Plasmon based biosensor for distinguishing different peptides mutation states
Gobind Das,1* Manohar Chirumamilla,1 Andrea Toma,1 Anisha Gopalakrishnan,1 Remo Proietti Zaccaria,1 Alessandro Alabastri1 and Enzo Di Fabrizio2,3
1Nanostructures, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy
2King Abdullah University Science and Technology (KAUST), PSE and BESE Divisions, Thuwal, 23955 -6900, Kingdom of Saudi Arabia
3BIONEM Lab,University of Magna Graecia, Campus Salvatore Venuta, Viale Europa 88100, Germaneto-Catanzaro, Italy
Supplementary Information
Sections:
- Process flow of plasmonic nanocuboid structure for SERS application
- Theoretical simulations
- Raman area mapping measurements
- Raman measurement of R6G molecule deposited over Au-bar
- SERS enhancement factor calculation
- SERS on myoglobin protein
- W1837R peptide of BRCT domain of BRCA1 protein
- Process flow of plasmonic nanocuboid structures for SERS application
The plasmonic nanocuboid structures were fabricated by means of EBL and lift-off techniques. After lift-off, a layer 80 nm thick of gold and sharp edge of cuboid nanostructures, with IPG of around 20 nm, was achieved. The molecules of interest were deposited using chemisorption technique by which a monolayer was achieved for SERS inspection. A complete process flow of the nanocuboid fabrication is shown in Fig. S1.
Figure S1: Process flow of fabrication of nanocuboid structures. The characterization of molecules by SERS was done after a depositionby chemisorption.
A magnified view of the nanocuboid matrices (2x2, 3x3 and 4x4), adopting the fabrication protocol herein explained, is reported in Fig. S2.
Figure S2: Representative SEM images of the nanocuboid structures; the square-like shape with slightly rounded corners is shown.
2. Theoretical simulations
Since the mesh density plays a crucial role to understand the relative performance of the device and to demonstrate even any minute difference, efforts were made to construct the mesh density as similar as possible for all the substrates. To this, adaptive mesh refinement was chosen to construct the mesh over the nanostructures (Fig. S3). The mesh density was constructed with high density at the edge while it reduces towards the center where the electric field variation is minimum. This mesh density provides us with the spatial resolution down to 0.5 nm.
Figure S3: Mesh construction of the nanocuboid SERS substrate.
In order to show the effect of sharp edge with respect to the real structures, the comparative simulation results are shown in Fig. S4, keeping all simulation parameters equal in both cases. It is very clear that in the case of sharp edge the surface plasmon confinement is much higher without any presence of electric field localization on the other two diagonal points. The electric field reaches up to 26.7 V/m for sharp edges cuboid whereas it was found 10.7 V/m for cuboid with truncated edges.
Figure S4: Comparative simulations for nanocubois with resembled/sharp edge corner.
3. Raman area mapping measurements
Mapping measurement for SERS device with IPG of 230 nm was performed with step size of 150 nm. The optical image of SERS device accompanied with the grid area is shown in Fig. S5a. SERS measurements for R6G molecule were performed on grid area. The mapping analysis for reference characteristic band@1360 cm-1 (baseline corrected) is performed and is shown in Fig. S5b blended with the grid area. The mapping analysis is also shown in Fig. S5c, for the sake of clarity.
Figure S5: a) Microscopic optical image of SERS device. In this figure, the grid area, where SERS mapping measurements were performed, and reference markers, are shown. b) Analyzed SERS imaging for R6G band at 1360 cm-1 blend over optical image and c) the SERS mapping image for the reference band, centered at 1360 cm-1.
4. Raman measurement of R6G molecule deposited over Au-bar
Raman measurements were performed for R6G molecules, deposited over Au-bar on the same device. In Fig. S6, it is shown the Raman spectrum of molecules, chemisorbed on gold-bar (inset of Fig. S6). It is evident from the figure that the R6G bands are observed with very low signal-to-noise ratio. It confirms that the molecular vibrations are not visible though the laser power and acquisition time are higher than the parameters used for SERS measurements (on cuboids).
Figure S6: SERS measurement of R6G (laser power: 1.75 mW, acquisition time: 50 sec, objective: 150x), deposited over Au bar. Au-bar is shown in inset.
5. SERS enhancement factor calculation
SERS enhancement factor can be calculated by using the following formula:
where, I, P, t and A are the intensity, laser power, acquisition time and active area to bind the molecules, respectively. The subscripts, SERS and Raman, are associated to the SERS and normal Raman measurements, respectively. For example, considering a single cuboid, ISERS, tSERS and PSERS are 210 counts, 50 sec and 0.17 mW, whereas IRaman, tRaman and PRamanare 30 counts, 30 sec and 1.70 mW, respectively. If the laser spot size is 1 m diameter, then the available active area for molecules to be chemisorbed on the flat Au surface will be 0.785 m2. However, for nanocuboid surface, since the major contribution in the SERS intensity is from the two corners of the nanocuboid, the SERS active area will be 0.628x10-3m2, assuming a radius of curvature of 10 nm. Solving the equation, the SERS enhancement factor is 1.40x105with respect to the flat gold surface.
6. SERS measurements on Myoglobin protein
Myoglobin (Mb) molecule is a metalloprotein of around 4.9 nm diameter and with aroughly spherical molecular structure, containing one planar Fe-protoporphyrin prosthetic heme group merged in polypeptide chains (around 75% of α-helix). The analyte molecule was deposited over the SERS device using chemisorption technique in which the sample was immersed in Mb solution for 20 min., then it was gently rinsed with water to wash out the excess molecules not attached to the metal surface. SERS measurements for myoglobin were carried out in the range of 1000-1800 cm-1 (region related to the ring binding vibrations) at different location of the SERS device. The measurements were carried out in this range because the first and second order of Si substrate. Part of second order of c-Si substrate can also be observed at around 1000 cm-1. Various characteristic bands related to Mb protein (which contain Fe-protoporphyrin prosthetic heme groupin polypeptide chain), centered at 1125, 1366 and 1560 cm-1 are observed which are attributed to the C-N stretching, an oxidation marker band of heme group and C-C vibrational band, respectively. Various broad bands in the range 1230-1300 cm-1 and 1630-1670 cm-1 are related to the amide III and amide I vibration modes, respectively, which are also providing the information about the secondary structures of protein. The band centered at 1450 cm-1 is associated to the C-Hx bending vibrations of all the aliphatic organic groups.
7. W1837R peptide of BRCT domain of BRCA1 protein
There are seven sequential aminoacids of BRCT which can be found in wild type BRCT domain. These seven locations were localized where the mutations can be observed, mostly causing the breast cancer. W1837R is one of them. W1837R peptide used in this study contains 16 amino acids. The synthetic wild type peptide with the sequence “EAPVVTREWVLDSVAL” of which tryptophan ‘W’ was substituted by arganine ‘R’. The wild type and mutated sequence of these peptides are shown in Fig S7. In the figure,it is clearly observed the substitution of tryptophan by arginine aminoacid, keeping all othersunchanged.
Figure S7: W1837R peptides (wild type and mutated) of BRCT domain of BRCA1 protein