Thin nanoporous alumina-based SERS platform for single cell sensing

Thin nanoporous alumina-based SERS platform for single cell sensing

Accepted Manuscript Title: Thin nanoporous alumina-based SERS platform for single cell sensing Author: C. Toccafondi R. La Rocca A. Scarpellini M. Sal...

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Accepted Manuscript Title: Thin nanoporous alumina-based SERS platform for single cell sensing Author: C. Toccafondi R. La Rocca A. Scarpellini M. Salerno G. Das S. Dante PII: DOI: Reference:

S0169-4332(15)01310-0 http://dx.doi.org/doi:10.1016/j.apsusc.2015.05.169 APSUSC 30495

To appear in:

APSUSC

Received date: Revised date: Accepted date:

19-3-2015 21-5-2015 28-5-2015

Please cite this article as: C. Toccafondi, R. La Rocca, A. Scarpellini, M. Salerno, G. Das, S. Dante, Thin nanoporous alumina-based SERS platform for single cell sensing, Applied Surface Science (2015), http://dx.doi.org/10.1016/j.apsusc.2015.05.169 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Highlights (for review)

Thin nanoporous alumina-based SERS platform for single cell sensing Highlights SERS-active surfaces based on Anodic Porous Alumina (APA) were fabricated;

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The Raman scattering enhancement is due to the presence of nanometric pores;

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Biocompatible thin APA films were used as cell seeding substrates;

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Enhanced Raman spectra of living cells were acquired;

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Thin nanoporous alumina-based SERS platform for single cell sensing Toccafondi C.*a,1, La Rocca R.a, Scarpellini A.a, Salerno M.a, Das G.a,2, Dante S.a Istituto Italiano di Tecnologia, via Morego, 30, Genova, 16163, Italy

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Present address: LPICM, Ecole Polytechnique, Palaiseau Cedex, France

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Present address: PSE, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

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* Chiara Toccafondi, Istituto Italiano di Tecnologia, via Morego, 30, Genova, 16163, Italy;

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phone: +39 01071781756; [email protected]

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1. Introduction

The development of living-cells based sensors probing the reactions to environmental stimuli

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is one of the most interesting topics in biotechnology. In fact, cells are the most complex biosensor, sensitive to changes in pH, temperature, to CO2 contents, and to several positive

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and negative factors appearing on both the adhesion substrate and the surrounding medium (molecules, nanoparticles, ions, bacteria, etc.). The achievement of cheap, large-area, reliable

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and fast response in vitro assays, which would decrease the amount of in vivo experiments, would provide enormous benefits to medical research, especially in the field of drug discovery [1,2]. The continuous progress in this area has led to outstanding results achieved

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through different techniques [3–8]. However, the need for specific labelling methods and/or the invasiveness of these techniques sometimes represent a significant disadvantage [9]. Raman spectroscopy, being a fast, label-free and non-destructive technique, offers a valid alternative to fluorescence microscopy when investigating living cells. Raman signals related to the constituents of the cell (proteins, lipids and nucleic acids) can be detected [10–14] and in principle the biochemical processes occurring in the cell can be monitored. However, this goal is prevented by some important drawbacks. First, each cell component is present in tiny concentrations inside the cell, such that high laser intensities or long irradiation times are

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needed to obtain detectable Raman signals [12,14,15]. Moreover, living cells and complex biomolecules are easily damaged by high power irradiation, leading to scarce feasibility of Raman investigation of biological processes [12]. To overcome this issue, in the past decades many efforts have been spent to find a way to enhance the sensitivity of the technique without

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increasing the irradiation intensity. One of the most promising approach is surface-enhanced Raman scattering (SERS) [12,16,17], where the Raman signal of a molecule can be enhanced

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by several orders of magnitude by placing the molecule in proximity of a plasmon resonant

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metal nanostructure [16–20]. For example, successful applications of SERS as a tool for in vitro studies have been obtained in targeting tumor cells with metal nanoparticles (mNPs)

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[21–24]. However, the requirement of antibody-conjugated mNPs implies several technological difficulties, such as non-specific bonding, interferences from the antibody and

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toxic effects on the cells [25,26]. In this perspective, we believe that the approach involving cells seeded on plasmonic substrates represents a valid alternative option [18,24,26]. In

