G. Gervinskas,1 P. Michaux,1 G. Seniutinas,1 J. S. Hartley,1 E. L. H. Mayes,2 R. Verma,3 B.D. Gupta,3 P. R. Stoddart,1 D. Morrish,1 N. F. Fahim,1 M. S. Hossain,1 S. Juodkazis1,4
1 Centre for Micro-Photonics and Industrial Research Institute Swinburne, Faculty ofEngineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, VIC
3122, Australia2 School of Applied Sciences, RMIT University, GPO Box 2476 V, Melbourne, Victoria 3001,
Australia3 Department of Physics, Indian Institute of Technology Delhi, New Delhi 110 016, India
4 Melbourne Centre for Nanofabrication, 151 Wellington Road, Clayton, VIC 3168, Australia
ABSTRACT
The nano-textured surface of black silicon can be used as a surface-enhanced Raman scattering (SERS) substrate.Sputtered gold films showed increasing SERS sensitivity for thicknesses from 10 up to 300 nm, with sensitivitygrowing nonlinearly from around 50 nm until saturation at 500 nm. At 50 nm, a cross over from a discontinuousto a fully percolated film occurs as revealed by morphological and electrical measurements. The roughness ofthe Au coating increases due to formation of nanocrystallites of gold. Structural characterization of the black-Si needles and their surfaces revealed presence of silicon oxide and fluoride. The sharpest nano-needles had atip curvature radius of ∼10 nm. SERS recognition of analyte using molecular imprinted gels with tetracyclinemolecules of two different kinds is demonstrated.
Keywords: black-silicon, SERS, tetracycline, food safety, water safety
1. INTRODUCTION
Surface area of surface-enhanced Raman scattering (SERS) sensor is one of contributing factors to an overalldetection sensitivity but usually it is not measured. Determination of the actual surface area by an electrochemicalcyclic voltammetry has been demonstrated using catodic peak of Au pasivation.1 It was shown that surface areaof Au sputtered over laser ablated SERS sensor on sapphire is up to 10 times larger as compared with surfacearea of Au on glass.1 Surface nano-roughening is contributing to the light scattering and, hence, to SERS.However, extremely rough surfaces produced by electrochemical, plasma, or laser assisted plasma etching such asblack Si (b-Si) have surface areas even larger. They appear black due to low ∼ 1% reflectivity at visible spectralrange. As SERS substrates, such surfaces are very sensitive to the numerical aperture of the focusing lens usedto collect Raman signal.2 At the optimized focusing (numerical aperture 0.7-0.9) for a fixed aspect ratio (< 5)black-Si, efficient SERS substrates can be prepared using Au sputtering. A natural question arises what is theoptimum reflectivity of the black-Si of a high surface area for a good overall sensitivity of analyte detection bySERS? Better understanding of b-Si surface properties, curvature and structure (amorphous vs crystalline) ofthe needles is required. Sputtering or evaporation of metal (Au, Ag) cause formation of none uniform layer,e.g., evaporation with a more directional metal flux tend to form a layer over top of the needles while sputteringcauses more even and conformal deposition.3
Black-Si can be used as a sensing platform with combination with Au-colloidal nanoparticles prepared by laserablation in water without surfactant;4 in water ablated Au colloidals are promising for more flexible chemicalfunctionalization since surface has now residual surfactant contamination. Bactericidal properties of black-Si5
due to mechanical puncture of the bacteria/cell membrane is promising for SERS of the interior of the cells, e.g., malaria parasites in red blood cells. SERS combination with molecular imprint technology6 is another very
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_
a) c)b) d)
Figure 1. Photos of the prepared b-Si SERS chips (∼ 0.5 × 0.5 cm2) with different molecular imprints of tetracycline: a)TC imprint, b) OTC imprint, c) TC imprint after immersion in OTC solution, d) TC imprint after immersion in TCsolution.
Figure 2. Tilted view (45◦) SEM image comparing two sample surfaces, when all fabrication conditions are the sameexcept for gas flows of gases: (a) 30 sccm SF6 and 50 sccm O2 and (b) 50 sccm SF6 and 30 sccm O2. Scalebars are 400 nmfor both images.
promising technology for sensing. Very strong discrimination between two most common tetracycline moleculesusing imprinted gels has been recently demonstrated using surface plasmon resonance (SPR) in optical fibers.7
Where a wavelength shift up to 30 nm was observed at ∼ 0.1 µM concentration of the matching analyte.7 SinceSPR via wavelength discrimination in fiber for white-light illumination has very broad spectral features thereis a limitation on sensitivity which can be further improved using SERS with molecular imprint investigated inthis study.
