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Accepted Manuscript Regular Article Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH Huilin Li, Dandan Men, Yiqiang Sun, Dilong Liu, Xinyang Li, Liangbin Li, Cuncheng Li, Weiping Cai, Yue Li PII: DOI: Reference:
S0021-9797(17)30690-2 http://dx.doi.org/10.1016/j.jcis.2017.06.034 YJCIS 22461
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
13 April 2017 12 June 2017 12 June 2017
Please cite this article as: H. Li, D. Men, Y. Sun, D. Liu, X. Li, L. Li, C. Li, W. Cai, Y. Li, Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH, Journal of Colloid and Interface Science (2017), doi: http://dx.doi.org/10.1016/ j.jcis.2017.06.034
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Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH Huilin Lia,b,+, Dandan Men a,+, Yiqiang Suna,b, Dilong Liu a, Xinyang Lia,b, Liangbin Li b
,Cuncheng Li c, Weiping Cai a, Yue Lia,*
a
Key Laboratory of Materials Physics, Anhui Key Laboratory of Nanomaterials and
Nanotechnology, Institute of Solid State Physics, Chinese Academy of Sciences, Hefei 230031, P. R. China, E-mail: [email protected]. b c
University of Science and Technology of China, Hefei 230026, P. R. China
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R.
China +
These authors contributed equally to this work.
Abstract: We developed an interesting route for preparing a poly (acrylamide-co-acrylic acid) (P(AAm-co-AA)) hydrogel microsphere with a coating of Au nanospheres (hydrogel microsphere @ Au nanospheres) through self-assembly based on electrostatic interaction. The fabricated composites could be used as highly sensitive enhanced Raman scattering substrates. The nanogaps between adjacent Au nanospheres were dynamically tuned by volume changes in the hydrogel microspheres in the semiwet state under different pH conditions. At pH 6, the hydrogel microsphere @ Au nanospheres demonstrated highly sensitive SERS with an enhancement factor of 10 9. The product could detect very low concentrations of analytes up to 10−12 M 4-aminothiophenol (4-ATP) molecules. This paper proposes a new method for detecting trace amounts of environmental organic pollutants by dynamically tuning the SERS enhancement in the semiwet testing state. Keywords: SERS, hotspots, Au nanoparticles, stimuli-responsive hydrogel, microfluidic
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1. Introduction Surface enhanced Raman scattering (SERS) is a promising technique in which Raman scattering signals can be greatly enhanced when molecules are adsorbed on SERS-active substrates, such as noble metal surfaces [1,2]. The SERS method can provide abundant spectroscopic fingerprint information and is thus an attractive tool for recognizing trace amounts of molecules in chemical and biological applications [3–5]. Two SERS enhancement mechanisms have been developed: an electromagnetic mechanism (EM) and a chemical or charge transfer mechanism (CT) [6 –8]. The EM utilizes light amplification by exciting a localized surface plasmon resonance, and it is mainly dependent on the nanogaps, sharp corners, or edges of plasmonic materials [1,6]. Nano-sized noble metals such as silver and gold are widely used as plasmonic materials. The SERS enhancement factor (EF) of the EM is theoretically calculated to reach 1010–10 11 in these materials [9]. The CT mainly involves charge-transfer excitation between molecules and the metal surface. The EF of this mechanism is lower than that of the EM, and the total EF of the SERS mainly relies on the EM. Hence, it is important to design and develop high-EF SERS substrates with nano-sized noble metals. These noble metal SERS substrates possess high-density “hot spots,” which especially depend on the shape, size, aggregation state, and nature of the metal materials. Hot spots are defined as highly localized regions with intense localized surface plasmon resonance, and these spots usually present in interstitial crevices in plasmonic materials [10,11]. The nanogaps between metal nanoparticles are more important for generating hot spots [12]. Gaps 10 nm between nanostructures can significantly enhance the electromagnetic field [13 –15], which in turn can significantly improve the SERS detection. Therefore, it would be very beneficial to design SERS substrates with well-controlled nanogaps between nanostructures. Two kinds of SERS substrates have previously been developed to control the nanogaps. The first type are traditional SERS substrates where the nanogaps between nanostructures are
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fixed once the substrates have been manufactured, such as highly ordered 1D [16,17], 2D [18,19] and 3D [20,21] nanostructures produced using a top-down or bottom-up method. In the other type, the nanogaps can be dynamically tuned by changing certain conditions during testing, such as the temperature and magnetism [10,22–28]. Yang and Liu et al. [27,28] evaporated a droplet of citrate–Ag solution on a fluorosilylated silicon wafer to generate a 3D hot spot substrate. When the average gap was 2 nm, the formed 3D SERS substrate generated many hot spots, which significantly enhanced the electric field. These substrates with dynamically tunable nanogaps had a great advantage in detecting trace-level pollutants. Moreover, nanogaps of 1.5 and 5 nm can also generate high SERS signals[29,30]. However, these tiny and uncertain nanogaps bring some difficulties for SERS detection using traditional SERS substrates, because they are fixed nanogaps. Therefore, there is still a need to develop new SERS substrates that allow the nanogaps between nanostructures to be dynamically tuned. In this study, we developed a new strategy for preparing a pH-responsive 3D poly (acrylamide-co-acrylic acid) (P(AAm-co-AA)) hydrogel microsphere with a coating of Au nanospheres (hydrogel microsphere @ Au nanospheres). The nanogaps between the adjacent Au nanospheres adsorbed on the hydrogel microsphere surfaces could be tuned by changing the pH value. The developed composite could be used as a highly sensitive SERS-active substrate. Uniform P(AAm-co-AA) hydrogel microspheres with negative charges were first fabricated using droplet-based microfluidic technique. Au nanospheres with positive charges were then prepared by a modified polyol route and chemical etching. The Au nanospheres self-assembled on the hydrogel microsphere through electrostatic interaction to form the hydrogel microsphere @ Au nanospheres. The hydrogel microsphere could be induced to swell by changing the pH value, thereby generating suitable nanogaps between the Au nanospheres on its surface. The as-prepared hydrogel microsphere @ Au nanospheres were used as 3D SERS substrates and demonstrated good sensitivity to probe 4-ATP molecules. -3-
This study provided a new means for detecting trace-level environmental organic pollutants by dynamic SERS enhancement based on the obtained responsive hydrogel-plasmonic materials.
2. Experimental Section 2.1. Materials Acrylamide (AAm, CP), acrylic acid (AA, CP), dimethyl sulfoxide (CP), paraffin liquid (CP), ethylene glycol (AR), and chloroauric acid hydrate (HAuCl4, AR) were purchased from the Sinopharm Chemical Reagent Corporation Ltd. N-N’-Methylenebisacrylamide (MBAAm,
CP)
was
acquired
from
the
Aladdin
Industrial
Corporation.
