Surface-enhanced Raman scattering (SERS) spectroscopy has been used for sensitive trace detection [1–3] for chemical analysis, archaeological identification, and biological research. SERS substrates are usually rigid, such as glass or silicon. Electron-beam lithography [4], reactive-ion beam etching [5], and focused-ion beam etching [6] have been used to fabricate metallic nanostructures on substrates to obtain high-performance SERS. However, wipeable sampling with flexible SERS substrates is more useful. [7] Recently, Tay [8] et al. prepared flexible SERS substrates via inkjet printing of gold nanoparticles on cellulose-based filter paper to detect fentanyl aerosols for drug screening. By controlling the printing cycle, Ma[9] et al. used screen printing to load “ink” containing graphene oxide and silver nanoparticles on cellulose paper to detect pesticide residues on fruit. Flexible and disposable SERS substrates still usually require various laboratory facilities that increase costs. Polavarapu et al. [10] used a pen-on-paper approach to write nanoparticle sols on plain photocopy paper with a fountain pen to prepare low-cost SERS test strips. They reported that gold nanoparticles, gold nanorods, or silver nanoparticles prepared via aqueous chemical reductions could be used as inks, and those containing Ag nanoparticles exhibited high SERS activity over a broad range of excitation wavelengths. The handwriting method significantly reduced the cost of flexible SERS test strips. In 2017, Han [11] et al. used pen-writing to load aqueous silver nanoparticles on modified cellulose paper for flexible SERS arrays that were used to detect melamine in liquid milk at a 0.27 mg/mL detection limit. However, all the strips noted above reported low laser damage thresholds and were readily ablated during applications. Moreover, the nanoparticle inks were made via chemically reducing Au (III) or Ag(I) in aqueous solutions. These nanoparticles were protected by adsorbed ionic agents that were unstable, which resulted in aggregation, clogged pens, and non-uniform writing. Polytetrafluoroethylene (PTFE) membranes have high laser damage thresholds [12] and stable chemical properties, and thus could be ideal flexible substrates. However, the super-hydrophobicity of PTFE makes it difficult to load aqueous dispersions of metal nanoparticles.
Here, we used oil-dispersed gold nanoparticles synthesized via a modified Brust-Schiffrin method [13] to handwrite, via a ballpoint pen, SERS strips on a PTFE membrane. We also synthesized gold-silver nanoparticle alloys with various Au and Ag ratios to optimize the SERS sensitivity. In this way, the localized surface plasmon resonance (LSPR) and the oxidation resistance of the metal nanoparticles were significantly improved. Overall, the flexible SERS test strips on PTFE substrates exhibited high performance, good stability, and large laser damage thresholds.
Preparation and Characterization of metallic nanoparticles
The metallic nanoparticles were prepared via a modified Brust-Schiffrin method [14]. A total of 1.5 g (2.75 mmol) of tetra-octylammonium bromide was added to a two-neck flask with 80 mL of toluene and magnetically stirred. Then 0.066 g (0.38 mmol) of AgNO3 dissolved in 2 mL of ultrapure water was added and stirred. The toluene solution became milky white with a pale-yellow precipitation. Then 0.088 g (1.52 mmol) of NaCl was added, and the pale-yellow flocculent precipitate disappeared within 5 min. A total of 0.16 g (0.38 mmol) of HAuCl4·4H2O was dissolved in 2 mL of ultrapure water and added to the solution, which then turned orange-red. After stirring for 5 min, 0.36 g (1.88 mmol) of hexanethiol was added, which caused the solution to become colorless. After stirring for 15 min, 0.28 g (7 mmol) NaBH4 dissolved in 20 ml of water was poured into the flask, and the solution became black. The reaction was finally stopped after stirring at room temperature for 4 h, and the aqueous phase was discarded. The black organic phase was distilled in a rotary evaporator at a temperature below 50°C to evaporate the solvent. The remaining black product was suspended in 30 mL of methanol, briefly sonicated to ensure complete dissolution of byproducts, collected via centrifugation, and then washed five times with 20 mL of methanol. Upon drying at room temperature in vacuo, alloy nanoparticles with a Au:Ag mole ratio of 1:1 were obtained. Various Au and Ag mole ratios, such as Au, Au1Ag1, Au1Ag2, Au1Ag4, Au1Ag8, and Ag, could be obtained by changing the AgNO3 and HAuCl4·4H2O stoichiometry. The metal nanoparticles synthesized by the Brust-Schiffrin method could be purified and dissolved in most organic solvents for their surfaces were modified by an alkylthiol monolayer through coordinate bond. Therefore, the metal nanoparticle ink concentrations could be precisely adjusted and the colloids did not aggregate.
