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Facile Fabrication of a Silver Nanoparticle Immersed, SurfaceEnhanced Raman Scattering Imposed Paper Platform through Successive Ionic Layer Absorption and Reaction for On-Site Bioassays Wansun Kim,† Yeon-Hee Kim,‡ Hun-Kuk Park,†,§ and Samjin Choi*,†,§ †
Department of Medical Engineering, Graduate School, Kyung Hee University, Seoul, 02447, Korea Department of Obstetrics and Gynecology, The Catholic University of Korea, Kyonggi-do, 11765, Korea § Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul, 02447, Korea ‡
S Supporting Information *
ABSTRACT: We introduce a novel, facile, rapid, low-cost, highly reproducible, and power-free synthesizable fabrication method of paper-based silver nanoparticle (AgNP) immersed surface-enhanced Raman scattering (SERS) platform, known as the successive ionic layer absorption and reaction (SILAR) method. The rough and porous properties of the paper led to direct synthesis of AgNPs on the surface as well as in the paper due to capillary effects, resulting in improved plasmon coupling with interparticles and interlayers. The proposed SERS platform showed an enhancement factor of 1.1 × 109, high reproducibility (relative standard deviation of 4.2%), and 10−12 M rhodamine B highly sensitive detection limit by optimizing the SILAR conditions including the concentration of the reactive solution (20/20 mM/mM AgNO3/NaBH4) and the number of SILAR cycles (six). The applicability of the SERS platform was evaluated using two samples including human cervical fluid for clinical diagnosis of human papillomavirus (HPV) infection, associated with cervical cancer, and a malachite green (MG) solution for fungicide and parasiticide in aquaculture, associated with human carcinogenesis. The AgNP-immersed SERS-functionalized platform using the SILAR technique allowed for high chemical structure sensitivity without additional tagging or chemical modification, making it a good alternative for early clinical diagnosis of HPV infection and detection of MG-activated human carcinogenesis. KEYWORDS: SILAR, SERS, paper substrate, AgNPs, HPV, malachite green, on-site bioassay
1. INTRODUCTION
Paper has been highlighted as an effective platform because it is economical, manageable, lightweight, and flexible, indicating its suitability for point-of-care (POC) applications.13−17 With the user-friendly advantages of paper, many groups have attempted to develop a SERS platform loaded with metallic nanoparticles.18−30 Most nanoparticles are synthesized in a colloidal phase and extracted by centrifugation, then deposited on paper using various methods including inkjet printing,19 screen printing,20,21 spraying,22 and others.23,24 Although the porous and three-dimensional (3D) structure of paper might have potential for enhancement of SERS EF, most nanoparticles in these printing methods are deposited only on the surface of the paper. To incorporate the nanoparticles into the paper, some studies have used a fabrication method based on dipping the paper into colloidal nanoparticles; however, this
Surface-enhanced Raman scattering (SERS) has become a representative technique for analysis and quantification of specific biological and chemical molecules.1−3 SERS activity is evaluated based on an enhancement factor (EF) that is commonly in the range of 104−108 and a high EF of >108, which is sufficient for single-molecule detection.4,5 Most studies to improve SERS activity have focused on modifying the surface of the substrate through nanomaterials and nanostructures synthesized using sophisticated techniques including lithography or a high-temperature process.6−8 In order to overcome these issues as well as the expensive, complicated, and cumbersome nature of the technique, metallic nanoparticles have been suggested as an alternative process that provides facile, low-cost fabrication with controllable size and shape through the reaction conditions, specifically those of gold and silver nanoparticles (AuNPs and AgNPs, respectively), resulting in the formation of surface plasmon resonance (SPR).9−12 © 2015 American Chemical Society
Received: October 20, 2015 Accepted: November 30, 2015 Published: November 30, 2015 27910
DOI: 10.1021/acsami.5b09982 ACS Appl. Mater. Interfaces 2015, 7, 27910−27917
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Novel Fabrication Method of the SERS Platform Based on AgNPs Synthesized Directly into Paper Using the SILAR Methoda
a