2-Hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (Irgacure 2959, RG) was obtained from the Tianjin Heowns Biochemical Technology Co., Ltd. Lauryl PEG-9 polydimethylsiloxyethyldimethicone (KF6038) was provided by the Shin-Etsu Chemical, Japan. Poly(diallyldimethylammonium chloride) solution (PDDA, 400000–500000, 20 wt.% in H2O) was supplied by Sigma–Aldrich (Shanghai) Trading Co., Ltd. These reagents were directly used without further purification. Water (18.2 MΩ·cm) was obtained from an ultrafilter system (Milli-Q, Millipore, Marlborough, MA). The chemical reagents had chemically pure (CP), analytical reagent (AR), and research grade (RG) purity level. 2.2 Preparation of Au nanospheres Monodispersed Au nanospheres were prepared using a modified polyol route followed by chemical etching [31,32]. A prepared gold precursor solution containing 20 µL of PDDA and 0.5 µL of a 1 M HAuCl4 aqueous solution per milliliter of ethylene glycol was placed in a glass vial. After stirring with a magnetic blender for 1–2 min at room temperature under ambient conditions, the glass vial was sealed and heated at 195 °C for 30 min in an oil bath. A HAuCl4 (0.25 µL, 1 M) solution was then added to the Au colloidal solution (1 mL). After 2 -4-
min of chemical etching, spherical Au nanoparticles were produced. The product was collected by centrifugation at 14000 rpm, washed repeatedly with pure water, and concentrated to obtain a 50 mM Au colloidal aqueous solution. 2.3 Preparation of hydrogel microspheres In our experiments, a cross-shaped microfluidic platform was used to produce hydrogel microspheres with tunable sizes [Fig. S1 (a)]. Paraffin liquid containing 2% (V/V) KF6038 as emulsifier was used as the continuous phase. A hydrogel precursor solution containing 200 mg of AAm, 7 mg of MBAAm, 120 µL of AA, and 100 µL of the photoinitiator solution per milliliter of water was used as the dispersed phase. The photoinitiator solution contained dimethyl sulfoxide and 30% (m/V) Irgacure 2959. When the continuous and dispersed phase solutions were simultaneously injected into the cross-shaped glass chip, the dispersed phase liquid was broken off by the strong shear force of the continuously dripping liquid, releasing uniform droplets in a cross-shaped pattern with a microfluidic cross-flow arrangement. After the formation of uniform droplets, the continuous phase served as a carrier to drive these droplets into the long polytetrafluoroethylene (PTFE) pipes. The hydrogel precursor droplets were solidified and polymerized into hydrogel microspheres through irradiation by 311 nm ultraviolet light (Philips, Narrow band TL 20W/01-RS) [Fig. S1 (a)]. After the oil and hydrogel microspheres were collected, the obtained microspheres were thoroughly washed with butyl alcohol to remove the paraffin liquid. 2.4 Preparation of 3D hydrogel microsphere @ Au nanospheres A specific amount (40–150 µL) of the 50 mM Au nanosphere colloidal solution was added to 10 µL of the aqueous solution of hydrogel microspheres (50 g/L). After 12 h, the Au nanospheres were adsorbed onto the surfaces of the hydrogel microspheres and formed 3D hydrogel microsphere @ Au nanospheres through self-assembly by the electrostatic -5-
interaction between the Au nanospheres and the hydrogel microspheres. After centrifugal separation at 4000 rpm and repeated washing with ethyl alcohol, the 3D hydrogel microsphere @ Au nanospheres were cleaned using an ultra-violet ozone cleaning systems (T10X10/OES/E) to remove the surfactant PDDA adsorbed on the Au nanosphere surfaces. The fabricated composite was used as a SERS substrate 2.5 Instrumentation and characterization A microfluidic device was acquired from the Suzhou Wenhao Chip Technology Co., Ltd. and used to prepare hydrogel microspheres (Fig. S1). The width and depth of the micro-channels were 120 and 50 µm, respectively, in a cross-shaped glass chip. A pump was applied to feed the dispersed phase into the middle micro-channels. The two other inlets of the cross-shaped chip were connected to a double-syringe pump, into which the continuous phase was fed. The morphology of the Au nanospheres was characterized by transmission electron microscopy (TEM; JEM-2010 transmission electron microscope). The morphologies of the hydrogel microspheres and 3D hydrogel microsphere @ Au nanospheres were observed using a field-emission scanning electron microscope (SEM; SU8020). Photographs of the hydrogel microspheres were obtained under an optical microscope (Axio Lab.A1) equipped with a digital camera (DLC300L, 3.0 M). Raman spectra were obtained through a confocal microprobe Raman spectrometer (Renishaw inVia Refl ex). The zeta potential of the hydrogel microsphere and Au nanospheres were obtained using a Zetasizer 2000/3000. Simultaneously, the average nanogaps between the Au nanospheres were characterized by in situ SAXS measurements at beamline BL16B of the Shanghai Synchrotron Radiation Facility. The optical absorption spectra were obtained using a microspectrometre system from Ideaoptics Corp. 2.6 SERS measurements SERS measurements were conducted using a laser beam with a wavelength of 785 nm, -6-
under 2 mW of power and 2 s of integral time. During the SERS measurements, the laser beam, with a size of 2 µm, was vertically projected onto the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres. The prepared P(AAm-co-AA) hydrogel microsphere @ Au nanospheres were immersed in 10 mL samples of 4-ATP aqueous solutions with different concentrations for 12 h and placed on clean silicon wafers. The composite microspheres were added to pH buffer solutions of different pH levels for 30 min. After removing pH buffer solutions with a filter paper or capillary, the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres were subjected to SERS measurements (Fig. S2). The water in the P(AAm-co-AA) hydrogel microspheres was volatilized under the heat generated by the light. After 15 s, the composite microsphere went from the wet state to the semiwet state, and finally became dry. The SERS spectrum was obtained in the semiwet state. The SERS effects of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres under different conditions were measured (Figs. S3-S5). Other molecules were detected using the composite microspheres in the semiwet state under a pH 6 condition, whereas the measurement conditions of the 2,4-dimethylbenzenethiol and 4-tert-butylthiophenol were the same as those for the 4-ATP, and those for the rhodamine 6G (R6G) were a wavelength of at 532 nm wavelength, 2 mW of power, and an integral time of 5 s integral time (Figs. S7 and S8).
3. Results and Discussion 3.1 Hydrogel microspheres Fig. 1 shows typical optical microscopy (OM) and scanning electron microscopy (SEM) images of the dried P(AAm-co-AA) hydrogel microspheres fabricated using the droplet-based microfluidic technique. In the OM image of the microspheres (Fig. 1 a), the P(AAm-co-AA) hydrogel particles have a spherical morphology, regular shape, and homogeneous particle size, which were confirmed from the SEM image of the hydrogel microspheres (Fig. 1 b). The size of the dried P(AAm-co-AA) hydrogel microspheres was approximately 16 µm. -7-
The uniform morphology of the hydrogel microspheres is related to the employed preparation method, the droplet-based microfluidic technique (Fig. S1), which consists of two processes: droplet generation and solidification. For the droplet-releasing mechanism, interfacial stress is generated on the surface, where the immiscible liquids interact with the microchannel. The stress is affected by the viscosity, liquid flow or velocity and channel size [33-35]. Uniform droplets of different sizes can be obtained by controlling these parameters. Generally, uniform droplets with tunable sizes can be synthesized by controlling the flow or velocity of the dispersed and continuous phases. In the present experiment, we tuned the flow of the dispersed phase flow without changing the continuous phase flow to obtain uniform droplets of different sizes (Fig. S1 b–e). The formed hydrogel precursor droplets were carried by the continuous phase and solidified under ultraviolet irradiation. The droplets were solidified using on-chip and off-chip (in the outer pipe) methods. On-chip solidification, such as instability emulsion is used more commonly than off-chip solidification. The former requires a special microfluidic device and expensive solidification equipment to rapidly solidify the polymers. In the latter, a continuous and dispersed liquid must form a stable emulsion and then be solidified by an ordinary ultraviolet lamp. In the present work, we used paraffin liquid as the continuous phase and KF6038 as emulsifier, which could form a water-in-oil (W/O) stable emulsion with the hydrogel precursor aqueous solution. The emulsion was solidified in the outer PTFE pipe (O.D.1.6 mm and I.D. 0.6 mm). The photoinitiator Irgacure 2959, AAm, AA, and MBAAm formed a water-soluble polymer with a network crosslinking structure under ultraviolet irradiation at 311 nm (Fig. S1 a). Uniform P(AAm-co-AA) hydrogel microspheres with a size of approximately 16 µm were obtained through solidification in the PTFE pipe for approximately 20 min under a 1000 µL/h continuous phase flow and 100 µL/h dispersed phase flow (Fig. 1). Carboxyl (–COOH) groups were derived from the AA in the P(AAm-co-AA) hydrogel microspheres. In the presence of the hydrogel microspheres in the aqueous solution, the -8-
