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A versatile nanoarray electrode produced from block copolymer thin films for specific detection of proteins
Accepted Manuscript A versatile nanoarray electrode produced from block copolymer thin films for specific detection of proteins Samira J. Fayad, Edson Minatti, Valdir Soldi, Sébastien Fort, Pierre Labbé, Redouane Borsali PII:
S0032-3861(17)30674-2
DOI:
10.1016/j.polymer.2017.07.015
Reference:
JPOL 19827
To appear in:
Polymer
Received Date: 30 May 2017 Revised Date:
27 June 2017
Accepted Date: 4 July 2017
Please cite this article as: Fayad SJ, Minatti E, Soldi V, Fort Sé, Labbé P, Borsali R, A versatile nanoarray electrode produced from block copolymer thin films for specific detection of proteins, Polymer (2017), doi: 10.1016/j.polymer.2017.07.015. 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.
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A Versatile NanoArray Electrode produced from Block Copolymer Thin Films for specific detection
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of proteins
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Samira J. Fayad,a,b Edson Minatti,b Valdir Soldi,b Sébastien Fort,a Pierre Labbé,c and Redouane Borsali*,a
Grenoble Alpes University, CNRS, CERMAV UPR 5301, 38000 Grenoble, France
b
Department of Chemistry, Universidade Federal de Santa Catarina, 88040-900 Florianópolis,
Grenoble Alpes University, CNRS, DCM UMR 5250, 38000 Grenoble, France
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Brazil
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a
Keywords: Self-Assembly, Thin Film, Single Molecule, Block Copolymer, Nanoelectrode Arrays, Cyclodextrin, electrochemistry
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ABSTRACT
This work describes how nanostructured thin films obtained from the self-assembly of block
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copolymers (BCPs) systems can be used as a nanoelectrode array (NEA) that can be programmed to specifically detect targeted molecules. Namely, poly(styrene-b-methacrylate) (PS-b-PMMA) thin films, after removal of PMMA phase, produced regular spaced 560 pores
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µm-2 with 16 nm of diameter. The nanopores were then chemically modified by the introduction of β-cyclodextrin (β-CD) molecules. By using the supramolecular interaction of β-CD and
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ferrocene (Fc), the pores could be programed by the introduction of molecules linked to Fc able to interact with target species. In the model system shown here, the linker had a biotin unit, aiming the detection of streptavidin. By changing the linker, other molecules can also be detected. This concept opens a window to many possibilities, including the development of
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1. INTRODUCTION
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devices for fast and versatile molecule detection.
The use of BCPs to obtain well-ordered nanostructures in the solid state is widely described in
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the current literature [1-6]. Distinct morphologies with different size and shapes can be easily tailored by changing the molecular characteristics of the BCP, such as molar mass, block volume fractions and block incompatibility [6-13]. The nano domains created by the self-assembly of BCPs can be transferred to solid substrates that can act as templates or scaffolds to the fabrication of highly ordered nanomaterials [14-18]. One of the fields taking advantages of such possibilities is electrochemistry, where BCPs have
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been used to obtain nanostructures for several applications, such as sensor materials and electrodes [19,20]. The fabrication and behavior of macrosized nanoelectrode ensembles (NEEs, where the nanoelectrodes are randomly spaced) and NEAs (where there is an order in the
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spacing between the individual nanoelectrodes) are very attractive [21].
It is well known that both the sensitivity and the selectivity of electrodes can be increased by
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using nanoelectrodes arrays, when compared to regular electrodes with planar surfaces [22-29]. They improve the signal-to-background current ratios and allow extremely low detection limits
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[30,31]. In a NEA, each single electrode is at the bottom of a nanopore; the size of the pore can be finely tuned, allowing single molecule detection. The mobility and detection of the target molecule are limited by the size and chemical characteristics of the nanopores [32]. In order to produce NEEs and NEAs, several strategies have been described in the literature and
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can be summarized as two general methods: the physical or chemical scratching of a previously regular surface in order to obtain the nano sized holes (by means of X-ray, electron nanolithography, plasma, and others) [33-35] or by using an already nano-organized surface, such as
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the use of nano-templates made from the self-assembly of block copolymers in thin films deposited onto the metallic surface of the electrode and followed by the selective removal of one
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of the phases (blocks) from the polymeric matrix [36-38]. The latter case is much more interesting, since the size and spacing of the pores can be easily adjusted by changing the molecular weight and block volume fraction of the polymers [39,40]. By playing with the variables in the equilibrium phase diagram of the block copolymer [10,41,42] one can choose the morphology of the self-assembly and obtain the desired pattern and, consequently, produce the required NEAs.