The gold or gold-silver alloy nanoparticles were dispersed in chloroform to prepare colloidal concentrations of 15 mg/ml, 35 mg/mL, and 55 mg/ml that were used to fill a ballpoint pen. A 0.5×0.5-cm SERS array was then written on the surface of the PTFE membrane. During the writing, the colloidal nanoparticles diffused along the membrane fibers via capillary effects and spread on the fiber surfaces as the solvent evaporated. Subsequently, the membrane was placed in a 200-°C muffle furnace for 20 min to remove the 1-hexanethiol ligands from the nanoparticle surfaces. At the same time, the nanoparticles fused into 10–20-nm diameter particles.
TEM images of Au-Ag alloy nanoparticles are shown in Fig. 1(a-d). In Fig. 1(a, b), the particles are spherical with 1–5-nm diameters. Au and Ag both have face-centered cubic fcc crystal structures and have similar lattice constants; hence, they can form perfect alloys in any proportion. From the high-resolution TEM images in Fig. 1(c, d), we calculated approximately 0.243-nm interplanar spacings of adjacent lattice fringes, which corresponded to Au (111) and Ag (111) planes. The absorption maxima for nanoparticles with various proportions of gold and silver are plotted in Fig. 1(e). The wavelength of the LSPR extinction peak was linearly blue-shifted with increased Ag content. Specifically, the Au1Ag1 absorption peak was at 502 nm, the Au1Ag2 peak was at 482 nm, the Au1Ag4 peak was blue-shifted to 470 nm, the Au1Ag8 peak was centered at 458 nm, and the Ag peak was blue-shifted to 449 nm.
Preparation of Handwritten Flexible SERS Strips
The process of writing on the surface of the PTFE membrane with a ballpoint pen filled with colloidal ink is shown in Fig. 2(a). As noted above, the colloidal metal nanoparticles wet and spread on the PTFE surface. By maintaining the approximately 1-mm distance between pen strokes, the metal nanoparticles could be smoothly coated on the fiber surface. Hence, the method was simple and convenient.
Distributions of nanoparticles (35 mg/mL) written on the PTFE membrane were characterized by SEM, as shown in Fig. 3(b). Compared with the blank PTFE membrane that had cracks caused by film stretching during fabrication [Fig. 3(a)], the surface of the written fiber was smoother in [Fig. 3(b)], due to the metal nanoparticles had filled these cracks. After annealing, the nanoparticles fused into larger particles with 5-20-nm diameters. A large number of gaps less than 10 nm formed between adjacent nanoparticles, as shown in the inset of Fig. 3(b). These gaps were SERS "hot spots," where adsorbed analytes exhibited Raman spectra with significantly increased intensities.
SERS Performances of Handwritten Flexible SERS Strips
SERS characterization was performed with a WITec Alpha300A confocal Raman spectrometer. Vibrational Raman spectra of the analyte were acquired in "mapping" mode to characterize the properties of the flexible SERS substrate. The Raman spectrometer was equipped with a synapse charge-coupled device (CCD) detector and a power tunable 50-mW incident 532-nm laser. A 50× objective (0.8 numerical aperture) was used, and the laser focal spot was approximately 811 nm in diameter. The scanning area was 25×25 µm2, and 2500 spectra were collected from the mapping image. The integration time for each point was 0.5 s. An averaged spectrum over the entire mapping area was calculated for the final test result.
We firstly investigated the effects of Au1Ag1 ink concentrations (15 mg/mL, 35 mg/mL, and 55 mg/mL) on SERS performance. In Fig. 4(a), the concentration has no significant effect on SERS performance; because of the good ink wettability, the PTFE fibers were like a reservoir. When the colloid concentration was low, the nanoparticles were mainly present on the surface after the solvent was volatilized. When the colloid concentration increased, there were particles both on the surface and inside the fiber. Hence, the SERS performance for the substrate prepared with 15 mg/mL ink was similar to that prepared with 55 mg/mL ink. Further increases in concentration (> 65 mg/mL) greatly decreased the performance because of excessive fusion of the metallic nanoparticles into solid blocks during annealing that prevented formation of hot-spots. Therefore, the optimal 35 mg/mL concentration was used to characterize the various Au-Ag alloys.