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nanostructured metal surfaces the analyte interacts with the metallic nanostructure or nanoparticles clusters at the surface, where SERS ‗hot spots‘ are formed. Very recently,

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examples of nanostructured SERS substrates capable to sense living cells have been presented, including nanoantennas [27] and black silicon [28]; nanoporous material, and in particular gold, [29–31] are also arising as novel SERS active substrates, but so far their

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applications for living cell sensing on large area active substrates are missing. Anodic porous alumina (APA) is a layered oxide that exhibits nanostructuring in the form of pores with nanometric size [32–38]. The possibility to control the pore size and density, the biocompatibility and the mechanical and chemical stability, make APA a promising candidate for applications both in orthopedic and dental implants [39–41] as well as in bio-sensing [42– 45]. It is widely recognized that surface morphology and roughness play an important role in the adhesion and proliferation of cells [1,46–48]. In this respect, assessing the influence of the

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surface porosity of APA on cell adhesion would provide valuable information. In addition, metal coated thick APA can be used as an efficient large-area SERS substrate [49–51]. In a few cases APA was already used for SERS applications, but mainly as a mold to fabricate plasmonic nanostructures, for example gold (Au) NPs arrays. Our concept is

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different: we exploit the nanoporosity of the surface to generate the plasmonic effect, and therefore the Au-APA assembly plays an active role in the SERS experiment.

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Additionally, the pores remain available for chemical loading and could be exploited for

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local interaction with cells.

We have therefore developed a SERS active substrate based on thin APA to sense single

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cells, which can be considered the first building block toward a biosensor to assess the status/functionality of living cells. We chose thin APA over standard thick APA (thickness >

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10 μm) in view of the future development of the device, based on manufacturing process on

2. Materials and methods

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known technological substrates of glass or Silicon.

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Thin APA fabrication: Aluminium thin films with thickness ~500 nm were deposited on glass substrates by electron-beam evaporation (PVD75 evaporator, Kurt J. Lesker Ltd., UK), at a base pressure of 10-6 Torr and with a deposition rate of 0.3-2 Å s-1. The films were

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potentiostatically anodized in a home-designed Teflon cell at 110 V in 0.4 M aqueous solution of phosphoric acid (Sigma-Aldrich, Italy) kept at 2°C. The selected voltage was reached through a voltage ramp at constant current to avoid any ―burning‖ damage to the sample. Anodization was carried out in a single step (about 15 min), yielding fully oxidized films, showing pores with diameter d~50 nm and pitch D~250 nm. Post-production etching was carried out using 9 wt% aqueous solution of phosphoric acid and increasing etching time and bath temperature.

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The surface morphology was analyzed by high-resolution scanning electron microscopy (SEM) carried out on a JEOL JSM-7500F (Jeol, Japan) equipped with a cold field emission gun. The instrument was operated at accelerating voltage of 15 or 5 kV on samples coated or not coated with a 10 nm carbon layer, respectively (coating system: Emitech K950X,

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Quorum Technologies, UK). The secondary electrons images were acquired using typical

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magnifications of 20,000X and 50,000X.

Cell cultures: The mouse neuroblastoma N2a cell line was obtained from the American Type

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Culture Collection (ATCC). The N2a cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, California) with high glucose, supplemented with

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10% fetal bovine serum (Invitrogen), 100 U mL-1 penicillin G (Invitrogen) and 100 mg mL-1

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streptomycin sulfate (Invitrogen). In all the experiments 5x104 cells in 2 mL of medium were grown at 37 °C in a humidified 5% CO2 atmosphere. The cells were cultured onto both APA

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and Au-APA substrates after UV sterilization (N=7).

Confocal imaging: N2a cells were plated on each thin APA sample and grown up to 2 days-

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in-vitro (DIV 2). Afterwards the cells were fixed with 2% paraformaldehyde for 40 min at room temperature (RT), permeabilized with 0.5% Triton X-100 for 10 min at RT and blocked with 1% of bovine serum albumin in phosphate buffered saline (PBS). The samples were

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stained for 30 min at RT with antibody against Vinculin (Abcam) and detected with a secondary antibody (chicken anti-mouse Alexa Fluor 488, Invitrogen). For F-Actin staining, the cells were incubated with Alexa Fluor 546 phalloidin for 30 min at 4°C in the dark. Finally the samples were mounted on glass slides using FluoroshieldTM (Sigma) containing DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) for nuclei staining. Confocal images were acquired with an inverted confocal microscope, equipped with confocal laser scanning