carboxyl groups could hydrolyze carboxylate radicals (−COO−) with negative electricity and hydrogen ions (H+) with positive electricity. Therefore, the P(AAm-co-AA) hydrogel microspheres were turned into negative particles in the aqueous solution. This finding was confirmed based on the zeta potential of −10.5 mV at 25 °C. 3.2 Au nanospheres Monodispersed Au nanospheres with a size of approximately 58 nm were successfully prepared using a modified polyol route and chemical etching (Fig. 2). The PDDA molecules were steadily coated on the surface of the Au nanospheres. When the PDDA-protected Au nanospheres were present in the aqueous solution, the PDDA surfaces could also be hydrolyzed, conferring positive surface charges on the Au nanospheres [36,37]. The Au nanospheres were positively charged and possessed a zeta potential of 56.7 mV at 25 °C. 3.3 Hydrogel microsphere @ Au nanospheres The Au nanospheres were self-assembled on the surface of the P(AAm-co-AA) hydrogel microspheres through electrostatic attraction because of their opposite zeta potentials, which produced the 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres (Fig. 3). The surfaces of the P(AAm-co-AA) hydrogel microspheres (concentration: 50 g/L, dosage: 10 µL) were gradually covered with an increasing amount of Au nanospheres (concentration: 50 mM, dosage: 40–150 µL), (Fig. 3 a–e). When using 150 µL of the Au colloidal solution, the P(AAm-co-AA) hydrogel microspheres were completely coated by the Au nanospheres, which formed black P(AAm-co-AA) hydrogel microsphere @ Au nanospheres and sunk to the bottom of the solution. With a further increase in the volume of the added Au colloidal solution, the upper solutionturned red. This finding indicated that in addition to the Au nanospheres coating the hydrogel microspheres, dissociated Au nanospheres were also present in the solution, and the quantity of Au nanospheres adsorbed on the hydrogel microspheres reached saturation through electrostatic attraction. Fig. 3 f displays a SEM image of the local -9-
surface of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres with saturation adsorption (corresponding to that in Fig. 3 e). The Au nanospheres were closely packed in a random manner on the hydrogel microspheres. 3.4 pH effect on SERS of hydrogel microsphere @ Au nanospheres Fig. 4 shows the Raman spectra of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the semiwet state at pH 2 to 10 for detecting 10−8 M 4-ATP molecules. Two noticeable peaks are present at 1082 and 1591 cm−1 in the SERS spectra (Fig. 4 a), and are attributed to the C (benzene ring)–S stretching vibration and C–C stretching vibration of the benzene rings of 4-ATP, respectively[38]. These two peaks can be fingerprint peaks for detecting 4-ATP through SERS enhancement, and are displayed as a function of pH, from pH 2 to 10 (Fig. 4 b). The peak intensity gradually increased with increasing pH up to 6 but declined when the pH exceeded 6. Hence, the SERS activity of the hydrogel microsphere @ Au nanospheres could be tuned by pH, and achieved maximum value at pH 6. The changes, which were the SERS effect on the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres under different pH conditions, originated from variations in the gaps between the adjacent Au nanospheres. This phenomenon was related to the semiwet state and the swelling degree of the P(AAm-co-AA) hydrogel microspheres at that pH value (Fig. 5 a and b). Hydrogel is a cross-linked polymer with a network structure, it can swell in water and hold a large amount of water while maintaining its network structure [39]. When the dry P(AAm-co-AA) hydrogel microsphere @ Au nanospheres was placed in the aqueous solution, the P(AAm-co-AA) hydrogel absorbed a large amount of water and swelled. However, the gaps between the adjacent Au nanospheres adsorbed on the surface of the P(AAm-co-AA) hydrogel microspheres were too large to enhance the Raman signals (Fig. 5 a I–II). Nevertheless, the composite microsphere could slowly lose some of water and gradually shrink (Fig. 5 a II–III and III–I) to reach the semiwet state after light exposure. In this case,
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the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres could be used as the 3D SERS substrate as result of the formation of nanogaps (less than 10 nm) between the adjacent Au nanospheres. During SERS measurements, the light beam was vertically projected onto the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres, and the water would volatilize with the heat generated by the light, which caused the microsphere to constantly shrink. When the P(AAm-co-AA) hydrogel microsphere was reduced to the semiwet state (about 15 s of light exposure), the gaps between the Au nanospheres were small, and the surface of the P(AAm-co-AA) hydrogel microsphere was fully covered by Au nanospheres. After reaching the semiwet state, the water volatilization in the hydrogel microsphere became very slow, and there was almost no change in the microsphere volume. Compared with the dry state, the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the semiwet state showed a strong SERS effect (Fig. S3). Meanwhile, P(AAm-co-AA) hydrogel containing numerous hydroxyl groups is a stimuli-responsive hydrogel, which responds to changes in environmental pH [39,40]. The pH-stimulated hydrogels can be classified into three types based on the presence of acidic and basic groups: acidic, basic, and amphiphilic hydrogels. P(AAm-co-AA) hydrogel is an acidic hydrogel and can be protonated under acidic conditions, as described in Equation 1. The charge density and content of mobile counterions decreases within the hydrogel, leading to gel shrinkage. Similarly, the acidic hydrogel is deprotonated under basic conditions, as described in Equation 2. Therefore, the number of similarly charged groups increases in the network structure, leading to electrostatic repulsion and hydrogel swelling. Interestingly, with a further increase in pH, the ionic strength increases but the osmotic pressure decreases. Thus, increasing the pH leads to hydrogel shrinkage [40]. Based on the volume change of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres (Fig. S2 b), we obtained its swelling capacity, which gradually increased larger with increasing pH in the acidic solution, slightly decreased stepwise with increasing pH in the - 11 -
basic solution, and reached the maximum result in the neutral solution (Fig. 5 b). This change was demonstrated in the absorption spectra of the single composite microsphere in the wet state under different pH conditions (Fig. 5 c). The composite microsphere had a visible absorbance in the visible region at pH = 2, and then the absorption peak at 530 nm became much sharper when the pH value increased from 2 to 7. However, this absorption peak became wide again when pH values larger than 7. When the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres shrunk, the gaps between the Au nanospheres became smaller. This allowed the Au nanoparticles to cause strong surface plasmon resonance coupling, with a very wide absorption peak, and vice versa. Thus, the pH value could be used to adjust the volume of the P(AAm-co-AA) hydrogel microsphere in the wet state. Similarly, when the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres was in the semiwet state, the P(AAm-co-AA) hydrogel could also be protonated or deprotonated with a small amount of water, which allowed a tiny change in the volume of the composite microsphere under the different pH condition. This tiny change could promote the movement of Au nanospheres, and produce the various nanogaps. Therefore, a suitable gap for the 3D substrate of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres was achieved at pH 6 in the semiwet state, which generated a strong SERS activity. The suitable gap was the average interparticle distance between adjacent Au nanospheres, which could be characterized by small-angle X-ray scattering (SAXS). Other pH conditions were compared, and we measured the values under pH = 5 ,6 and 7 (Fig. 5 d). From these SAXS curves, we know that q pH5 = 0.104 nm-1, qpH6 = 0.098 nm-1 and qpH5 = 0.092 nm-1, with the follow gaps: gpH5 = 10 nm, gpH6 = 6 nm and gpH10 = 4 nm. Therefore, at pH 6, a small nanogap (6 nm) between the adjacent Au nanospheres on the hydrogel microspheres was achieved, which was the main contributor to the strong SERS activity, although some aggregates composed of Au nanospheres might have existed in the swelling or shrinking process. (1) - 12 -