Here, we report on a structural characterization of a low-aspect-ratio b-Si which has the needle heightcomparable with the axial extent. SERS was carried out at typical focusing with a numerical aperture ofNA = 0.8 ± 0.1 which correspond to the conditions when axial length of the focus is comparable with the b-Sineedle height. We show for the first time that molecular imprint is compatible with SERS on black-Si using themost simple drop cast preparation of senors for the two varieties of tetracycline used in the tests. High rejectionrate of non-matching analyte molecules is promising for food safety applications.
2. EXPERIMENTAL: SAMPLES AND PROCEDURES
The final goal of this study was creation of SERS substrates on b-Si for sensing with the molecular imprint.This is promising due to a very time efficient substrate preparation which requires minutes of dry etching andAu sputtering. The gel preparation by drop-casting and baking is a fast process. The final samples are shownin Fig. 1. Below, the used procedures of sample preparation, characterization and SERS measurements aredescribed in more details.
2.1 Fabrication of black-Si
Plasma dry etching procedures are described for nano-texturing the Si surface via a controlled flow of SF6:O2
mixture, with voltages of the bias and inductively-coupled plasma that influence the anisotropy of surface etchingtogether with a chamber pressure. The formation of nano-pillars of aspect ratio 2.2 ± 0.3 takes 10 - 15 min ofplasma etching, with full area coverage of 3- and 4-inch wafers.
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50 -
45 -
40 -
-
35
30 -
Pillar height:highesthigh
A mediumlowlowest
Ai i
30 35 40 45 50
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Figure 3. A graphical representation of pillar height distribution dependence on SF6 and O2 gas flow rates. Red dotsindicate pillars higher than 1 µm, green triangles represent dots between 1 µm and 800 nm and grey squares show pillarswith height less than 800 nm.
Figure 4. (a) SEM image of a top view of black silicon (b-Si) coated with 200 nm of gold, (b) tilted (45 ◦) SEM image ofb-Si coated with 200 nm of gold, (c) tilted (45 ◦) SEM image of b-Si with 400 nm gold coating. Scalebars are 200 nm.
Black-Si was fabricated from a single side polished p-type 〈100〉 orientation silicon wafer. Fabrication wasperformed using dry reactive ion etching (DRIE) method. A RIE-101iPH (SAMCO Inc.) tool capable of per-forming reactive ion etching (RIE) and inductively coupled plasma (ICP) assisted RIE was used in experiments:150 W ICP and 15 W (for the RIE) bias powers were typically used, flows were set 35 sccm for SF6 and 45 sccmfor O2, chamber pressure was 1 Pa and etching time was 15 min. Typical sample is shown in Fig. 2 (height vspreparation conditions summary is shown in Fig. 3). Magnetron sputter AXXIS (Kurt J. Lesker Ltd) then wasused for gold deposition with well controlled thickness (Fig. 4).
2.2 Structural and surface characterization
X-ray photoelectron spectroscopy (XPS) using a Thermo Scientific K-alpha XPS system equipped was Al Kαsource (1487.6 eV). Spectra were collected with the flood gun active to alleviate sample charging.
2.3 SERS measurements and preparation of molecular imprints
Fabricated b-Si was used as a substrate for SERS. The surface was coated by 200 nm Au layer to form plasmonichot-spots for electric field enhancement. Coated b-Si wafer was diced into 0.5 × 0.5 cm2 chips which wereprepared for SERS by molecular imprint procedures described below.
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Selective layer was formed on top of the coated black silicon. Two selective layers for different molecules weremade by imprinting target molecules in acrylamide/N,N-methyllenebisacrylamide (AM/BIS) matrix. In our casethe target molecules were widely used antibiotics: tetracycline hydrochloride (TC) and oxytetracycline hydrochlo-ride (OTC) Phosphate buffer of 0.1 M and pH 7 was prepared using (Na2HPO4)2H2O and (NaH2PO4)2H2O inMillipore water. The master solution was prepared by mixing AM/BIS (4 g AM + 0.2 g BIS) and 0.8 g TC (0.8 gOTC for OTC molecular imprints) molecules in Millipore water and stirring for 10 min in nitrogen atmosphere.Polymerization medium was made by adding 2.5 ml master solution, 4.5 ml buffer, 30 wt % Acrylic acid (AA),7.5 mg ammonium persulfate (APS) and 20 µl of N-tetramethylenthylenediamide (TEMED) in 10 ml Milliporewater. The diced black silicon chips then was drop coated by the prepared solution and kept in oven at 50 ◦Cfor 3 hours for polymerization. After polymerization has completed the substrates were taken out and dipped inaqueous solution of 10 wt% sodium lauryl sulphate (SDS)and 1 ml acetic acid for 2 hours at room temperaturefor the removal of the target molecules (TC or OTC). It was then washed with de-ionized water. For SERSdetection 10 mM TC and OTC aqueous solutions were prepared separately. The SERS substrates designed forTC or OTC molecules were immersed in both solutions for 1 hour at room temperature for target molecules tointer inside imprint layer and reach the surface of coated gold. Then samples were rinsed with de-ionized waterand dried. Fig. 1 showns images of the tested samples.