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system A1 (Nikon, Japan), using excitation wavelength of 495, 556 or 350 nm for the different fluorophores above. Images were analyzed using the freeware program ImageJ [52]. Cells Fixation for SEM: N2a cells were plated on each substrate and grown for 2 days at 37ºC and 5% CO2. Afterwards, the cells were fixed with glutaraldehyde 2% solution in deionized

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water for 40 min a RT. Then the cells were dehydrated with a series of 10 min incubations in

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rising concentrations of ethanol in water solutions (from 50% to 100%). Finally, the samples were coated with a 10 nm thick gold layer to perform SEM measurements (JSM-7500F, Jeol,

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Japan).

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SERS measurements: A 25 nm-thick layer of Au was evaporated on thin APA substrates by electron beam evaporation (PVD75 evaporator, Kurt J. Lesker Ltd., UK). These substrates

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(Au-APA) were used in preliminary SERS investigations with Rhodamine 6G (R6G, Sigma, Italy). Au-APA substrates were incubated in 1 μM R6G aqueous solutions for 30 minutes at

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RT, and then gently rinsed in water and dried under nitrogen flow. Additional tests with 4-

Information).

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Mercaptobenzoic acid (MbA, Sigma, Italy) were performed (Section S5 of the Supplementary

A micro-Raman spectrometer (inVia Raman microscope, Renishaw) equipped with the software program WiRE 3.2 was used to perform SERS investigations. A 785 nm laser line

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(output power 100 mW, power on the sample ranging from 0.5 to 50 mW), a grating with 1200 grooves mm-1 and a 50X objective (NA 0.75) were used. Spectra were collected in the 200-2000 cm-1 spectral range. In order to verify the uniformity of the samples, 2D Raman mapping were also carried out. A 13 x 11 pixels grid with a 4 µm step was used, resulting in a scanned area of 2288 µm2. SERS on cells: The Raman spectra arising from living N2a cells cultured on Au-APA substrates were collected with the same setup used for R6G. After 2 DIV the cells were

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washed three times with PBS and Raman measurements were acquired in PBS using a 60X immersion objective. Control measurements were performed on flat Au films evaporated on mica (Phasis, Switzerland) and Raman transparent CaF2 substrates.

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3. Results and discussion 3.1. Thin APA fabrication

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The as-fabricated thin APA films on glass show uniformly distributed vertical pores, with

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diameter of ~60 nm (Figure 1b). The mean values of the morphological parameters (Figure 1 a) such as the size of pores (d), the pore pitch (D) and the thickness of the wall between two

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adjacent pores (w) were estimated from SEM images using grain analysis software tools.

Figure 1. SEM images (50,000X) of thin APA substrates (thickness ~500 nm); a) Close-up of a single pore unit cell and sketch defining the pore size d, the pore pitch D and the wall thickness w; b-e) SEM images showing the increase of d (at constant D~250 nm) with different conditions of pore widening process, from asprepared APA60 (b), to APA75 (c), APA90 (d), and APA120 (e). The table at the bottom reports the values of

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pore size d, pore pitch D, wall thickness w, pore coverage and pore density of the APA substrates as derived from the analysis of SEM images.

The limited thickness of the films prevented us from performing a two-step anodization

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process which yields hexagonally ordered arrays of pores in thick APA [34,37] grown from Al foils. However, thin APA surfaces with irregular pore arrangement were also demonstrated

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to provide SERS effect [50]. The reduced thickness is an advantage in the perspective of

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developing a device, because it allows integration with standard lithographic methods. Moreover, growing the porous alumina from an almost flat Al film assures a higher planarity

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of the surface.

The capability of tuning the ratio wall thickness/pore size (w/d) is of strategic importance

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for SERS applications [49,50]. Post-production etching allowed to obtain APA surfaces with increasing pore size and constant pitch. Figure 1 (b-f) shows SEM images of thin APA films

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with increasing d and D~250 nm. The effect of acid etching on the APA surface morphology is evident when comparing the image of the as-anodized sample in panel b with those in

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panels c-e, corresponding to increasing etching time and/or temperature. The average pore size d was estimated to be 63±12 nm after fabrication (Figure 1b) and increased with progressive post-fabrication etching up to 72±14 nm (c), 90±15 nm (d) and 120±18 nm (e).