(2)
3.5 Hydrogel microsphere @ Au nanospheres as SERS substrates In order to demonstrate the detection limit of the 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres, 4-ATP molecules of 10-8–10-12 M were probed at pH 6. As shown in Fig. 6 a, the characteristic peaks of 4-ATP were distinctly observed at a low concentration of 10 -12 M. It was demonstrated that the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres could serve as a highly sensitive 3D SERS substrate in the semiwet state. Ten SERS spectra for ten composite microspheres were obtained randomly and are shown in Fig.6 b. To quantitatively evaluate the SERS reproducibility, we calculated the mean, standard deviation (SD), and relative standard deviation (RSD, the ratio of the standard deviation to the mean) of the noticeable peaks at 1082 and 1591 cm-1 (Fig. 6 b). It can be observed that the 10 measurements are highly reproducible and stable. Therefore, analytes could be sensitively and stably detected by the SERS technique using the hydrogel and Au composite materials. The EF is an important parameter used to evaluate the activity in a new type of SERS substrate. We calculated the EF for the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres. The analytical EF is related to the SERS enhancement of analytes of a known concentration and can be calculated as follows [41] (3)
where ISERS and IRS denote the intensities of the SERS and Raman scattering (RS) signal of the same mode for a given analyte, respectively; and CSERS and CRS represent the analyte concentrations for the SERS and the RS signals, respectively. To obtain the Raman spectrum of 4-ATP, it was immersed in a 10-2 M 4-ATP solution under the same conditions as the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres (Fig. S6). As listed in table 1, we estimated the EF at 1082 and 1591 cm-1 under different 4-ATP concentrations according to - 13 -
the Equation 3; the SERS and RS intensities were taken from Fig. 6 and Fig. S6. The EF values were between 4.9 × 108 and 9 × 109, and most could reach up to 109. The 3D SERS substrate had the highest EF compared to the other SERS substrates, i.e., the Ag nanostructured array (EF: 107) [1,42] and Au film (EF: 108) [14]. In addition, we conducted an investigation on detection of another common indicator of SERS, R6G, and the result are shown in Fig. S7 and Table S1. Compared with 4-ATP, the EF for R6G was low and unstable for different concentrations as a result of weak adsorption capacity of R6G on the surface of the Au nanospheres. In general, to enhance the R6G detection ability, a method is employed that involves dropping, some amount of the R6G solution onto the SERS substrate at an extremely low concentration [43–45]. However, this method was not suitable for the 3D hydrogel microsphere @ Au nanospheres. The composite substrate of hydrogel @ Au nanospheres was more suitable for detecting some molecules containing the sulfydryl group (-SH) like 4-ATP (Figs. S8), because the sulfydryl group can be strongly adsorbed on the surface of Au. For other composite systems of hydrogel and Au nanoparticles, the Au nanoparticles were mostly embedded in the hydrogel, but the SERS activity was low [24,25]. In contrast, the as prepared 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres had a high SERS activity with the Au nanospheres exposed to the outside.
4. Conclusion In conclusion, an interesting composite material consist of a P(AAm-co-AA) hydrogel microsphere and Au nanospheres was successfully prepared by self-assembly based on electrostatic interaction. Uniform P(AAm-co-AA) hydrogel microspheres with negative charges were fabricated using a microfluidic method and oppositely charged Au nanospheres were prepared using a chemical route. The P(AAm-co-AA) hydrogel microsphere @ Au nanospheres demonstrated a substantial 3D SERS effect, wherein the nanogaps could be - 14 -
dynamically tuned by changing the pH value during testing. At pH 6, the 3D SERS substrate produced a strong SERS activity, which could detect the complete set of SERS signals at 10 -12 M 4-ATP, and the EF reached up to 109 because of the presence of some nanogaps smaller than 10 nm. The results showed that the detection sensitivity of SERS could be dynamically tuned using the pH value in the semiwet state of the hydrogel microsphere @ Au nanospheres. This dynamic tuning method in the semiwet state is also called state translation nanoparticle enhanced Raman spectroscopy, and compared to the wet and dry testing methods, it can obtain a high SERS activity [27]. In contrast to other dynamic SERS substrates of Ag sols or noble nanoparticle encapsulated hydrogels [22–25, 27,28], the 3D SERS substrate of the hydrogel microsphere @ Au nanospheres could achieve high SERS performance and maintain it for a long time. Therefore, the 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres could be used as a 3D SERS substrate for trace analyses in medicine, biology, chemistry and other fields. However, this 3D substrate could also be further improved in future work. For example, the number and distribution of the Au NPs on the hydrogel microsphere could be controlled.
Acknowledgements The authors acknowledge the financial support provided by the Natural Science Foundation of China (grant no. 51371165, 51571189), State Key Program of National Natural Science Foundation of China (grant No. 51531006), Anhui Provincial Natural Science Foundation (grant no. 1508085JGD07), Cross-disciplinary Collaborative Teams Program in CAS, and CAS/SAFEA International Partnership Program for Creative Research Teams.
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Fig. 1. (a) Typical OM and (b) SEM images of P(AAm-co-AA) hydrogel microspheres obtained on microfluidic platform (continuous phase flow: 1000 µL/h, dispersed phase flow: 100 µL/h). Scale bars are 20 µm.
Fig. 2. Typical TEM image of Au nanospheres fabricated by modified polyol route and chemical etching. Scale bar is 50 nm.
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Fig. 3. SEM images of P(AAm-co-AA) hydrogel microspheres (50 g/L, 10 µL) that adsorbed different amounts Au nanoparticles (50 mM): (a) 40, (b) 60, (c) 80, (d) 100, and (e)–(f) 150 µL. Scale bars, (a)–(e): 5 µm and (f): 500 nm.
Fig. 4. (a) SERS spectra of P(AAm-co-AA) hydrogel microsphere @ Au nanospheres after incubation in 10 −8 M 4-ATP solution under various pH conditions. (b) Two fingerprint bands and the error bars [in (a)] plotted at 1082 and 1591 cm−1.
Fig. 5. (a) Schematic of volume variation of P(AAm-co-AA) hydrogel microsphere @ Au nanospheres with added water and lost water. (b) Schematic of the variation in the gaps between the Au nanospheres on the P(AAm-co-AA) hydrogel microspheres with various swelling capacities under different pH conditions. (c) Absorption spectra of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the wet state under different pH conditions. (d) One dimensional integrated SAXS curves for P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the wet state under pH = 5, 6 and 7 conditions; inset shows portions of SAXS curves after subtracting baseline. Center spacing between Au nanospheres can be obtained by d = g+R = 2π⁄q [28], where q is the module of the scattering vector, g is the gap and R is the diameter of the Au spheres.
Fig. 6. (a) SERS spectra of different concentrations of 4-ATP solution at pH 6 and (b) ten SERS spectra of 10 -10M 4-ATP at pH 6 on P(AAm-co-AA) hydrogel microsphere @ Au nanospheres. Table 1. Intensity and EF values at 1082 and 1591 cm−1 under different 4-ATP concentrations.
RS SERS
4-ATP (M) 10-2
1082 cm-1 I (a.u.) EF 200 -
1591 cm-1 I (a.u.) EF 120 -
10-8
98068
4.9×108
63588
5.3×108
10-10
7815
3.9×109
6359
5.3×109
10-11
1614
8.1×109
1084
9×109
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH Huilin Li a,b,+, Dandan Men a,+, Yiqiang Sun a,b, Dilong Liu a, Xinyang Li a,b, Liangbin Li b, Cuncheng Li c, Weiping Cai a, Yue Lia,*
An interesting route was developed for preparing P(AAm-co-AA) hydrogel microsphere @ Au nanospheres through self-assembly based on electrostatic interaction. The nanogaps between adjacent Au nanospheres on the surfaces of P(AAm-co-AA) hydrogel microsphere could be tuned dynamically to obtain high SERS effect by controling volume of the hydrogel microsphere under different pH condition in the semiwet state.