SERS, surface-enhanced Raman spectroscopy. AgNPs, silver nanoparticles. SILAR, successive ionic layer absorption and reaction. The paper substrate consisting of AgNP-depositable hydrophilic and wax-printed hydrophobic areas was successively treated in four steps for 30 s each, and one SILAR cycle was performed for 2 min. Silver nitrate (AgNO3) and sodium borohydride (NaBH4) were used as reagents, and water was used for rinsing. The floating on solution method was used instead of a conventional dipping method. This modified SILAR approach was based on the porous structure of the paper. The SILAR method led to direct synthesis of AgNPs on the reactive site such that the presence of AgNPs was observed at the exterior and interior of the paper. This 3D architecture of AgNPs led to high Raman intensities associated with interparticle plasmon coupling. Paper might be the best substrate for a facile, low-cost, high-sensitivity SERS fingerprinting platform using the SILAR technology. Sylgard 186 silicone elastomer kit purchased from Dow Corning (Cortland, NY, USA) was prepared by mixing a resin-to-curing agent of 10:1 at 80 °C for 2 h. Other substrates, such as glass, silicon (Si) wafer, polyethylene terephthalate (PET) film, and aluminum (Al) foil, were purchased from a local market. 2.2. Instrumentation. The morphologies of the AgNPs on the substrates were investigated using a S-4700 field emission scanning electron microscope (FE-SEM; Hitachi, Tokyo, Japan) at an accelerating voltage of 5 kV. The AgNP size was analyzed using ImageJ software (NIH, Bethesda, MD, USA). The Raman spectra were obtained using a SENTERRA confocal Raman system (Bruker Optics, Billerica, MA, USA). A 785 nm diode laser source with a 10 mW power was focused to a spot size of ∼2.4 μm with a 20× objective lens. The spectrum of each point was recorded in the range of 417−1782 cm−1 with a spectral resolution of 5 cm−1 and twice the acquisition time of 30 s. To ensure reliability, a Raman spectrum of each sample was collected at 10 random points on the paper substrate. A 1 μL analytic sample droplet was used to evaluate the Raman spectrum. 2.3. Wax Treatment of the Paper Platform. The body of the SERS paper platform consisted of two parts: a AgNP-synthesizable hydrophilic area and a wax-printed hydrophobic area (Figure S1). A 2 mm hydrophilic circle used to measure the Raman signal and the hydrophobic region in order to prevent AgNPs from depositing onto the substrate was designed using AutoCAD (Autodesk, San Rafael, CA, USA) and wax-printed onto 10 × 20 mm2 or 30 × 35 mm2 paper using a Xerox ColorQube 8570N printer (Fuji Xerox, Tokyo, Japan). Uniform impregnation of wax into the paper was performed in a drying oven at 130 °C for 45 s, followed by drying at room temperature.17 2.4. Fabrication of the SERS Paper Platform Using SILAR Approach. AgNPs were directly synthesized and grown on the surface of the paper as well as within the paper using the SILAR technique (Scheme 1), in which AgNO3 and NaBH4 solutions interacted by forming AgNPs through the reaction (eq 1). The paper substrate was treated in a AgNO3 solution of metal salt and rinsed with water. The substrate was then treated in a NaBH4 reducing solution and rinsed with water to remove the residual nonreacted reagents. The treatment time of each solution was fixed to 30 s and one SILAR cycle consisting of four successive steps for 2 min. To optimize the SERS activity, the AgNPs size was controlled by the concentration of reactive solutions and the number of cycles. In order to deposit the nanoparticles onto a substrate, most SILAR approaches dip the substrate in a solution. However, repeated SILAR cycles using this method might lead to swelling and destruction of the paper substrate. The modified SILAR approach of floating the paper on a solution was used to selectively deposit AgNPs onto the hydrophilic area due to the capillary action of the porous paper. Additionally, the wax-printed