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For clarity these sample sets will be addresses as APA60, APA75, APA90 and APA120 throughout the paper. The values of all the morphological parameters of thin APA shown in Figure 1 as derived from grain analysis are summarized in the table at the bottom of the same figure. Similar values were obtained for several other samples prepared under the same conditions. Looking at the results, one can observe that the increase of d results in a thinning of the pore walls, i.e. a decrease of w, due to the fact that the density of pores remains

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constant at 109 cm-2. In addition, the etching determines a more rounded shape of the pores and in some cases the merging of two adjacent pores. The SEM images reported in Figure 1 point out the good control achieved in both mean pore size and its dispersion, and prove the long-range uniformity and good reproducibility of

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the surfaces.

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3.2. Thin APA as a cell seeding substrate

The feasibility of a living cell sensor is based on the possibility to culture the cells directly on

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the active surface. For this reason, biocompatibility tests aiming to assess cell adhesion and

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proliferation on thin APA and Au-APA were performed using the N2a cell line. Standard thick APA has been extensively studied as a cell substrate [39–41]and its

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biocompatibility is recognized; however, due to the differences in the anodization process, thin APA may show strong variation in pore size and density and, since cell

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adhesion is strongly affected by surface roughness and morphology [46,53–55] the assessment of its biocompatibility was necessary. The biocompatibility was not

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systematically tested for Au-APA, since the morphological influence of Au deposition on thin APA is negligible (see Fig.S3) and Au biocompatibility is well-known; therefore, no major effect on cell viability is expected.

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Confocal fluorescence microscopy was used to investigate the adhesion and proliferation of N2a cells on thin APA surfaces, to also assess the possible influence of pore size on cell growth. In order to investigate the effect of pore size, we fabricated samples with increasing d and fixed pore pitch D~250 nm (as in Figure 1). N2a cells were seeded on porous APA substrates without treatment with polylysine or other adhesion factors, and let to proliferate up to 2 DIV. After cell fixation and selective staining with fluorescent dyes, confocal fluorescence images were acquired on different zones of the samples. Figure 2 a-b shows two typical images acquired on stained N2a cells adhering

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on as-fabricated APA60 (a) and on analogous films with widened pores, APA120 (b). The cell nuclei were stained with DAPI (blue), the actin filaments with phalloidin (red), and vinculin protein with specific antibodies (green). In Figure 2 a-b one can see the vinculin expression and F-actin filaments distribution inside N2a cells, which both play an important

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role in cell morphology, cell motility and muscle function [56]. In all the acquired images the distribution of vinculin, which is associated with focal adhesions, indicates good cell adhesion

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and proliferation, in analogy to the glass control (images not shown). This result is also

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confirmed by the F-actin distribution, evidencing flat adherent cells with extended filopodia.

Figure 2. a-b) Representative confocal fluorescence images of N2a cells at DIV 2 on as-fabricated APA60 (a) and the APA120 set with largest pores (b) (10 μm scale bar). N2a cells were stained for F-actin (red), vinculin protein (green) and nuclei (blue) using phalloidin, anti-vinculin/Alexa 488 and DAPI respectively, as evidenced by the inset in panel a. c-f) SEM images acquired on N2a cells fixed at DIV 2 after seeding on as-fabricated thin

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APA (APA60) (c,e) and APA120 (d,f). The cells are well-spread on both substrates and show a flat morphology with well-visible filopodia.

The number of cells nuclei adhering on the bare APA was counted and the cell density was estimated after averaging the counts of all images. The estimated normalized cell densities are

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plotted in Figure S1 of the Supporting Information. The sets with the smallest and largest pores (APA60 and APA120) showed significantly higher cell densities (p<0.01) and were

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therefore selected as the substrates for the Raman experiments on living cells. However,

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it is worth noting that the relevant result here is that N2a cells grew on all the thin APA substrates, even in the absence of adhesion layer. These findings, which prove the good

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biocompatibility of the substrates, are in agreement with previous investigations on thick APA [40,41,57,58] and our recent work addressing the growth of fibroblasts on 100 nm-thick

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APA films [50]. N2a cells, being a neuroblastoma cell line, was chosen in view of the possible application of our Au-APA platform for neuronal cells, which is of interest in our

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group [59].