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S0021-9797(17)30690-2 http://dx.doi.org/10.1016/j.jcis.2017.06.034 YJCIS 22461
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
13 April 2017 12 June 2017 12 June 2017
Please cite this article as: H. Li, D. Men, Y. Sun, D. Liu, X. Li, L. Li, C. Li, W. Cai, Y. Li, Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH, Journal of Colloid and Interface Science (2017), doi: http://dx.doi.org/10.1016/ j.jcis.2017.06.034
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH Huilin Lia,b,+, Dandan Men a,+, Yiqiang Suna,b, Dilong Liu a, Xinyang Lia,b, Liangbin Li b
,Cuncheng Li c, Weiping Cai a, Yue Lia,*
a
Key Laboratory of Materials Physics, Anhui Key Laboratory of Nanomaterials and
Nanotechnology, Institute of Solid State Physics, Chinese Academy of Sciences, Hefei 230031, P. R. China, E-mail: [email protected]. b c
University of Science and Technology of China, Hefei 230026, P. R. China
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R.
China +
These authors contributed equally to this work.
Abstract: We developed an interesting route for preparing a poly (acrylamide-co-acrylic acid) (P(AAm-co-AA)) hydrogel microsphere with a coating of Au nanospheres (hydrogel microsphere @ Au nanospheres) through self-assembly based on electrostatic interaction. The fabricated composites could be used as highly sensitive enhanced Raman scattering substrates. The nanogaps between adjacent Au nanospheres were dynamically tuned by volume changes in the hydrogel microspheres in the semiwet state under different pH conditions. At pH 6, the hydrogel microsphere @ Au nanospheres demonstrated highly sensitive SERS with an enhancement factor of 10 9. The product could detect very low concentrations of analytes up to 10−12 M 4-aminothiophenol (4-ATP) molecules. This paper proposes a new method for detecting trace amounts of environmental organic pollutants by dynamically tuning the SERS enhancement in the semiwet testing state. Keywords: SERS, hotspots, Au nanoparticles, stimuli-responsive hydrogel, microfluidic
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1. Introduction Surface enhanced Raman scattering (SERS) is a promising technique in which Raman scattering signals can be greatly enhanced when molecules are adsorbed on SERS-active substrates, such as noble metal surfaces [1,2]. The SERS method can provide abundant spectroscopic fingerprint information and is thus an attractive tool for recognizing trace amounts of molecules in chemical and biological applications [3–5]. Two SERS enhancement mechanisms have been developed: an electromagnetic mechanism (EM) and a chemical or charge transfer mechanism (CT) [6 –8]. The EM utilizes light amplification by exciting a localized surface plasmon resonance, and it is mainly dependent on the nanogaps, sharp corners, or edges of plasmonic materials [1,6]. Nano-sized noble metals such as silver and gold are widely used as plasmonic materials. The SERS enhancement factor (EF) of the EM is theoretically calculated to reach 1010–10 11 in these materials [9]. The CT mainly involves charge-transfer excitation between molecules and the metal surface. The EF of this mechanism is lower than that of the EM, and the total EF of the SERS mainly relies on the EM. Hence, it is important to design and develop high-EF SERS substrates with nano-sized noble metals. These noble metal SERS substrates possess high-density “hot spots,” which especially depend on the shape, size, aggregation state, and nature of the metal materials. Hot spots are defined as highly localized regions with intense localized surface plasmon resonance, and these spots usually present in interstitial crevices in plasmonic materials [10,11]. The nanogaps between metal nanoparticles are more important for generating hot spots [12]. Gaps 10 nm between nanostructures can significantly enhance the electromagnetic field [13 –15], which in turn can significantly improve the SERS detection. Therefore, it would be very beneficial to design SERS substrates with well-controlled nanogaps between nanostructures. Two kinds of SERS substrates have previously been developed to control the nanogaps. The first type are traditional SERS substrates where the nanogaps between nanostructures are
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fixed once the substrates have been manufactured, such as highly ordered 1D [16,17], 2D [18,19] and 3D [20,21] nanostructures produced using a top-down or bottom-up method. In the other type, the nanogaps can be dynamically tuned by changing certain conditions during testing, such as the temperature and magnetism [10,22–28]. Yang and Liu et al. [27,28] evaporated a droplet of citrate–Ag solution on a fluorosilylated silicon wafer to generate a 3D hot spot substrate. When the average gap was 2 nm, the formed 3D SERS substrate generated many hot spots, which significantly enhanced the electric field. These substrates with dynamically tunable nanogaps had a great advantage in detecting trace-level pollutants. Moreover, nanogaps of 1.5 and 5 nm can also generate high SERS signals[29,30]. However, these tiny and uncertain nanogaps bring some difficulties for SERS detection using traditional SERS substrates, because they are fixed nanogaps. Therefore, there is still a need to develop new SERS substrates that allow the nanogaps between nanostructures to be dynamically tuned. In this study, we developed a new strategy for preparing a pH-responsive 3D poly (acrylamide-co-acrylic acid) (P(AAm-co-AA)) hydrogel microsphere with a coating of Au nanospheres (hydrogel microsphere @ Au nanospheres). The nanogaps between the adjacent Au nanospheres adsorbed on the hydrogel microsphere surfaces could be tuned by changing the pH value. The developed composite could be used as a highly sensitive SERS-active substrate. Uniform P(AAm-co-AA) hydrogel microspheres with negative charges were first fabricated using droplet-based microfluidic technique. Au nanospheres with positive charges were then prepared by a modified polyol route and chemical etching. The Au nanospheres self-assembled on the hydrogel microsphere through electrostatic interaction to form the hydrogel microsphere @ Au nanospheres. The hydrogel microsphere could be induced to swell by changing the pH value, thereby generating suitable nanogaps between the Au nanospheres on its surface. The as-prepared hydrogel microsphere @ Au nanospheres were used as 3D SERS substrates and demonstrated good sensitivity to probe 4-ATP molecules. -3-
This study provided a new means for detecting trace-level environmental organic pollutants by dynamic SERS enhancement based on the obtained responsive hydrogel-plasmonic materials.
2. Experimental Section 2.1. Materials Acrylamide (AAm, CP), acrylic acid (AA, CP), dimethyl sulfoxide (CP), paraffin liquid (CP), ethylene glycol (AR), and chloroauric acid hydrate (HAuCl4, AR) were purchased from the Sinopharm Chemical Reagent Corporation Ltd. N-N’-Methylenebisacrylamide (MBAAm,
CP)
was
acquired
from
the
Aladdin
Industrial
Corporation.