requires a process time longer than 24 h or has complex iterations.25,26 This paper introduces a facile and innovative fabrication technique for paper substrates for sensitive SERS detection in which the nanoparticles are directly synthesized within the paper without external processing. This approach is based on the successive ionic layer absorption and reaction (SILAR) method, which involves self-assembly for synthesis of a thin film on a solid support through spontaneous deposition of nanoparticles of oppositely charged positive and negative ions, frequently used in the fabrication of solar cells and capacitors.31−36 This method allowed the formation of a multilayer and control of the size of the film or particle via multiple repetitions of the chemical treatments. A few studies have used a similar method, such as layer-by-layer deposition to paper;37,38 however, they used presynthesized nanoparticles and were for purposes other than a SERS platform. Although Stamplecoskie and Manser39 used the SILAR method to fabricate a SERS platform, the SERS effect was not observed, and the substrate was not paper. In the present study, we demonstrate the fabrication of a SERS-functionalized platform based on AgNPs synthesized directly into paper using the SILAR method. Silver was selected because it has superior SERS performance compared to gold.40 The SILAR approach provides a facile, rapid, low-cost, and highly reproducible SERS platform compared to other methods (Supporting Information, Table S1).19−28 Additionally, it does not require external equipment for synthesis of the nanoparticles. The potential of this platform was evaluated through two samples of human cervical fluid for the clinical diagnosis of human papillomavirus (HPV) infection associated with cervical cancer and a malachite green (MG) solution for fungicide and parasiticide in aquaculture, associated with human carcinogenesis.
2. EXPERIMENTAL SECTION 2.1. Reagents and Materials. Silver nitrate (AgNO3, >99%), sodium borohydride (NaBH4, >96%), rhodamine B (RhB, >95%), malachite green chloride (>96%), octadecyltrichlorosilane (OTS), and toluene (C6H5CH3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were of analytical grade, and all solutions were prepared using 18.3 MΩ·cm−1 distilled water. Whatman cellulose chromatography paper (grade 1) with a 0.18 mm thickness was purchased from Sigma-Aldrich. Polydimethylsiloxane (PDMS) from a 27911
DOI: 10.1021/acsami.5b09982 ACS Appl. Mater. Interfaces 2015, 7, 27910−27917
Research Article
ACS Applied Materials & Interfaces hydrophobic area was used to prevent AgNPs from depositing on the substrate. 2AgNO3 + 2NaBH4 → 2Ag + 2NaNO3 + H 2 + B2H6
(1)
3. RESULTS AND DISCUSSION 3.1. Substrate Selection. We investigated the influence of substrate on the distribution of SILAR-synthesized AgNPs. Figure S2 shows the distribution of AgNPs synthesized through five SILAR cycles on four representative substrates of glass, PDMS, a Si wafer, and paper. AgNP dispersion was not observed on the glass, PDMS, or Si wafer substrates (Figure S2A−C), while AgNPs were well-dispersed on paper (Figure S2D). This difference was due to a distinctive feature of the SILAR chemical process; once the precursor in the solution formed seeds on the surface, a synthesis reaction occurred at the seeds through multiple repetitions. This cycle was used to control particle size and layer thickness. Therefore, the rough and porous surface of the paper substrate led to the formation of seeds of SILAR-synthesized AgNPs, while the smooth and nonporous surfaces of the glass, PDMS, and Si wafer substrates did not result in seed formation. This was consistent with the result associated with a coffee ring effect. As the nonporous solid substrates did not absorb the aqueous solution, the sample suspension concentrates around the perimeter of the droplet during evaporation.26 This effect did not occur to paper, because it was the porous structure and its cellulose material absorbed the reactive aqueous solution. Next, the SERS performance according to type of substrate using SILAR fabrication was demonstrated (Figure S3). Position-to-position Raman spectra in five zones of a dried 1 μL 1 mM RhB droplet on each substrate were compared in the prominent RhB-characterized Raman peaks at 620 cm−1 (aromatic bending), 1201 cm−1 (aromatic C−H bending), and 1356 cm−1 (aromatic C−C stretching).8 The paper substrate showed highly reproducible and intense Raman spectra regardless of position, while the other substrates showed variable Raman intensities due to the coffee ring effect of samples and the peaks of their own substrate materials. The position-independent high Raman intensity of the paper was likely responsible for the SERS effect of uniform AgNPs synthesized on the cellulose fibers. In addition, since the SILAR method led to direct synthesis of AgNPs on the reactive site, the presence of nanoparticles was observed at the exterior and interior of the paper (Figure 1). The high Raman intensities of the paper were responsible for the interparticle plasmon coupling in the 3D architecture of the SILAR-synthesized AgNPs due to the porous structure of the paper. Therefore, paper is the best substrate for a low-cost, highly sensitive SERS fingerprinting platform fabricated through SILAR-synthesized AgNPs. 