The good biocompatibility of thin APA has been also confirmed by viability tests, whose

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results (Supplementary Information: section S2) indicate a large majority of living cells on all the investigated samples, similarly to the positive glass control. The morphology of the N2a cells cultured on thin APA films of different pore size d was

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further investigated by SEM. Figure 2 c-f shows representative SEM images at different magnifications acquired on as-fabricated APA60 (c, e) and on APA120 (d, f). All the images show cells well spread on the substrate with typical morphology. Lamellipodia and filopodia extending on the substrate are clearly visible and well developed. Since the adhesion of cells to APA was achieved without using any adhesion factor, these results confirm thin APA to be an excellent substrate for living cell applications.

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3.3. Enhanced Raman spectroscopy SERS testing of Au-APA Verifying the capability of thin APA to enhance the Raman scattering of molecules placed in the proximity of its surface is an essential prerequisite to the measurement on living cells. To

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this aim, thin APA substrates were coated with a 25 nm thick Au layer, providing a

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nanostructured metal surface (Au-APA) where localized surface plasmons can be generated. This Au thickness was carefully chosen not only to hinder the substrate in the scattered

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beam signal but also to induce only negligible alteration to the surface morphology

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(Supplementary Information: section S3).

Figure 3 – SERS measurements of R6G molecules incubated on thin Au-APA substrates; a) representative SERS average spectra with standard deviation showing the characteristic peaks of R6G (purple) and reference spectra on bare Au-APA (black); a zoom of the 1360 cm-1 peak region is shown in the inset; b) optical image showing the region (grid) where the Raman mapping was performed; c) 2D SERS mapping centered at the R6G band at 1360 cm-1, showing extended signal over all the considered area.

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Experiments and theoretical calculations performed previously on ordered thick Au-APA revealed that hot-spots occurred on the edge of Au-APA walls, causing remarkable enhancement of the Raman scattering of a molecule placed in their vicinity [49]. Such an effect has also been observed in similar experiments involving Au-coated thin APA (~100

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nm) over Si substrates showing smaller pores (d=20-50 nm) and thus higher pore density (~10 times higher than the one considered here [50]). In this work we use APA films with different

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to verify the capability of such surfaces to provide SERS effect.

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morphological parameters (see section 3.13.1. Thin APA fabrication) and thus it is necessary

SERS test measurements were performed using a 785 nm laser line on selected test

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molecules, namely MbA and R6G, adsorbed on different thin Au-APA substrates. The mean SERS spectrum of R6G adsorbed on Au-APA (purple curve) is shown in Figure 3 a

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together with a reference spectrum of bare Au-APA (black curve), which demonstrates that there are no features associated to Au-APA itself. The Raman band located at ~ 612

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cm-1 can be attributed to the C-C-C ring bending, while the peaks centered at 1360 and 1509 cm-1 originate from the stretching of the aromatic ring [60]. A close-up of the 1360 cm-1 peak

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spectral range is reported in the inset and helps to appreciate the features of the spectrum and its standard deviation.

A key feature of an efficient SERS substrate is the presence of the signal enhancement

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provided over the whole area. The SERS activity of the substrates was therefore verified by 2D mapping measurements, as shown in Figure 3 b-c. A 2D map was acquired in the region highlighted by the grid in the optical image in Figure 3 b. The grid is 13x11 pixels wide, with 4 µm steps, resulting in a total area of 2300 µm2. The SERS mapping measurements were acquired using the R6G reference band centered at 1360 cm-1. The intensity map in Figure 3 c shows only limited intensity variations (the point-to-point

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standard deviation is 23%), which confirms that thin Au-APA is an efficient large-area SERS substrate with hot-spots distributed everywhere on its surface. The SERS enhancement factor G was calculated according to the formula reported in Section S6 of the Supplementary Information. The G calculated for Au-APA substrates

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with respect to the flat Au surface can reach 2×104. This is a remarkable result considering that this enhancement was achieved employing a facile and inexpensive electrochemical

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technique, providing large areas of nanostructured surface with respect to literature reports

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involving expensive facilities such as electron beam lithography systems [20,61]. It should be stressed that our goal is not to obtain the best possible (i.e. highest enhancement) SERS

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substrate, as compared to traditional and also inexpensive SERS substrates such as casted colloidal gold nanoparticles. We rather aim to demonstrate good SERS functionality in a

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trade-off with the additional advantages of our Au-APA platform, which are i) the good adhesion properties of cells in the absence of external adhesion factors, such as the coating of

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adhesion or ECM proteins or peptides; and ii) the possible use of the peculiar nature of the Au-APA roughness, namely the porosity, for biofunctionalization by pore loading with

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appropriate nanoparticles or biomolecules, i.e. the potential drug delivery effect of Au-APA morphology [62,63].

