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We report on the nonlinear photochemistry fabrication of three-dimensional silver (Ag) microstructures in microfluidic channels for volumetric surface-enhanced Raman scattering (3D SERS). The fabrication of high resolution 3D Ag microstructures is obtained by a two-photon induced reduction process of silver cations, which is restricted at the focal point of a Q-switched Nd:YAG microlaser (sub-nanosecond pulses at 1064 nm). Firstly, 3D Ag micro-pillars made on cover glass showed a 3D SERS detection limit of Oxazine 720 as low as 10−8 M. Secondly, we directly fabricated 3D microstructures within microfluidic channels, and demonstrated their 3D SERS capability. The micro-cube geometry gave a significantly larger 3D SERS signal than the micro-pillar geometry. This result demonstration is paving the way for further optimization routes by varying the geometry, the size, and the density of complex 3D structures which can be obtained by direct laser writing based on two-photon induced chemistry.
© 2016 Optical Society of America
1. Introduction
Since surface-enhanced Raman scattering (SERS) was discovered in 1974 [1], it has been extensively employed for the investigation of molecular composition in a non-destructive manner. It enables an ultra-sensitive and real-time detection of biological or trace chemical analytes, and provides with high accuracy spectral fingerprint signature of analytes [2–4]. In SERS, the hot-spots created at the gaps of colloidal aggregates, between interconnected nanoparticles or from the roughened metal nanostructures provide intense electromagnetic fields to promote a tremendous increase in the Raman intensity of molecules located in these regions, making possible to detect a SERS signal even from single molecules [2]. As a result, SERS has recently become a very appealing nanotechnology tool for multiplexed detection using a single laser excitation.
In order to improve the SERS performance, various strategies have been developed for the fabrication of active metal nanostructures, ranging from silver nano or microflowers [5,6], and dendritic gold nanostructures [7] to silver nanospheres [8]. It is important to note that most of the SERS substrates are 2D planar systems which reduce their versatility. The building of 3D SERS substrates is a challenge to increase the Raman detection in volume, allowing thus more analyte molecules to be adsorbed and implicitly detected [9]. It is particularly needed for the detection of analyte molecules in microfluidics devices which have laminar flow.
Microfluidic devices are very appealing systems for sensitive detection due to the decreased risks of contaminating the biological samples, the extremely small sample volumes required (10−9-10−8 L) and -more exciting- the possibility for developing high-throughput, parallel and multiplexed analyses [10,11]. Despite the existence of several reported papers implementing SERS detection in microfluidic platforms, it is still a challenge to integrate SERS plasmonic substrate inside microchannels in a controlled manner [10–12].
The fabrication of high resolution 3D microstructures by using the help of a laser is possible owing to the process that is restricted to the focal point of the laser beam, where its intensity is strong enough to generate the simultaneous absorption of two photons in a single event [13–16]. By moving the focused laser beam spot, various 3D microstructures can be fabricated. The microfabrication of 3D metallic microstructures by two-photon induced photoreduction has been previously reported [17–19]. Recently, the two-photon fabrication of 3D functional microstructures with polymers, and biomaterials inside microfluidic channels has been reported [20–28].
Here, we present the fabrication of 3D SERS active silver (Ag) microstructures by a Q-switched 1064 nm Nd:YAG microlaser directly within the microfluidic channel. In order to evaluate the performance of the fabricated- SERS sensor, Oxazine 720 was chosen as probe molecule. Raman results show that the detection limits for Oxazine 720 on 3D Ag pillars are as low as 10−8 M. Micro-cubes gave a significant larger 3D SERS signal, which demonstrates the possibility for further optimization by varying the microstructure geometry.
2. Experimental details
Poly(4-styrenesulfonic acid) (C8H8O3S, PSS, 18% Wt), Trifluoroacetic acid silver salt (CF3COOAg, 220.88 g/mol), Ruthenium(II)tris(bipyridine) ([Ru(bipy)3]2 + ) and 3-Mercaptopropyl)methyldimethoxysilane (95%) were purchased from Sigma-Aldrich and used as received. Oxazine 720 (C21H22N3O) was obtained from Exciton. Polydimethylsiloxane (PDMS) was obtained from Dow Corning (Sylgard 182, Midland, MI, USA).
For the microfabrication solution, a PSS (18 wt %) aqueous solution containing Ruthenium (1 mg) was prepared and stirred for 24 hours. Then, 50 mg of CF3COOAg was dissolved in 60 µl of distilled water and added to 500 µl of the previous solution.
For the fabrication of active-SERS microstructures on glass substrate, a Q-switched Nd-YAG laser (Team Photonics Inc.; wavelength 1064 nm, repetition rate 10 kHz, pulse length: 0.5 ns, power: 1 mW and writing speed 50 μm/sec) was tightly focused on the prepared sample by a 100x oil-immersion objective lens with a high numerical aperture (Zeiss, A-Plan, NA: 1.25) of a Zeiss Axiovert 200 inverted microscope. Different 3D Ag microstructures were firstly fabricated on glass substrate using Simpoly control software. After the fabrication process of Ag microstructures, the samples were rinsed in ethanol and deionized water to clean up the excess of PSS matrix, leaving thus the Ag pattern on the top of the microscope cover glass.