3.2. Optimization of the SILAR Conditions. The concentration of SILAR reagents and number of SILAR cycles were optimized in order to improve the SERS efficiency on the paper substrate. On the basis of the literature,31,39,41 the concentrations of silver salt (AgNO3) and reducing agent (NaBH4) were chosen in the range of 10−40 mM. In order to prevent the particles from forming a thin film and to establish a facile cost-effective fabrication, we set a limit of 10 SILAR cycles. As a result, five SILAR cycles were fixed for optimizing the concentration of the reactive solutions. Figure 2A and Figure 2B show the Raman spectra and intensities of the RhBcharacterized peaks at seven different conditions (Table S2). All
Figure 1. (A) Cross-sectional FE-SEM images of the SILAR-fabricated SERS paper platform. Scale bar = 50 μm. SILAR technique led to direct synthesis and growth of AgNPs on the surface of the paper (B) as well as within the paper (C, D). Scale bar = 250 nm.
conditions showed high Raman intensities with prominent RhB-characterized peaks at 620, 1201, and 1356 cm−1. Generally, a higher concentration of reducing agent is required for complete reduction of the adsorbed metal cations;41 however, a small number of cycles, such as five cycles (Figure 2B), does not allow complete reduction. As a result, the optimal number of cycles needed to be estimated. Therefore, the two conditions with the highest Raman intensity, condition 4 (20/ 10 mM/mM AgNO3/NaBH4) and condition 5 (20/20 mM/ mM AgNO3/NaBH4), were preferentially selected to determine the optimal number of SILAR cycles. Figure 2C and Figure 2D show the Raman intensities of RhB-characterized peaks at 2−10 SILAR cycles under the two chosen conditions (Figure S4 and Table S3). The signal intensity was enhanced with increasing SILAR cycle due to the increasing size of the AgNPs synthesized on the paper. This SERS activity reached the highest intensity at four cycles with condition 4 (36 411 at 1356 cm−1) and six cycles with condition 5 (33 363 at 1356 cm−1), followed by saturation or a decrease in activity. This finding was caused by the localized SPR (LSPR) that occurred a few nanometers between the particles due to the incident energy. If the size of the particles increases and the interparticle distance decreases, the SPR is enhanced. However, if the size of the particles is too large or if the particles aggregate, they act as a film rather than separate particles. Under such a condition, the interparticle resonance decreases and leads to a decrease in Raman intensity. Since four cycles with condition 4 (relative standard deviation RSD = 23%) showed low reproducibility with a high Raman intensity compared to six cycles with condition 5 (RSD = 11%), six cycles with 20/20 mM/mM AgNO3/NaBH4 were selected as the optimal SILAR conditions. 3.3. Evaluation of the Optimized SILAR Condition: Structural Property. Figure 3 shows FE-SEM images of SILAR-synthesized AgNPs on the paper substrate with 2−10 SILAR cycles and 20/20 mM/mM AgNO3/NaBH4. The RGB intensity of AgNPs deposited on paper darkened with increasing number of cycles (Figure S5). The presence of disordered cellulose fibers and a porous structure was observed in bare paper (Figure S6). AgNPs smaller than 5 nm formed as seeds on the surface of the cellulose fibers or at the gap between the cellulose fibers early in the cycle. An increasing number of SILAR cycles led to an insignificant increase in the size of AgNPs (1 nm per cycle, Figure 3F) and a decrease in the 27912
DOI: 10.1021/acsami.5b09982 ACS Appl. Mater. Interfaces 2015, 7, 27910−27917
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ACS Applied Materials & Interfaces
Figure 2. (A) Raman spectra of 1 mM RhB and (B) Raman intensities of three RhB-characterized peaks at 620, 1201, and 1356 cm−1 with seven different concentrations of AgNO3 and NaBH4 reducing agent. Condition 4 (20/10 mM/mM AgNO3/NaBH4) and condition 5 (20/20 mM/mM AgNO3/NaBH4) with the highest Raman intensities were preferentially selected. Shown are Raman intensities of three RhB-characterized peaks with 2−10 SILAR cycles at (C) 20/10 and (D) 20/20 mM/mM AgNO3/NaBH4. Six cycles showed high reproducibility among the SILAR cycles with 20/20 mM/mM AgNO3/NaBH4.
