 60 mm. Note that the width and height of the strip allow efficient lateral flow without the edge effect and paper chromatography, respectively. This wafer-level low-temperature fabrication of plasmonic alloys on paper substrate enables plasmonic paper strips to be applied in clinic such as lateral flow assay and paper chromatography column. The concurrent thermal evaporation of Au and Ag apparently forms the physical nanocomposites of Au and Ag (Fig. 2b). The element mapping images of energy dispersive X-ray spectroscopy (EDS) on transmission electron microscope (TEM) image clearly show the element distribution of each metal as well as their exact compositions (see Experimental Section for detailed information). The measured Au/Ag fractions are 0/1, 0.12/0.88, 0.52/0.48, 0.81/0.19, and 1/0 with excluding the emissions from carbon, oxygen, and copper from TEM grid. Note that Au and Ag have similar lattice constant and surface energy, which allow the Au/Ag nanocomposites with uniform distribution of Au and Ag.40


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Figure 2. (a) Paper-based plasmonic alloy strips fabricated in wafer-level. The plasmonic alloy with different composition was fabricated in wafer-level, and the dynamic color change implies high tunability in PRW. (b) The EDS mapping images for plasmonic alloy of different compositions based on TEM images. The low-temperature concurrent thermal evaporation of Au and Ag enables the uniform mixing of Au and Ag.

The plasmonic alloy of Au/Ag nanocomposites allows broad-range tuning of PRW as shown in the measured extinction spectra depending on the composition of each metal (Fig. 3a). In this experiment, the composition of each metal was precisely controlled by regulating the deposition conditions such as deposition thickness and deposition rate. The extinction peak becomes red-shifted from the PRW of Ag nanoislands as the Au fraction increases. This peak shift in broad range from 499 nm to 606 nm is largely based on the PRW of each metal.33,35 Note that the single extinction peak of PRW for each composition clearly confirms the alloyed plasmonic properties although the Au/Ag nanocomposites were physically mixed. The linear correlation between PRW and molar fraction of each metal is highly in accord with the calculated values based on the finite


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domain time difference (FDTD) method (Fig. 3b and S1). Both the numerically calculated and the experimentally measured PRWs become red-shifted as the Au fraction increases. The composition of each metal directly affects the PRW of Au/Ag nanocomposites as shown in both the numerically calculated and the experimentally measured PRWs. This linear correlation strongly suggests huge potential in targeting the PRW of plasmonic alloy to the excitation laser wavelength for additional enhancement in SERS and MEF. The paper-based plasmonic alloy substrate provides about two-fold enhancement in MEF signals owing to matching PRW to fluorescence excitation wavelength of each dye. The fluorescence signals measured by using the paper-based plasmonic substrate of five different compositions were compared to those measured by using Whatman chromatography paper substrate (Fig. 3c). The fluorescence signals from fluorescein isothiocyanate (FITC), Rhodamine 6G (R6G), and Congo red (CR) become maximized for the PRWs matched to the fluorescence excitation wavelengths of each dye component. In addition, the matching of the PRW to the fluorescence emission wavelength also provides the enhancement of MEF signals as shown in the second largest enhancement for FITC and R6G. For CR, the MEF signal becomes slightly increased against the tendency when the PRW matches to the fluorescence emission wavelength. The plasmonic alloy reveals that the matching of the PRW to the fluorescence excitation wavelength provides significant enhancement in MEF signals as well as the matching to the fluorescence emission wavelength is also be partially helpful to enhance MEF signals. Furthermore, selective MEF detection of dye component in mixture phase has been achieved by using hygroscopic cellulose layer as paper chromatography column (Fig. 3d). The mixture solution of safranin O (SO), toluidine blue (TB), and CR was spotted at the


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end of the plasmonic alloy strip, and then each dye component was clearly partitioned by ascending chromatography with elution by ethanol. The MEF signals from each dye component were separately measured at different positions depending on each retention factor of 0.20, 0.38, and 0.96 for CR, TB, and SO, respectively. This selective fluorescence detection in mixture phase suggests huge potential in biosensing of fluorescent biomolecules in body fluid.

Figure 3. PRW and fluorescence measurements. (a) Extinction spectra and plasmonic color change depending on the fraction of Au/Ag nanocomposites. The PRW becomes red-shifted as the Au fraction increases. The Au/Ag nanocomposites act as plasmonic alloy, which is shown by the clear single extinction peaks. (b) The numerically calculated and the practically measured


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PRW. The experimentally measured PRW has highly linear relationship to the FDTD-calculated PRW. (c) Fluorescence signal enhancement from FITC, R6G, and CR depending on the PRW. The MEF signals were increased about two-fold compared to the Whatman chromatography paper substrate, and they were maximized by matching of the PRW to the fluorescence excitation wavelength. (d) Chromatographic MEF. The dye mixture solution of TB, SO, CR was separated by using paper chromatography, and then each component was selectively detected.