The microfluidic devices were fabricated using the optically transparent elastomer-polydimethylsiloxane (PDMS) bonded on a glass microscope slide, through soft lithography and replica molding techniques, as we previously described [22]. Experimentally, the fabrication of PDMS microchannels involves the following steps. The negative photoresist (SU-8, GM 1060, Gersteltec) was patterned onto a silicon wafer to form a mold structure. The PDMS, obtained by mixing a polymer base and a curing agent in 10:1 ratio, was poured into the mold and cured at 65 °C for 1 h. After curing, the microchannel was removed from the mold. The design of microfluidic channel is shown in Fig. 1.
The channel is 100 μm wide and 50 μm deep. The PDMS and glass surfaces were plasma treated at P ~0.2 mbar for 40 s using a plasma cleaner (Harrick Plasma, Ithaca, NY, USA) and immediately brought into contact to create a permanent bond [29]. After the microchannel fabrication, 50 μL microfabrication solution was injected through inlet 1 into the microfluidic channel via tubing connected to a syringe controlled by a syringe pump (KD Scientific 100). Then, 3D Ag microstructures were directly fabricated on the glass substrate by focusing the laser beam into the microfabrication solution within the PDMS microchannel. After the fabrication of the 3D Ag microstructures within the microchannel, the circuit was repeatedly washed with ethanol and deionized water to remove the excess of PSS matrix.
All Raman measurements were performed on a Agiltron Raman (QEB061) system equipped with a 785 nm laser source. This system was connected to the microscope through a 2.5X objective (Rolyn, Germany, NA: 0.10). First, the SERS efficiency of the fabricated Ag microstructures was evaluated by using Ozaxine 720 aqueous solution, as probe molecule. In particular, Ozaxine 720 with concentrations ranging from 10−4 to 10−8 M was deposited onto the laser fabricated microstructures on glass substrate. Then, for SERS detection measurements performed inside microfluidic channels, the Oxazine 720 solution was loaded into the syringe and then injected through inlet 2 into the channel. The spectra were collected by using a 20x microscope objective, which gave a focus diameter of approximately 4 μm. The integration time for each Raman spectrum was 3 s. The maximum laser power was 3 mW.
3. Results and discussion
3.1 Fabrication of 3D silver microstructures
First, we fabricated different morphologies of 3D Ag microstructures on glass substrate in order to demonstrate the versatility of the proposed two-photon microfabrication method. Figure 2 presents the three different examples of 3D Ag microstructures that we fabricated by two-photon photoreduction with the Q-switched microlaser at 1064 nm. We have previously reported the advantages of using a low cost Nd:YAG microlaser over femtosecond laser sources [16]. The first is a 4x4 array of 3D micro-pillars, i.e. micro-cylinder with a flat top. Each micro-pillars has a 2 µm diameter and a 10 µm height. The second consists of a 2x2 array of Ag micro-stars. Each micro-star has a 20 µm width and a 2 µm height. The third is a 3 x 3 array of micro-cubes with a side of 5 µm. As shown in Fig. 2(d), we can observe a good reflectivity of the fabricated 3D micro-cubes, indicating the metallic nature of the structures.
Fig. 2 Examples of 3D silver microstructures fabricated with by two-photon reduction with a Nd:YAG Q-switched microlaser at 1064 nm. Optical transmission images of (a) 4x4 array of Ag micro-pillars, (b) 2x2 Ag micro-star array, (c) 3x3 array of Ag micro-cubes and (d) the metallic reflectivity image of 3x3 array of Ag micro-cubes. Scale bar: 20 μm.
3.2 3D SERS detection with 3D micro-pillars
In order to evaluate the 3D SERS performance of the fabricated Ag microstructures, the 3D micro-pillars (see Fig. 3(a)) were used as a model architecture for the subsequent detection of Oxazine 720. The optical transmission image of the pillar array is presented in Fig. 3(b). The metallic nature of the 3D micro-pillars was confirmed by the reflectivity image presented in Fig. 3(c). Typical SERS spectra of Oxazine 720 at different concentrations ranging from 10−8 M to 10−4 M in aqueous solution are presented in Fig. 3(d).
Fig. 3 (a). CAD model of one 3D Ag micro-pillar; (b) Optical transmission image of a 5x5 3D Ag micro-pillars array, and (c) its corresponding metallic reflectivity image; (d) Concentration-dependent SERS spectra of Oxazine 720 dispersed on 3D Ag micro-pillars array. The Oxazine 720 concentration ranges from 10 −4 to 10−8 M. Excitation: 785 nm laser line. The spectrum marked by (i) represents the Raman signal obtained from outside of the 3D Ag micro-pillars.