2-Hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (Irgacure 2959, RG) was obtained from the Tianjin Heowns Biochemical Technology Co., Ltd. Lauryl PEG-9 polydimethylsiloxyethyldimethicone (KF6038) was provided by the Shin-Etsu Chemical, Japan. Poly(diallyldimethylammonium chloride) solution (PDDA, 400000–500000, 20 wt.% in H2O) was supplied by Sigma–Aldrich (Shanghai) Trading Co., Ltd. These reagents were directly used without further purification. Water (18.2 MΩ·cm) was obtained from an ultrafilter system (Milli-Q, Millipore, Marlborough, MA). The chemical reagents had chemically pure (CP), analytical reagent (AR), and research grade (RG) purity level. 2.2 Preparation of Au nanospheres Monodispersed Au nanospheres were prepared using a modified polyol route followed by chemical etching [31,32]. A prepared gold precursor solution containing 20 µL of PDDA and 0.5 µL of a 1 M HAuCl4 aqueous solution per milliliter of ethylene glycol was placed in a glass vial. After stirring with a magnetic blender for 1–2 min at room temperature under ambient conditions, the glass vial was sealed and heated at 195 °C for 30 min in an oil bath. A HAuCl4 (0.25 µL, 1 M) solution was then added to the Au colloidal solution (1 mL). After 2 -4-
min of chemical etching, spherical Au nanoparticles were produced. The product was collected by centrifugation at 14000 rpm, washed repeatedly with pure water, and concentrated to obtain a 50 mM Au colloidal aqueous solution. 2.3 Preparation of hydrogel microspheres In our experiments, a cross-shaped microfluidic platform was used to produce hydrogel microspheres with tunable sizes [Fig. S1 (a)]. Paraffin liquid containing 2% (V/V) KF6038 as emulsifier was used as the continuous phase. A hydrogel precursor solution containing 200 mg of AAm, 7 mg of MBAAm, 120 µL of AA, and 100 µL of the photoinitiator solution per milliliter of water was used as the dispersed phase. The photoinitiator solution contained dimethyl sulfoxide and 30% (m/V) Irgacure 2959. When the continuous and dispersed phase solutions were simultaneously injected into the cross-shaped glass chip, the dispersed phase liquid was broken off by the strong shear force of the continuously dripping liquid, releasing uniform droplets in a cross-shaped pattern with a microfluidic cross-flow arrangement. After the formation of uniform droplets, the continuous phase served as a carrier to drive these droplets into the long polytetrafluoroethylene (PTFE) pipes. The hydrogel precursor droplets were solidified and polymerized into hydrogel microspheres through irradiation by 311 nm ultraviolet light (Philips, Narrow band TL 20W/01-RS) [Fig. S1 (a)]. After the oil and hydrogel microspheres were collected, the obtained microspheres were thoroughly washed with butyl alcohol to remove the paraffin liquid. 2.4 Preparation of 3D hydrogel microsphere @ Au nanospheres A specific amount (40–150 µL) of the 50 mM Au nanosphere colloidal solution was added to 10 µL of the aqueous solution of hydrogel microspheres (50 g/L). After 12 h, the Au nanospheres were adsorbed onto the surfaces of the hydrogel microspheres and formed 3D hydrogel microsphere @ Au nanospheres through self-assembly by the electrostatic -5-
interaction between the Au nanospheres and the hydrogel microspheres. After centrifugal separation at 4000 rpm and repeated washing with ethyl alcohol, the 3D hydrogel microsphere @ Au nanospheres were cleaned using an ultra-violet ozone cleaning systems (T10X10/OES/E) to remove the surfactant PDDA adsorbed on the Au nanosphere surfaces. The fabricated composite was used as a SERS substrate 2.5 Instrumentation and characterization A microfluidic device was acquired from the Suzhou Wenhao Chip Technology Co., Ltd. and used to prepare hydrogel microspheres (Fig. S1). The width and depth of the micro-channels were 120 and 50 µm, respectively, in a cross-shaped glass chip. A pump was applied to feed the dispersed phase into the middle micro-channels. The two other inlets of the cross-shaped chip were connected to a double-syringe pump, into which the continuous phase was fed. The morphology of the Au nanospheres was characterized by transmission electron microscopy (TEM; JEM-2010 transmission electron microscope). The morphologies of the hydrogel microspheres and 3D hydrogel microsphere @ Au nanospheres were observed using a field-emission scanning electron microscope (SEM; SU8020). Photographs of the hydrogel microspheres were obtained under an optical microscope (Axio Lab.A1) equipped with a digital camera (DLC300L, 3.0 M). Raman spectra were obtained through a confocal microprobe Raman spectrometer (Renishaw inVia Refl ex). The zeta potential of the hydrogel microsphere and Au nanospheres were obtained using a Zetasizer 2000/3000. Simultaneously, the average nanogaps between the Au nanospheres were characterized by in situ SAXS measurements at beamline BL16B of the Shanghai Synchrotron Radiation Facility. The optical absorption spectra were obtained using a microspectrometre system from Ideaoptics Corp. 2.6 SERS measurements SERS measurements were conducted using a laser beam with a wavelength of 785 nm, -6-
under 2 mW of power and 2 s of integral time. During the SERS measurements, the laser beam, with a size of 2 µm, was vertically projected onto the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres. The prepared P(AAm-co-AA) hydrogel microsphere @ Au nanospheres were immersed in 10 mL samples of 4-ATP aqueous solutions with different concentrations for 12 h and placed on clean silicon wafers. The composite microspheres were added to pH buffer solutions of different pH levels for 30 min. After removing pH buffer solutions with a filter paper or capillary, the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres were subjected to SERS measurements (Fig. S2). The water in the P(AAm-co-AA) hydrogel microspheres was volatilized under the heat generated by the light. After 15 s, the composite microsphere went from the wet state to the semiwet state, and finally became dry. The SERS spectrum was obtained in the semiwet state. The SERS effects of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres under different conditions were measured (Figs. S3-S5). Other molecules were detected using the composite microspheres in the semiwet state under a pH 6 condition, whereas the measurement conditions of the 2,4-dimethylbenzenethiol and 4-tert-butylthiophenol were the same as those for the 4-ATP, and those for the rhodamine 6G (R6G) were a wavelength of at 532 nm wavelength, 2 mW of power, and an integral time of 5 s integral time (Figs. S7 and S8).
3. Results and Discussion 3.1 Hydrogel microspheres Fig. 1 shows typical optical microscopy (OM) and scanning electron microscopy (SEM) images of the dried P(AAm-co-AA) hydrogel microspheres fabricated using the droplet-based microfluidic technique. In the OM image of the microspheres (Fig. 1 a), the P(AAm-co-AA) hydrogel particles have a spherical morphology, regular shape, and homogeneous particle size, which were confirmed from the SEM image of the hydrogel microspheres (Fig. 1 b). The size of the dried P(AAm-co-AA) hydrogel microspheres was approximately 16 µm. -7-
The uniform morphology of the hydrogel microspheres is related to the employed preparation method, the droplet-based microfluidic technique (Fig. S1), which consists of two processes: droplet generation and solidification. For the droplet-releasing mechanism, interfacial stress is generated on the surface, where the immiscible liquids interact with the microchannel. The stress is affected by the viscosity, liquid flow or velocity and channel size [33-35]. Uniform droplets of different sizes can be obtained by controlling these parameters. Generally, uniform droplets with tunable sizes can be synthesized by controlling the flow or velocity of the dispersed and continuous phases. In the present experiment, we tuned the flow of the dispersed phase flow without changing the continuous phase flow to obtain uniform droplets of different sizes (Fig. S1 b–e). The formed hydrogel precursor droplets were carried by the continuous phase and solidified under ultraviolet irradiation. The droplets were solidified using on-chip and off-chip (in the outer pipe) methods. On-chip solidification, such as instability emulsion is used more commonly than off-chip solidification. The former requires a special microfluidic device and expensive solidification equipment to rapidly solidify the polymers. In the latter, a continuous and dispersed liquid must form a stable emulsion and then be solidified by an ordinary ultraviolet lamp. In the present work, we used paraffin liquid as the continuous phase and KF6038 as emulsifier, which could form a water-in-oil (W/O) stable emulsion with the hydrogel precursor aqueous solution. The emulsion was solidified in the outer PTFE pipe (O.D.1.6 mm and I.D. 0.6 mm). The photoinitiator Irgacure 2959, AAm, AA, and MBAAm formed a water-soluble polymer with a network crosslinking structure under ultraviolet irradiation at 311 nm (Fig. S1 a). Uniform P(AAm-co-AA) hydrogel microspheres with a size of approximately 16 µm were obtained through solidification in the PTFE pipe for approximately 20 min under a 1000 µL/h continuous phase flow and 100 µL/h dispersed phase flow (Fig. 1). Carboxyl (–COOH) groups were derived from the AA in the P(AAm-co-AA) hydrogel microspheres. In the presence of the hydrogel microspheres in the aqueous solution, the -8-