Figure 3. FE-SEM images of AgNPs deposited on paper with (A) two, (B) four, (C) six, (D) eight, and (E) 10 SILAR cycles and (F) size variations at the optimized concentration of 20/20 mM/mM AgNO3/NaBH4 SILAR reagents. Insets are photographs of the paper surface according to number of SILAR cycles (Figure S7). Scale bar = 200 nm.
poor reproducibility.8,46 However, 10 different SERS platforms at an optimized SILAR condition showed high reproducibility with a 4.2% RSD in the present study (Figure S9). This excellent reproducibility was responsible for the formation of the AgNP-stacked 3D structure generating the hot spot effect. Therefore, a 3D stack structure of nanoparticles is likely to achieve good SERS performance and an increase in the sensitivity of detection. 3.4. Evaluation of the Optimized SILAR Conditions: SERS Activity. The sensitivity of a paper-based SERS platform fabricated at the optimized SILAR conditions was evaluated at various concentrations of 100 fM to 1 mM RhB (Figure 4). The SILAR-synthesized AgNPs SERS platform provided information to clearly detect the presence of RhB solution up to a 1 pM concentration; however, we could not detect the presence of 1356 cm−1 RhB-characterized peak at a 100 fM concentration, due to low signal-to-noise ratio (Figure S10). All concentrations exhibited prominent RhB-characterized Raman peaks, which could be used as an analytical marker. Since an RhB peak near 1356 cm−1 was more representative and the intensity was
number of AgNPs per unit area (Figure S7). Herein, in spite of increasing number of cycles, the presence of tiny AgNPs might be caused by synthesis on the other site (Figure S8). Therefore, although the paper fabricated with two cycles had the relatively small size and sparse distribution of the AgNPs (Figure 3A), it showed relatively higher Raman intensity (21 149 at 1356 cm−1, Figure 2D), caused by hot spots occurring between the tiny particles. As previously mentioned, the SPR phenomenon strongly coupled the gap regions of a pair of AuNPs or AgNPs, i.e., a hot spot. The formation of hot spot plays an important role in SERS activity. Since the interparticle distance of these tiny AgNPs and lager particles decreased with increasing the number of SILAR cycle, the SERS activity was enhanced until six SILAR cycles. At the following cycle, these particles were aggregated or formed as a thin film, and the SERS effect decreased. Although computer-based simulation or expensive sophisticated nanotechnologies have also been used to illustrate this effect, the mechanism for SERS enhancement is still not completely understood.9,42−45 Most SERS-active platforms explained by hot spot theory have a critical problem with 27913
DOI: 10.1021/acsami.5b09982 ACS Appl. Mater. Interfaces 2015, 7, 27910−27917
Research Article
ACS Applied Materials & Interfaces
conditions (Table S4). The SERS platform showed a logarithmic characteristic of intensity variation within a range of 10−12−10−3 M RhB (Figure 4) with a sensitivity of 0.97. Additionally, the characteristic curve showed good agreement with the concentrations in three different ranges: picoscale, 1− 100 pM; nanoscale, 1−100 nM; microscale, 1−100 μM (Figure S11). In order to quantify the SERS activity, the Raman spectra of SILAR-synthesized AgNPs SERS paper and bare paper were investigated at 1 pM and 1 mM RhB concentrations, respectively (Figure S12). In general, SERS EF is acquired from an average number of adsorbed molecules (N) in the scattering volume of SERS and non-SERS areas (eq S1 in Supporting Information). It assumes N = cV, where c is the concentration and V is the scattering volume, which were the same because the probe molecules were uniformly distributed on paper.47 Therefore, the SERS EF was calculated using the following equation:
⎛I ⎞⎛ c ⎞ EF = ⎜ SERS ⎟⎜ bare ⎟ ⎝ Ibare ⎠⎝ cSERS ⎠
(2)
where cSERS is the concentration of RhB on SILAR-synthesized AgNP SERS paper and cbare is that on bare paper, respectively. The Raman intensities of SERS paper and bare paper at 1356 cm−1 were ISERS = 87.5 and Ibare = 78.9, respectively. The corresponding SERS EF to satisfy the requirement for single molecule detection was 1.1 × 109. Therefore, the AgNP paper platform fabricated at the optimized SILAR conditions demonstrated superior and stable performance in SERS activity for further practical applications. 3.5. Application of On-Site Bioassays. The applicability of the SILAR-optimized AgNPs SERS paper platform was evaluated using two samples including real HPV-infected human cervical fluid (clinical application) and carcinogenesisassociated MG solution (environmental application). HPV infection is one of the most common sexually transmitted diseases. HPV-16 and HPV-18 are prevalent types worldwide, while South Korea, Malaysia, Vietnam, Singapore, and
Figure 4. Representative SERS spectra with (A) different RhB concentrations (10−13−10−3 M) and (B) low concentrations of RhB on SILAR-synthesized AgNPs deposited on SERS paper. (C) Variation in Raman intensity at 1356 cm−1 with different RhB concentrations. The characteristic curve was y = −96730 + 9366 ln(x + 35960) with a sensitivity of 0.97.
very sensitive according to the spectra, it was selected as the representative RhB peak to evaluate the sensitivity of a paperbased SERS platform fabricated at the optimized SILAR
Figure 5. (A) Representative SERS spectra and (B) difference among HPV types. (C) Representative SERS spectra and (D) Raman intensities at 1613 cm−1 of different MG concentrations (10−10−10−6 M) on SILAR-synthesized AgNP-deposited SERS paper. The characteristic curve was y = −21860 + 2150 ln(x + 34200) with a sensitivity of 0.99. 27914
DOI: 10.1021/acsami.5b09982 ACS Appl. Mater. Interfaces 2015, 7, 27910−27917
Research Article
ACS Applied Materials & Interfaces
applicability of other techniques, including silica coating9 and bimetallic deposition,56,57 using the SILAR approach.
Philippines demonstrate HPV-16 as the most commonly observed type, followed by HPV-52 and HPV-58.48 Our previous study demonstrated that Raman spectroscopy might be a good alternative method for early clinical diagnosis of HPV infection.49 Figure 5A shows Raman spectra according to HPV subtype (Figure S13). Overall, HPV-16 and HPV-52 showed similar Raman bands but different intensities, while normal (HPV noninfected patient) and HPV-58 showed completely different Raman peaks and intensities compared to the other types. There was a difference in behavior among the three highrisk HPV types (Figure 5B), indicating that these Raman peaks of a SILAR-synthesized AgNPs paper platform can be used as a novel marker to detect the presence of HPV and to determine the specific type (Table S5). MG is a triphenylmethane dye and a highly effective parasiticide and fungicide in aquaculture. It is absorbed by fish tissue and remains during transport. However, it is banned in the use in edible fish due to its carcinogenic potential in humans.50 MG is conventionally inspected using liquid chromatography, which is a meticulous instrumental assay for on-site applications. Some studies have reported nanoparticlebased Raman investigations for screening the concentration of MG.24,28,51 Figure 5C shows the Raman spectra of different concentrations of 10 pM to 1 μM MG solution. All concentrations showed prominent Raman peaks at 438, 798, 916, 1172, 1365, and 1613 cm−1 (Figure S14, Table S6). The Raman peak at 1613 cm−1 was selected as a representative marker of the presence of MG. The SERS platform achieved an MG detection as low as 10 pM (