The paper-based plasmonic alloy substrate also enables highly sensitive SERS detection of biomolecules such as adenine and picomolar detection of folic acid. Adenine is one of the most representative DNA base molecule, and folic acid is well-known as vitamin B9 as well as cancer biomarker related to nucleic acid synthesis. Prior to SERS detection of biomolecule, the plasmonic alloy provides additional enhancement in SERS signals of dye molecules by matching of the PRW to the excitation laser wavelength (Fig. 4a and S2). The Ar laser and HeNe laser were used for measuring the SERS signals of crystal violet (CV) and R6G, respectively. The major SERS peak intensities from CV at 1595 cm-1 and R6G at 1507 cm-1 become maximized when the PRW matches to the excitation wavelength. In particular, plasmonic alloy of Au0.12Ag0.88 with 514 nm excitation shows the best SERS performance among the paper-based plasmonic substrates with five different compositions, which is largely due to not only the multiple hotspot generation but also the matching of the PRW to the excitation wavelength. Moreover, the plasmonic alloy substrate provides SERS detection of adenine under Ar laser excitation with 514 nm (Fig. 4b). The SERS peaks from adenine at 567, 684, and 738 cm-1 become clearly distinguishable by using plasmonic alloy substrate with the PRW of 522 nm. The


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SERS peak at 567 cm-1 refers to out-of-plane vibrational mode, C-H bending, and wagging of C-H and N-H.41 The other SERS peaks of adenine at 684 and 738 cm-1 relate to out-of-plane vibrational mode and in-plane vibrational modes including ring breathing, respectively.41 The specific SERS peaks from adenine are apparently unclear by using Au or Ag nanoislands on paper substrate due to the low accordance of PRW with excitation wavelength. However, the major SERS

peaks from adenine become clearly

distinguishable by using the plasmonic alloy substrate with the PRW of 522 nm due to its highly matched PRW to the excitation laser wavelength. The paper-based tuneable plasmonic alloy substrate provides huge opportunities for SERS detection of DNA base components, and further development in bioassay will suggest DNA sequencing by using the plasmonic alloy substrate. Finally, the paper-based plasmonic alloy substrate successfully demonstrated SERS detection of folic acid in picomolar level (Fig. 4c). The plasmonic alloy of Au0.12Ag0.88 was used under 514 nm excitation of Ar laser. The specific SERS peak of folic acid at 1604 cm-1 refers to scissoring of NH2, asymmetric stretching of C=N in pteridine, symmetric stretching of C=C, and rocking of C-H in paminobenzoic acid.42 Note that the specific SERS peak at 1604 cm-1 is still clearly distinguishable even at 1 pM concentration, which is the lowest limit of detection (LOD) for folic acid reported up to now. The broad-range tuning of the PRW enables the significantly low SERS LOD by matching of the PRW to the excitation laser wavelength. This detection limit of plasmonic alloy substrate inspires highly sensitive SERS detection of biomolecules in picomolar level, and the plasmonic alloy substrate is also readily applicable to various fields such as physiology, genetics, cancer research, and clinical diagnosis.


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Figure 4. SERS measurements. (a) The SERS peak enhancement by matching of PRW to the excitation laser wavelength. The SERS peak enhancement is based on both the generation of multiple hotspot and the matching of the PRW to the excitation wavelength. (b) SERS detection of adenine. The measurements were carried out under 514 nm Ar laser excitation. The most distinguishable SERS peaks of adenine were shown by using plasmonic alloy substrate with the PRW of 522 nm. (c) Picomolar SERS detection. The Au0.12Ag0.88 alloy substrate was used under 514 nm excitation of Ar laser, and the major SERS peaks are clearly distinguishable even at 1 pM concentration. The LOD of plasmonic alloy becomes 1 pM for folic acid.


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CONCLUSION : In summary, this work has successfully demonstrated low-temperature of plasmonic alloy of Au/Ag nanocomposites on paper substrate and its biosensing application such as chromatographic MEF and picomolar SERS detection. Paper substrate provides hierarchical cellulose micro-/nanofiber matrices as three-dimensional backbone securing Au/Ag nanocomposites to generate highly intense electromagnetic field in terms of LSPR for highly sensitive MEF and SERS detection of biomolecules. The concurrent thermal evaporation of Au and Ag forms Au/Ag nanocomposites, which feature plasmonic alloy with a single PRW. The compositions of each metal were delicately controlled by regulating the deposition conditions, and it resulted in tailoring of the PRW in broad range. This broad-range tuning of PRW allows matching of the PRW to the excitation wavelength for additional enhancement in MEF and SERS signals. Furthermore, the hygroscopic nature of paper substrate provides selective MEF detection of each dye component in mixture phase after paper chromatography. Finally, the plasmonic alloy also provides picomolar SERS detection of folic acid. The low-temperature plasmonic alloy fabrication allows broad-range tuning of PRW for highly sensitive fluorescence detection and SERS detection, and further it provides potential applications for healthcare monitoring and disease diagnosis.


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ASSOCIATED CONTENT Supporting Information Experimental Methods : Detailed information for materials, fabrication of plasmonic alloy, element analysis, numerical analysis, extinction and plasmon resonance wavelength, fluorescence measurement and chromatographic MEF, and SERS measurement. Figure S1 : FDTD simulation results for extinction wavelengths of Au/Ag nanocomposites Figure S2 : SERS signals of crystal violet (CV) and Rhodamine 6G (R6G)

AUTHOR INFORMATION Corresponding Author *E-mail : [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2016919193, 201613061). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2016919193, 201613061).


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Table of Contents (TOC) Graphics


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Ag Nanocomposites on Paper Substrate

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