Raman microscope (Renishaw) was used for sample characterization. Microscope objective with 0.75 NA and50 times magnification was used to deliver 785 nm laser light to the substrate.
3. RESULTS
Samples of b-Si were characterized by SEM and XPS for their morphology and surface properties. Then, SERSmeasurements were carried out using molecular imprints.
3.1 Morphology of b-Si
The morphology of b-Si surface greatly depends on all the conditions used in fabrication.8 This is demonstrated inthe SEM image Fig. 2, taken at a 45◦ angle, where only the flow rates of process gases SF6 and O2 were changed.An increase in SF6 gas flow from 30 to 50 sccm creates a larger separation between the formed pillars. WhenICP and RIE powers are varied, the etching time and/or a process pressure is changed, surface morphologiesvary even more greatly: forming pyramids, pillars or tube-like structures on the sample surface. It is noteworthythat an increase of the RIE voltage cause a more anisotropic etching while a large bias voltage of ICP causefavors isotropic etching. Usually there is a limitation for application of simultaneous high RIE and ICP voltages.
3.2 Surface characterization
Figure 5 shows an XPS survey spectrum of the black Si surface. The adventitious surface carbon peak is centeredat 285.3 eV indicating that minimal sample charging occurred. Several surface containments are present fromthe black Si fabrication process, in addition to a native surface oxide. The presence of strong oxygen and fluorinepeaks is due to residual contaminates from the dry etching process, in addition surface oxide will continue to formonce the sample is exposed to atmosphere. Sulphur (S) from the dry etching process is also present, however therelatively low intensity of this peak indicates that S as most likely not formed a surface compound like fluorineand oxygen. The Al seen likely originates from the Al2O3 sample holder, during the dry etching process some Alis removed and will redeposit on the sample surface. The doublet in the Si 2p peak indicates the formation of asecond Si species, this second peak can be attributed to SiO2
9 but likely has a component that can be attributedto SiFx.10
Table 1 shows the composition of the black Si surface as determined from the XPS survey spectrum. Thepresence of a significant amount of F further reinforces that the fluorine present has formed SiFx other authorshave attributed the fluorine peak to a combination of SiFx and Si-O-F bonds.11
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i
Al 2pS 2p
Si 2p
/ 1sCN 1s
Ois
0
' I '200
I ' I ' I ' I ' I
400 600 800 1000 1200Binding Energy (eV)
Figure 5. XPS survey scan of black silicon created with 15 min etch time. Photoelectron peaks have been labeled.
Table 1. The composition of black Si surface as determined by XPS measurements.
Element Atomic %Si 24.94O 35F 20.34S 2.2C 7.32Al 10.7N 1.71
3.3 SERS substrate preparation
Black-Si samples coated with Au showed a change from a non-continuous metal island film to distinct nano-gapsformation at the thicknesses of Au from 10 to 200 nm. At thicker coatings of ∼400 nm Au (Fig. 4) a continuouslayer of Au with apparent grains was formed. For the best SERS performance we used 200-nm-thick coatingwhen nano-gaps are present. For the higher aspect ratio b-Si, we have observed formation of conformal coatingof Au sputtered at similar conditions.3
To test the dependance of a formed pillar height on b-Si surface versus gas flow rates used in fabrication, theflow of SF6 and O2 was varied from 30 to 50 sccm in 5 sccm increments. Figure 3 demonstrates a schematicrepresentation of the obtained results; the flow of 50 sccm of O2 and 30 sccm of SF6 produced the highest pillarsof ∼1300 nm. Interplay of ICP and RIE voltages are the main control knobs to change the balance between moreanisotropic (by RIE) vs isotropic (by ICP) etching. Usually, it is not possible to set both bias voltages high dueto hardware and electronic constraints.