carboxyl groups could hydrolyze carboxylate radicals (−COO−) with negative electricity and hydrogen ions (H+) with positive electricity. Therefore, the P(AAm-co-AA) hydrogel microspheres were turned into negative particles in the aqueous solution. This finding was confirmed based on the zeta potential of −10.5 mV at 25 °C. 3.2 Au nanospheres Monodispersed Au nanospheres with a size of approximately 58 nm were successfully prepared using a modified polyol route and chemical etching (Fig. 2). The PDDA molecules were steadily coated on the surface of the Au nanospheres. When the PDDA-protected Au nanospheres were present in the aqueous solution, the PDDA surfaces could also be hydrolyzed, conferring positive surface charges on the Au nanospheres [36,37]. The Au nanospheres were positively charged and possessed a zeta potential of 56.7 mV at 25 °C. 3.3 Hydrogel microsphere @ Au nanospheres The Au nanospheres were self-assembled on the surface of the P(AAm-co-AA) hydrogel microspheres through electrostatic attraction because of their opposite zeta potentials, which produced the 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres (Fig. 3). The surfaces of the P(AAm-co-AA) hydrogel microspheres (concentration: 50 g/L, dosage: 10 µL) were gradually covered with an increasing amount of Au nanospheres (concentration: 50 mM, dosage: 40–150 µL), (Fig. 3 a–e). When using 150 µL of the Au colloidal solution, the P(AAm-co-AA) hydrogel microspheres were completely coated by the Au nanospheres, which formed black P(AAm-co-AA) hydrogel microsphere @ Au nanospheres and sunk to the bottom of the solution. With a further increase in the volume of the added Au colloidal solution, the upper solutionturned red. This finding indicated that in addition to the Au nanospheres coating the hydrogel microspheres, dissociated Au nanospheres were also present in the solution, and the quantity of Au nanospheres adsorbed on the hydrogel microspheres reached saturation through electrostatic attraction. Fig. 3 f displays a SEM image of the local -9-
surface of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres with saturation adsorption (corresponding to that in Fig. 3 e). The Au nanospheres were closely packed in a random manner on the hydrogel microspheres. 3.4 pH effect on SERS of hydrogel microsphere @ Au nanospheres Fig. 4 shows the Raman spectra of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the semiwet state at pH 2 to 10 for detecting 10−8 M 4-ATP molecules. Two noticeable peaks are present at 1082 and 1591 cm−1 in the SERS spectra (Fig. 4 a), and are attributed to the C (benzene ring)–S stretching vibration and C–C stretching vibration of the benzene rings of 4-ATP, respectively[38]. These two peaks can be fingerprint peaks for detecting 4-ATP through SERS enhancement, and are displayed as a function of pH, from pH 2 to 10 (Fig. 4 b). The peak intensity gradually increased with increasing pH up to 6 but declined when the pH exceeded 6. Hence, the SERS activity of the hydrogel microsphere @ Au nanospheres could be tuned by pH, and achieved maximum value at pH 6. The changes, which were the SERS effect on the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres under different pH conditions, originated from variations in the gaps between the adjacent Au nanospheres. This phenomenon was related to the semiwet state and the swelling degree of the P(AAm-co-AA) hydrogel microspheres at that pH value (Fig. 5 a and b). Hydrogel is a cross-linked polymer with a network structure, it can swell in water and hold a large amount of water while maintaining its network structure [39]. When the dry P(AAm-co-AA) hydrogel microsphere @ Au nanospheres was placed in the aqueous solution, the P(AAm-co-AA) hydrogel absorbed a large amount of water and swelled. However, the gaps between the adjacent Au nanospheres adsorbed on the surface of the P(AAm-co-AA) hydrogel microspheres were too large to enhance the Raman signals (Fig. 5 a I–II). Nevertheless, the composite microsphere could slowly lose some of water and gradually shrink (Fig. 5 a II–III and III–I) to reach the semiwet state after light exposure. In this case,
- 10 -
the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres could be used as the 3D SERS substrate as result of the formation of nanogaps (less than 10 nm) between the adjacent Au nanospheres. During SERS measurements, the light beam was vertically projected onto the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres, and the water would volatilize with the heat generated by the light, which caused the microsphere to constantly shrink. When the P(AAm-co-AA) hydrogel microsphere was reduced to the semiwet state (about 15 s of light exposure), the gaps between the Au nanospheres were small, and the surface of the P(AAm-co-AA) hydrogel microsphere was fully covered by Au nanospheres. After reaching the semiwet state, the water volatilization in the hydrogel microsphere became very slow, and there was almost no change in the microsphere volume. Compared with the dry state, the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the semiwet state showed a strong SERS effect (Fig. S3). Meanwhile, P(AAm-co-AA) hydrogel containing numerous hydroxyl groups is a stimuli-responsive hydrogel, which responds to changes in environmental pH [39,40]. The pH-stimulated hydrogels can be classified into three types based on the presence of acidic and basic groups: acidic, basic, and amphiphilic hydrogels. P(AAm-co-AA) hydrogel is an acidic hydrogel and can be protonated under acidic conditions, as described in Equation 1. The charge density and content of mobile counterions decreases within the hydrogel, leading to gel shrinkage. Similarly, the acidic hydrogel is deprotonated under basic conditions, as described in Equation 2. Therefore, the number of similarly charged groups increases in the network structure, leading to electrostatic repulsion and hydrogel swelling. Interestingly, with a further increase in pH, the ionic strength increases but the osmotic pressure decreases. Thus, increasing the pH leads to hydrogel shrinkage [40]. Based on the volume change of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres (Fig. S2 b), we obtained its swelling capacity, which gradually increased larger with increasing pH in the acidic solution, slightly decreased stepwise with increasing pH in the - 11 -
basic solution, and reached the maximum result in the neutral solution (Fig. 5 b). This change was demonstrated in the absorption spectra of the single composite microsphere in the wet state under different pH conditions (Fig. 5 c). The composite microsphere had a visible absorbance in the visible region at pH = 2, and then the absorption peak at 530 nm became much sharper when the pH value increased from 2 to 7. However, this absorption peak became wide again when pH values larger than 7. When the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres shrunk, the gaps between the Au nanospheres became smaller. This allowed the Au nanoparticles to cause strong surface plasmon resonance coupling, with a very wide absorption peak, and vice versa. Thus, the pH value could be used to adjust the volume of the P(AAm-co-AA) hydrogel microsphere in the wet state. Similarly, when the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres was in the semiwet state, the P(AAm-co-AA) hydrogel could also be protonated or deprotonated with a small amount of water, which allowed a tiny change in the volume of the composite microsphere under the different pH condition. This tiny change could promote the movement of Au nanospheres, and produce the various nanogaps. Therefore, a suitable gap for the 3D substrate of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres was achieved at pH 6 in the semiwet state, which generated a strong SERS activity. The suitable gap was the average interparticle distance between adjacent Au nanospheres, which could be characterized by small-angle X-ray scattering (SAXS). Other pH conditions were compared, and we measured the values under pH = 5 ,6 and 7 (Fig. 5 d). From these SAXS curves, we know that q pH5 = 0.104 nm-1, qpH6 = 0.098 nm-1 and qpH5 = 0.092 nm-1, with the follow gaps: gpH5 = 10 nm, gpH6 = 6 nm and gpH10 = 4 nm. Therefore, at pH 6, a small nanogap (6 nm) between the adjacent Au nanospheres on the hydrogel microspheres was achieved, which was the main contributor to the strong SERS activity, although some aggregates composed of Au nanospheres might have existed in the swelling or shrinking process. (1) - 12 -
