Multifunctional Substrates of Thin Porous Alumina for Cell Biosensors

Supplementary information

Chiara Toccafondi1,2,*, Sanjay Thorat1,3, Rosanna La Rocca2, Alice Scarpellini4, Marco Salerno1, Silvia Dante1, Gobind Das2

1 Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, Genova I-16163, Italy;

2 Nanostructures, Istituto Italiano di Tecnologia, via Morego 30, Genova I-16163, Italy,

3 Università di Genova, viale Causa 13, Genova I-16145, Italy

4Nanochemistry, Istituto Italiano di Tecnologia, via Morego 30, Genova I-16163, Italy.

*Corresponding author: chiara.toccafondi@iit.it, Tel.: +39-(0)10-71.781.756, Fax: +39-(0)10-72.03.21

Section 1: Anodization

Fig. S1 shows the chrono-amperometric curves obtained during the anodization of two Al thin films (500 and 100 nm).

. Fig. S1 Chrono-amperometric curves recorded during anodization of 500 nm-thick (red circles) and 100 nm-thick (black triangles) Al

films.

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It is worth underlining that the anodization conditions were different for the two processes, as mentioned in the APA fabrication section. However the values of the current densities appear to be similar, even though the two processes are carried out in different time scales.

Section 2: Pore loading

Fig. S2 A) Molecular structure of sodic fluorescein (FITC) dye. B) Excitation and emission spectra of FITC molecule. C) Visible image

acquired just after the deposition of an array of R6G micro-drops made by liquid micro-spotting. E) Fluorescence image acquired

using the FITC filters.

Additional tests of molecular patterning have been performed employing fluorescein (FITC) molecules (Fig. S2 A-B). Diluted (500 pM) aqueous solutions were spotted into 5x5 ordered arrays on a representative thin APA substrate by liquid micro-spotting. The optical image (Fig. S2 C) acquired from the instrument camera just after the deposition and the fluorescence image collected on the same spots after some time (Fig. S2 D) are reported. The fluorescence image shows evidence of the penetration of the FITC solution inside the pores, analogously to R6G.

Section 3: Calculation for SERS enhancement factor

SERS enhancement factor ‘G’ was estimated using following expression:

G=( I SERSI Raman )∗( PRaman∗tRaman∗ARamanPSERS∗t SERS∗ASERS )

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where, I, P, t and A are intensity at 1360 cm-1, laser power, accumulation time and active surface area available for the molecules to be attached, respectively. The subscript indicates regarding the measurement type: SERS for AuAPA substrate and Raman for flat Au surface. The available active area for AuAPAd=50 and AuAPAd=20 is 0.7578 µm2 in 0.785 µm2 total area and 0.5578 µm2 in 0.785 µm2 total area, respectively. The power for SERS was kept 100 times lower than for Raman measurements. The accumulation time ‘t’ is kept 50 sec for Raman whereas for SERS it is 30 sec. The band intensity is around 2800 and 300 for AuAPAd=50 and for AuAPAd=20

substrate, respectively, whereas it is around 70 for Raman measurement. Keeping all these values mentioned above, the SERS enhancement factor is estimated to be 0.94x104 and 7.44x102 for AuAPAd=50 and AuAPAd=20

substrate, respectively.

Section 4: Contact angle

Fig. S3 Water contact angle measured on a Petri dish, an Al foil both as rolled and after electropolishing, a thin film of evaporated Al

and a thin APA film immediately after the release of the drop (black bars) and after 4 minutes (red bars).

Water contact angle measurements were performed on different substrates (Petri dish, as-rolled thick Al foil, electropolished Al foil, as-deposited Al thin film and a typical thin APA substrate). The obtained results are presented in Fig. S3. Thin Al and APA show hydrophobic behaviour (110-120°), at difference from the slightly hydrophilicity of the thick Al foil after electropolishing (80°).

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Section 5: Live/dead assay

The cell viability on APA surfaces has been investigated employing a LIVE/DEAD Viability/Cytotoxicity Kit (see paragraph 2.3).

Fig. S4 Typical images obtained from a live/dead assay performed on the Petri dish control (b) and an APA substrate. In all cases the

number of live cells (green) is considerably higher than that of the dead ones (red).

The results obtained indicate a good cell viability (after 48h of incubation) on all the investigated samples, showing a large majority of live cells (green) and only few dead cells (red). Fig. S4 shows two representative images obtained on the glass control (a) and a APA substrate (b).

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