The SERS-active thin APA substrates were also tested with MbA molecules

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(Supplementary Figure S5).

Enhanced Raman from living cells on Au-APA Thin Au-APA substrates were demonstrated to be efficient large-area SERS surfaces for small molecules. However the main purpose of this work is to demonstrate that Raman scattering enhancement can be observed also from cells seeded on Au-APA. Figure 4 shows microRaman spectra acquired on a single N2a cell at 2 DIV on a thin Au-APA. In panels from a) to c) the effect of focusing the laser spot at increasingly higher distances from the metal surface

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is presented: curve a) was obtained by focusing the laser on the substrate and thus represents the enhanced Raman spectrum of the cell. Various Raman peaks associated to the cell constituents can be observed throughout the 700-1800 cm-1 spectral range [11,14,19,64],

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which have been highlighted by vertical dashed lines in Figure 4.

Figure 4 – Micro-Raman spectra acquired on a single N2a cell adhering on a thin Au-APA at increasingly

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higher distances from the metal surface (the curves are shifted for clarity). Curve a) was obtained by focusing the laser on the substrate and therefore represents the enhanced Raman spectrum of the cell. The typical Raman

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peaks associated to the cell‘s constituents are highlighted by vertical dashed lines. By progressively moving the focus out above the cell, the intensity of the peaks decreases (b-c).

The Raman band at 1001 cm-1 is associated to the symmetric ring breathing mode of phenylalanine (Phe) [10,11,19,65,66] and its presence is considered a typical signature of

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cells. In particular, there appear sharp bands at 1150 cm-1, attributed to C-C/C-N stretching modes in proteins [10], and the band at 1447 cm-1, attributed to CH2 and CH3 deformations [10,11,14,19] and likely due to membrane phospholipids. A weak signal at 841 cm-1 can be disentangled and is attributed to O-P-O backbone vibrations [10,66], occurring in nucleic acids and in phospholipids [10,66]. Most of the detected peaks are likely attributed to the cell membrane, as this is the cell element closest to Au. In fact, the plasmonic effect due to the nanotexture of the metal surface and causing the enhancement of the Raman signal is only

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active in the close proximity of the surface. For this reason, the signal intensity should not be affected by cells possible sitting one onto another, in case of cultures that have overcome cell confluence. However, this was not occurring for the considered culture time (DIV 2). Similarly, each Raman spectrum is a punctual signal, coming from a laser spot in a lateral

Therefore, no effect of surface concentration of cells is expected.

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position on the Au-APA substrate which is of similar size or smaller that that of a single cell.

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By progressively moving the focus outwards, the intensity of the peaks soon decreases

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until no signal is recorded (Figure 4 b-c). However, we can reasonably assume that there are other effects which contribute to the final enhanced spectrum, such as multi-scattering of

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enhanced Raman signal within the cell.

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Figure 5 – Comparison between Raman spectra acquired on N2a cells adhering on a) nanostructured Au-APA and b) flat Au on mica. The Raman signal enhancement achieved in Au-APA thanks to the presence of nanometric pores in the metal structure is high, considering that higher laser power and irradiation times (10 mW power, 100 s) were required in the case of flat Au in order to achieve peak intensities comparable to Au-APA (1 mW power, 10 s).