(2)
3.5 Hydrogel microsphere @ Au nanospheres as SERS substrates In order to demonstrate the detection limit of the 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres, 4-ATP molecules of 10-8–10-12 M were probed at pH 6. As shown in Fig. 6 a, the characteristic peaks of 4-ATP were distinctly observed at a low concentration of 10 -12 M. It was demonstrated that the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres could serve as a highly sensitive 3D SERS substrate in the semiwet state. Ten SERS spectra for ten composite microspheres were obtained randomly and are shown in Fig.6 b. To quantitatively evaluate the SERS reproducibility, we calculated the mean, standard deviation (SD), and relative standard deviation (RSD, the ratio of the standard deviation to the mean) of the noticeable peaks at 1082 and 1591 cm-1 (Fig. 6 b). It can be observed that the 10 measurements are highly reproducible and stable. Therefore, analytes could be sensitively and stably detected by the SERS technique using the hydrogel and Au composite materials. The EF is an important parameter used to evaluate the activity in a new type of SERS substrate. We calculated the EF for the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres. The analytical EF is related to the SERS enhancement of analytes of a known concentration and can be calculated as follows [41] (3)
where ISERS and IRS denote the intensities of the SERS and Raman scattering (RS) signal of the same mode for a given analyte, respectively; and CSERS and CRS represent the analyte concentrations for the SERS and the RS signals, respectively. To obtain the Raman spectrum of 4-ATP, it was immersed in a 10-2 M 4-ATP solution under the same conditions as the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres (Fig. S6). As listed in table 1, we estimated the EF at 1082 and 1591 cm-1 under different 4-ATP concentrations according to - 13 -
the Equation 3; the SERS and RS intensities were taken from Fig. 6 and Fig. S6. The EF values were between 4.9 × 108 and 9 × 109, and most could reach up to 109. The 3D SERS substrate had the highest EF compared to the other SERS substrates, i.e., the Ag nanostructured array (EF: 107) [1,42] and Au film (EF: 108) [14]. In addition, we conducted an investigation on detection of another common indicator of SERS, R6G, and the result are shown in Fig. S7 and Table S1. Compared with 4-ATP, the EF for R6G was low and unstable for different concentrations as a result of weak adsorption capacity of R6G on the surface of the Au nanospheres. In general, to enhance the R6G detection ability, a method is employed that involves dropping, some amount of the R6G solution onto the SERS substrate at an extremely low concentration [43–45]. However, this method was not suitable for the 3D hydrogel microsphere @ Au nanospheres. The composite substrate of hydrogel @ Au nanospheres was more suitable for detecting some molecules containing the sulfydryl group (-SH) like 4-ATP (Figs. S8), because the sulfydryl group can be strongly adsorbed on the surface of Au. For other composite systems of hydrogel and Au nanoparticles, the Au nanoparticles were mostly embedded in the hydrogel, but the SERS activity was low [24,25]. In contrast, the as prepared 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres had a high SERS activity with the Au nanospheres exposed to the outside.
4. Conclusion In conclusion, an interesting composite material consist of a P(AAm-co-AA) hydrogel microsphere and Au nanospheres was successfully prepared by self-assembly based on electrostatic interaction. Uniform P(AAm-co-AA) hydrogel microspheres with negative charges were fabricated using a microfluidic method and oppositely charged Au nanospheres were prepared using a chemical route. The P(AAm-co-AA) hydrogel microsphere @ Au nanospheres demonstrated a substantial 3D SERS effect, wherein the nanogaps could be - 14 -
dynamically tuned by changing the pH value during testing. At pH 6, the 3D SERS substrate produced a strong SERS activity, which could detect the complete set of SERS signals at 10 -12 M 4-ATP, and the EF reached up to 109 because of the presence of some nanogaps smaller than 10 nm. The results showed that the detection sensitivity of SERS could be dynamically tuned using the pH value in the semiwet state of the hydrogel microsphere @ Au nanospheres. This dynamic tuning method in the semiwet state is also called state translation nanoparticle enhanced Raman spectroscopy, and compared to the wet and dry testing methods, it can obtain a high SERS activity [27]. In contrast to other dynamic SERS substrates of Ag sols or noble nanoparticle encapsulated hydrogels [22–25, 27,28], the 3D SERS substrate of the hydrogel microsphere @ Au nanospheres could achieve high SERS performance and maintain it for a long time. Therefore, the 3D P(AAm-co-AA) hydrogel microsphere @ Au nanospheres could be used as a 3D SERS substrate for trace analyses in medicine, biology, chemistry and other fields. However, this 3D substrate could also be further improved in future work. For example, the number and distribution of the Au NPs on the hydrogel microsphere could be controlled.
Acknowledgements The authors acknowledge the financial support provided by the Natural Science Foundation of China (grant no. 51371165, 51571189), State Key Program of National Natural Science Foundation of China (grant No. 51531006), Anhui Provincial Natural Science Foundation (grant no. 1508085JGD07), Cross-disciplinary Collaborative Teams Program in CAS, and CAS/SAFEA International Partnership Program for Creative Research Teams.
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Fig. 1. (a) Typical OM and (b) SEM images of P(AAm-co-AA) hydrogel microspheres obtained on microfluidic platform (continuous phase flow: 1000 µL/h, dispersed phase flow: 100 µL/h). Scale bars are 20 µm.
Fig. 2. Typical TEM image of Au nanospheres fabricated by modified polyol route and chemical etching. Scale bar is 50 nm.
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Fig. 3. SEM images of P(AAm-co-AA) hydrogel microspheres (50 g/L, 10 µL) that adsorbed different amounts Au nanoparticles (50 mM): (a) 40, (b) 60, (c) 80, (d) 100, and (e)–(f) 150 µL. Scale bars, (a)–(e): 5 µm and (f): 500 nm.
Fig. 4. (a) SERS spectra of P(AAm-co-AA) hydrogel microsphere @ Au nanospheres after incubation in 10 −8 M 4-ATP solution under various pH conditions. (b) Two fingerprint bands and the error bars [in (a)] plotted at 1082 and 1591 cm−1.
Fig. 5. (a) Schematic of volume variation of P(AAm-co-AA) hydrogel microsphere @ Au nanospheres with added water and lost water. (b) Schematic of the variation in the gaps between the Au nanospheres on the P(AAm-co-AA) hydrogel microspheres with various swelling capacities under different pH conditions. (c) Absorption spectra of the P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the wet state under different pH conditions. (d) One dimensional integrated SAXS curves for P(AAm-co-AA) hydrogel microsphere @ Au nanospheres in the wet state under pH = 5, 6 and 7 conditions; inset shows portions of SAXS curves after subtracting baseline. Center spacing between Au nanospheres can be obtained by d = g+R = 2π⁄q [28], where q is the module of the scattering vector, g is the gap and R is the diameter of the Au spheres.
Fig. 6. (a) SERS spectra of different concentrations of 4-ATP solution at pH 6 and (b) ten SERS spectra of 10 -10M 4-ATP at pH 6 on P(AAm-co-AA) hydrogel microsphere @ Au nanospheres. Table 1. Intensity and EF values at 1082 and 1591 cm−1 under different 4-ATP concentrations.
RS SERS
4-ATP (M) 10-2
1082 cm-1 I (a.u.) EF 200 -
1591 cm-1 I (a.u.) EF 120 -
10-8
98068
4.9×108
63588
5.3×108
10-10
7815
3.9×109
6359
5.3×109
10-11
1614
8.1×109
1084
9×109
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Surface Enhanced Raman Scattering Properties of Dynamically Tunable Nanogaps between Au Nanoparticles Self-assembled on Hydrogel Microspheres Controlled by pH Huilin Li a,b,+, Dandan Men a,+, Yiqiang Sun a,b, Dilong Liu a, Xinyang Li a,b, Liangbin Li b, Cuncheng Li c, Weiping Cai a, Yue Lia,*
An interesting route was developed for preparing P(AAm-co-AA) hydrogel microsphere @ Au nanospheres through self-assembly based on electrostatic interaction. The nanogaps between adjacent Au nanospheres on the surfaces of P(AAm-co-AA) hydrogel microsphere could be tuned dynamically to obtain high SERS effect by controling volume of the hydrogel microsphere under different pH condition in the semiwet state.
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