The Raman intensity increases with the increasing concentration of Oxazine 720, as expected, every Raman peak intensity being linear correlated with the target molecule concentration. Notably, 10−4 M Oxazine 720 measured at 3 mW displays distinct characteristic Raman peaks at 1643 cm−1, 1408 cm−1, 1351 cm−1, 1188 cm−1, 683 cm−1, 597 cm−1, 563 cm−1, which are in good agreements with previous reports [30,31]. Two main characteristic bands at 597 cm−1 and 1643 cm−1 are clearly observed for 10 −8 M Oxazine 720, indicating the high sensitivity of such microstructure. No Raman bands of Oxazine 720 are visible in the spectrum, when the laser spot is focalized outside of the 3D Ag micro-pillars (Fig. 3(d) – spectrum i).
To reveal the origin of SERS signal enhancement, we performed 3D FDTD simulation of light interaction with a square array of vertical parallel Ag micro-pillars with length, full diameter and wall thickness of 10 μm, 2 μm and 0.3 μm, respectively, using the commercially available FDTD Solutions software from Lumerical Inc [32]. We have considered for Ag the optical constants described by CRC data Tables [33]. The lattice constant was 5 μm, and the micro-pillars were placed in water (n = 1.33). We considered an incident light radiating at 785 nm that propagates parallel to the pillars axis being polarized parallel to one of the square lattice axis. The perfect matched layers (PMLs) approach was chosen on the x direction, at the top and bottom of the structure for providing absorption boundary conditions. The mesh step size was 20 nm. Figures 4(a) and 4(b) illustrates the distribution of relative electromagnetic field intensity ((E/E0)2) in xy-plane along the pillar axis (Fig. 4(a)) and in xz-plane at the top of the pillar (Fig. 4(b)) calculated under 785 nm laser line excitation.
Fig. 4 Relative field enhancement ((E/E0)2 distribution in two different planes. (E0 the intensity of electromagnetic field of incident light is considered unity).
As shown in Fig. 4(a), the electromagnetic field enhancement along the pillar wall shows a series of modulated interference peaks with features characteristic of Fabry-Pérot resonators, as a result of coupling between different plasmonic wave modes propagating at the metal–dielectric interface. The wavelength of these resonances is considerably shorter than the wavelength of the excitation light (785 nm) and can be calculated from the resonance condition where a positive integer multiple (m) of surface plasmon half wavelengths (λSP/2) equals the length L of the pillar (λSP = 2L/m). Similar results were reported by Giloan et al. who studied the light transmission in arrays of Ag nanocylinders [34].
3.3 SERS detection with Ag micro-cubes in microfluidics channels
To test 3D SERS in a microfluidic channel, Ag micro-cubes array were directly fabricated on the glass substrate within the PDMS microchannel (Fig. 5(a), marked area). The side of each micro-cube is 5 µm. A concentration of 10−8 M Oxazine 720 was loaded into the microfluidic channel via a syringe pump. The SERS spectrum (Fig. 5(b), spectrum ii) was acquired under the continuous flow condition when the laser spot was focused on the fabricated 3D Ag micro-cubes. The integration time for spectra acquisition is 3 sec. The SERS signal on the 3D Ag micro-cubes inside microfluidic channel is significantly improved compared to the signal on 3D micro-pillars, under the same experimental conditions (Fig. 5(b), spectrum iii). Specifically, we can clearly distinguish all the characteristic Raman peaks of Oxazine 720 (Fig. 5(b) spectrum (ii)), highlighting an increase in the number of SERS hot-spots provided by larger microstructures. Additional contribution to SERS from the edges of micro-cubes or from the sharp tip (so called lighting effect) of the square pyramid capping the micro-cubes, clearly seen in metallic reflectivity image in Fig. 2(d), cannot be excluded. In contrast, it is worth to mention that no Raman signal was detected near 3D Ag microstructures inside microfluidic channel (Fig. 5(b), spectrum (i)).
Fig. 5 (a). Optical transmission image of the fabricated 3D Ag micro-cubes array in the microfluidic channel and (b) SERS spectra of Ozaxine 720 solution beside (spectrum i) and on the 3D Ag micro-cubes array (spectrum ii), compared to SERS spectrum on 3D Ag micro-pillar inside microfluidic channel. Excitation: 785 nm laser line.
4. Conclusions
In conclusion, we have demonstrated the fabrication, and the SERS efficiency of 3D Ag microstructures in microfluidic channels by two-photon induced reduction using a Q-switched Nd:YAG microlaser (subnanosecond pulses at 1064 nm). A concentration of 10−8 M Oxazine 720 was easily detected. The micro-cube geometry appears more efficient than the micro-pillar geometry. This demonstration indicates the possibility to obtain ultrasensitive molecular detection by 3D SERS in microfluidic channels with further optimization of 3D structure geometry, size, and density.
Acknowledgments
This work was supported by CNCS-UEFISCDI Romania, under the projects number PN-II-PT-PCCA-2013-4-1961, PN-II-CT-ROFR-2014-2-0049 and PHC Brancusi 32656UE. We acknowledge Pr. Olivier Stephan, and Laetitia Gredy (LIPHY, Université Grenoble-Alpes, France) for the development of the two-photon reduction process used in this work.
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