In order to confirm that the enhancement of the Raman signal from the N2a cells is due to the thin Au-APA, we performed micro-Raman experiments on cells seeded onto flat Au and on calcium fluoride (CaF2). The comparison between Raman measurements on N2a cells

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adhering on both flat and nanostructured Au is shown in Figure 5. In panel a) a typical spectrum of a cell acquired on thin Au-APA irradiated with the 785 nm laser line (1 mW power, 10 s) is presented, showing the bands at 1001 and 1447 cm-1 attributed to Phe and CHx modes, respectively [10,11,19]. It is worth noting that some variations among the N2a

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SERS spectra occur, for instance some bands that appear in the reference spectrum on flat Au surface are not visible in the spectrum recorded on Au-APA and vice versa. This

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can be explained by considering the fact that the measurements are performed on living

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cells, therefore the orientation of the molecules with respect to the gold surface could be different from spot to spot, and also that the focal position of the laser within the cell is

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generally not the same for different measurements [67]. However, the same experimental conditions (785 nm, 1 mW, 10 s) used with cells adhering on flat Au did

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not provide any signal. In order to achieve Raman peak intensities close to those obtained with thin Au-APA (Figure 5 b), we had to increase both laser intensity and

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irradiation time (up to 10 mW power, 100 s), thus increasing the risk of cell damage. Analogous results were obtained using the transparent CaF2 substrate (see Supplementary

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Figure S7). This confirms that the cell Raman signal is enhanced on Au-APA thanks to its

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characteristic nanostructure.

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Figure 6 – Micro-Raman spectra were acquired on N2a cells seeded on a half-coated Au-APA sample (see image in the inset). The two spectra reported were acquired on the same cell grown at the boundary between bare APA and Au-APA: the black curve refers to the portion of the cell adhering to bare APA (black spot in the inset), while the blue one refers to the portion of cell attached to the metal surface (blue spot). The contour of the cell in the small inset has been highlighted by a dashed yellow line.

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A further confirmation of this evidence is provided by the results shown in Figure 6. Here a

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thin APA sample half-coated with a 25 nm-thick Au film was used as a seeding substrate for N2a cells. Then, micro-Raman measurements were performed on the cells on both sides of the

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sample (inset of Figure 5). Spectra acquired on the Au-APA side show the typical fingerprints of cells, while on the bare APA side no signal was recorded using the same irradiation

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conditions. We have acquired spectra even on a single cell grown across the boundary

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between APA and Au-APA, which are reported in Figure 6. Sharp Raman bands related to cell components are found (blue curve) when the laser spot was focused on the portion of cell

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attached to the metal surface (blue spot). On the contrary, when the laser spot was focused over the portion of cell adhering to bare APA, only background noise was recorded (black

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spot and black curve). Therefore, Raman signals related to cell constituents were detected thanks to the signal enhancement provided from the nano-texture of the metal surface, without any invasive cell treatment, such as injection and endocytosis of mNPs. The increased Raman

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sensitivity together with the possibility of easily tuning the morphological features of the APA surface paves the way towards the development of a large-area cell-based sensor. 4. Conclusions

We have demonstrated the fabrication of thin Au-APA films for single cell sensing, which could be used as a platform for the development of living cell biosensors. The fabrication of thin APA through anodization of ~500 nm-thick Al films was optimized in order to obtain oxide films with pore size ranging between 50 and 120 nm. Biocompatibility tests using N2a

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cells were performed to verify cell adhesion and proliferation on thin APA. Preliminary SERS investigations with R6G proved Au-APA to intensify the molecule Raman signals, making this substrate suitable for the development of SERS sensors for the detection of cells presence, status (healthy/unhealthy) and response to possible environmental factors (both from the

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medium and from the substrate, e.g. the biomolecule loaded APA pores). In our devices the Au-APA template acts both as a cell seeding substrate and as a SERS-active surface, and will

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eventually work as a drug delivery carrier after appropriate pore loading.

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Raman measurements on living N2a cells in liquid buffer cultured on Au-APA substrates were performed, and sharp signals assigned to different components of the cells were

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collected. By comparing Raman spectra gathered from cells adhering on different substrates, we proved that the presence of nanometric pores in the metal surface not only allows for

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optimal cell growth and proliferation but also determines an enhancement of the Raman spectra associated to the cells. Remarkably, the improved sensitivity of the Raman technique

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was reached without commonly used invasive cell treatment such as injection of mNPs. These results prove Au-APA to be an inexpensive, large-area surface viable for SERS

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sensors screening the cell status / functionality. The future steps towards the realization of such a device should also include the monitoring through SERS the effect of localized release

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into the cells of drugs previously loaded in the pores. Acknowledgements

The authors acknowledge the support of the IIT Interdepartmental project P29406. The authors also thank Marco Leoncini from IIT Nanostructures Dpt. for metal evaporations. References [1]

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