3.4 SERS sensing
Molecular imprinted gels for tetracycline recognition on Au-coated b-Si were tested as SERS platforms. Ramanresponse spectra from 200 cm−1 to 2000 cm−1 were measured. Figure 6(a) shows spectra from TC imprint itselfand TC imprint after immersion into a TC solution. The molecules and imprint match each other and thisallows TC molecules enter into the imprint polymer and reach proximity of the Au nano-rough regions with hotspots. The measured TC imprint spectra was subtracted from TC imprint with TC molecules data to improvea TC peak recognition. The subtracted spectrum is plotted in Fig. 6(b). Reference spectra of TC powderwas also measured and compared with the SERS data. Distinct TC peaks between 1200 cm−1 and 1400 cm−1
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a) b)
d)c)
Figure 6. SERS spectra of TC imprint with matching and mismatching molecules: (a)&(b) TC molecules in TC imprint,(c)&(d) OTC molecules in TC imprint.
clearly indicates recognition of the matching molecules. A crosscheck of the substrate was done by immersingTC imprint in the OTC solution. In this case OTC molecules should not be detected as they can not enter intothe polymer and reach vicinity of gold layer. Figure 6(c,d) shows recorded and subtracted spectra, respectively.The spectrum does not show well expressed peaks of the OTC molecules due to mismatch between imprint andmolecules as one would expect.
The same procedure was repeated with OTC imprint ant results were similar: OTC molecules were detectedwith the OTC imprint and very low signal from TC molecules were obtained on the OTC imprint.
4. DISCUSSION
The concentration of the solution for molecular recognition was considerably high 10 mM and this allowedshortened time for permeation of the gel of the imprint. Typical thickness of the drop-casted gel used in theimprint was varied from 20 to 60 µm, obtained by mixing gel components in different ratios. In all cases SERSmeasurements were successful demonstrating a good permeability of the gel matrix.
In future work spin coating of gel will be made over larger areas of b-Si and this should improve performanceof SERS recognition due to thinner gel film. By intentionally shifting the focal spot from the Au surface intogel by ∼ 2 µm caused almost total loss of a SERS signal (doubled Rayleigh length of the beam was ∼ 1.6 µm).Hence, only the pre-surface regions are active for SERS. This favors b-Si with needle height of 1.5 µm as a goodmatch for SERS measurements with NA = 0.8− 0.9 objective lenses.
Black-Si is a promising SERS sensing platform2 and can find applications along SERS sensors made bysurface nano-texturing by ablation.12–14 Laser fabrication with ultra-short pulses has unique capability to create
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textures from tens-of-nanometers to wavelength scale,15–26 however, b-Si has also potential for tuning surfacemorphologies via processing parameters and is a faster parallel method as compared with laser direct write.Future work is required to investigate potential of b-Si as a metal island SERS substrate at thinner Au coatingswhen additional light enhancement effects can be created due to Fresnel enhancement effect.27 The proposedsensor can be integrated into micro-fluidic sensing platforms28,29 and be used with laser/plasmonic tweezers30–32
and to explore gel phase transitions for concentration of analyte.33
5. CONCLUSIONS
Black-Si of the aspect ratio ∼ 2.2± 0.2 closely matching the axial extent of the light beam with NA = 0.8± 0.1objective lens widely implemented for SERS measurements was fabricated and characterized. Au coating of∼ 200 nm was chosen for SERS measurements using tetracycline molecular imprints in gel.
Recognition of tetracyclines by molecular gel imprints is a promising result of this work. It is noteworthy,that gel imprints were prepared and SERS measurements were carried out in collaborating labs in Delhi andMelbourne. We used the same chemicals ordered from different suppliers. Considering courier shipping conditionsand approximately an one week duration from gel preparation to the actual measurements, the demonstratedSERS recognition using molecular imprints on b-Si has a promising future for real life applications. Moreover,gels of different thicknesses of tens of micrometers performed well in SERS measurements demonstrating goodpermeability.
ACKNOWLEDGMENTS
We are grateful for support via Australian Research Council Discovery DP130101205 and DP120102980 grants.The PhD scholarship of GS is funded via the ARC Linkage grant LP120100161 with Raith-Asia. SJ acknowledgesthe Australian Academy of Science senior fellowship support for a visit of Prof. Banshi Gupta laboratory at theIndian Institute of Technology, Delhi. The authors would like to gratefully acknowledge the RMIT microscopyand microanalysis facility for use of their XPS system.
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