SERS spectra of 10− 6-mol/L R6G analyte on SERS strips with various Au-Ag alloys are shown in Fig. 4. The SERS intensity increased with Ag content in the alloys. The intensity from the Au1Ag8 alloy strips was approximately ten times that from the Au strips. This was attributed to electromagnetic (EM) enhancement and charge-transfer enhancement mechanisms. With the increased Ag content on the SERS substrate, the LSPR wavelength blue-shifted, which was better for 532-nm excitation. This generated a strong localized EM field between adjacent nanoparticles. Because the Raman intensity is proportional to the fourth power of the EM field intensity, the R6G Raman scattering was greatly enhanced. In the other mechanism, hot electrons from LSPR excitation decays enhanced charge-transfer between the plasmonic substrate and the adsorbed R6G, which changed the local electron density and increased R6G Raman scattering.
Using the Au1Ag8 alloy, we characterized the SERS performance for various R6G concentrations (10− 6–10− 12 M), as shown in Fig. 5. The SERS intensity decreased with decreasing of R6G concentration; characteristic peaks were still visible at 10− 11 M. However, when the concentration was 10− 12 M, the SERS spectrum was not uniformly detected at all mapping locations. Therefore, for the Au1Ag8 alloy and a 5-µL loading volume, 10− 11 M R6G was the detection limit. With Eq. (1), we calculated the number of R6G molecules loaded on a single mapping point at any concentration.
$$\frac{{S}_{l}}{{S}_{A}}=\frac{{N}_{E}}{{N}_{A}},$$
1
where Sl was the area of the laser spot, SA was the distribution area of the solution to be detected, NE was the number of molecules detected in a single laser spot, and NA was the total number of molecules detected. For the 10− 11-M R6G concentration, there were 6 molecules in a single laser spot; and for 10− 12 M R6G, it was 0.6 molecules. This explained why the 10− 12-M solution could not be uniformly detected. The flexible SERS strips had a high R6G sensitivity. From Eq. (2), we can also calculate the enhancement factor (EF) of the substrate.
$$EF=\left(\frac{{I}_{SERS}}{{N}_{SERS}}\right)/\left(\frac{{I}_{REF}}{{N}_{REF}}\right),$$
2
where ISERS was the intensity of a specific analyte Raman band, and NSERS was the number of molecules contributing to ISERS. Similarly, IREF was the intensity of the same Raman band from a reference solution, and NREF was the number of molecules contributing to IREF. The EF was 5.4×108 for the 10− 11−M R6G 613-cm− 1 peak from Au1Ag8 alloy strips, which was one of the best reported enhancement factors for flexible SERS substrates.
To demonstrate the good uniformity of the Au1Ag8 alloy SERS strips, spectra for more than 2500 points were collected in a 25×25-µm2 mapping area, with 0.5-s integration times at each point. The intensity of the 613-cm− 1 Raman peak is plotted in Fig. 6(a), and the average SERS spectrum over the entire mapping area is shown as red in Fig. 6(b). The maximum and minimum spectral intensities were plotted as blue and black curves, respectively, in Fig. 6(b). The high-intensity (bright) areas and the low-intensity (dark) areas were randomly distributed in the 2D mapping image, and the minimum-intensity spectrum still exhibited characteristic R6G peaks. Figure 6(c) shows cross-sections of the mapping image in Fig. 6(a), which reflect intensity fluctuations of 613-cm− 1 and 1362-cm− 1 peaks. From Fig. 6(c), we calculated 18% and 31% relative standard deviations for the 613-cm− 1 and 1362-cm− 1 peak intensities, respectively. The Raman signals were enhanced at all spots, indicating excellent uniformity in SERS activity.
To test the laser damage threshold of the SERS strips, the 532-nm laser power was increased to 50 mW, and was focused on the strips with a 100× microscope objective (NA = 0.9), at which the laser power was attenuated to 5.14 mW. Both optical microscope and SEM images indicated no changes in the irradiated areas. Thus, the SERS strips had high laser damage thresholds.
Applications to On-Site Sampling and Detecting
Malachite green (MG) was once widely used as a fungicide in aquaculture. However, because of its carcinogenic, teratogenic, and mutagenic hazards,[15] most countries have banned it since the 1990s. Nevertheless, because of its low cost and bactericidal effects, MG can still be found in aquaculture.
Using Au1Ag8 SERS strips and beakers, we simulated MG sampling of a fish tank surface. We added various concentrations of aqueous MG solutions to the beakers, and let them stand for 4 h. We then poured out the solutions and wiped the beakers with SERS test strips that were wetted with ethanol, to mimic sampling of MG residues on the surface of fish tanks in a market. The test results for various MG concentrations are shown in Fig. 7. The laser wavelength was 532 nm, the power was 0.6 mW, and the integration time was 1s. Residual MG on the inner surface of the beaker was still clearly detected when the 10− 7-M MG solution was contained in the beaker. This demonstrated that the SERS substrates could be used as test strips for on-site sampling and detection.