For instance, if the thin film is obtained using the conditions that favor
cylindrical domains of one block perpendicularly oriented to the electrode surface, the removal
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of this phase will produce NEAs [19]. Another advantage of the use of BCPs templates is the larger density of pores compared to traditional lithography. In addition, by using BCPs the pores
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can be regularly spaced and have long range order. Usually, when removing one of the phases of a thin film made from a self-assembled BCP, the remaining phase is left with some residual and reactive groups at the boundary of the now extinct
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phase [43]. This behavior can be exploited to add new chemical functionalities to the pore walls — a way to input detection selectivity to the obtained NEA [44-46]. For instance, the inclusion
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of biomolecules, such as proteins or antibodies, can lead to specific interactions with target molecules; the specific interactions in enzyme-substrate or antibody-antigen coupling are then transferred to the NEAs, making the detection very specific and selective. The PS-b-PMMA is one of the BCPs most often used to produce nano-templates [47-50]. The
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segregation phase diagram is well known and the influence of variables such as temperature, molar mass and volume fractions are thoroughly described in the literature [38-40,51]. One of the most important characteristics of this copolymer is its behavior when irradiated with UV
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light: the PS block is cross-linked and the PMMA block is degraded [44, 52]. Thus, in a selfassembled PS-b-PMMA thin film, the PMMA phase can be removed by UV radiation. If the film
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is made by perpendicularly arranging the cylindrical PMMA domains in a PS matrix, nano-pores will then be obtained in which the size and spacing of the pores can be controlled by the molar mass and block composition of the BCP. In addition, the PS boundary is left with residuals and reactive carboxyl acid groups as previously observed by Li et al. [43]. The introduction of supramolecular chemistry in NEAs is particularly attractive, because it allows controllable molecular recognition and structural modifications at specific areas of the
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nanopores. In this work, we used PS-b-PMMA to obtain thin films with PMMA hexagonally packed cylinders on a PS matrix deposited on gold surfaces. After the removal of the PMMA phase, the nanopores walls were chemically modified by the introduction of β-CD molecules.
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The redox behavior of Fc is well-known for its reversibility in host–guest supramolecular systems with β-CD [53,54]. By taking advantage of these interactions, the pores could be easily changed by introduction of other molecules covalently bound to Fc that are able to interact with
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target molecules. In this way, we demonstrate a versatile way to obtain NEAs that have specific interactions with analytes, allowing single molecule detection. To prove the method, we chose a
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protein-ligand model system, namely an orthogonal bifunctional tetravalent Fc-biotin linker, in which a cyclodecapeptide scaffold is coupled to one biotin molecule at one end and four Fc functionalities at the other end [54]. The supramolecular attachment of Fc to β-CD leaves the biotin anchored at the surface of the nanopores walls. The biotin has a very strong and specific
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interaction with streptavidin (SA), therefore allowing the NEA to detect the presence of this
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protein in the aqueous medium.
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2. EXPERIMENTAL SECTION
The proposed method can be summarized as: (1) casting of nano-organized diblock copolymer thin film onto a conductive gold subtract; (2) removal of one of the copolymer phases by means of UV radiation to obtain regularly spaced nanopores; (3) chemical modification of the nanopores to include selective binding molecules; (4) detection of target molecule due to the reduction of pore entrance.
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2.1. Chemicals and Materials.
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All solutions were prepared with water having a resistivity of 18 MΩ cm or higher (Millipore system, France). PS-b-PMMA (Mn 53 000 g mol-1 for PS and 20 500 g mol-1 for PMMA and Mw/Mn 1.08) was purchased from Polymer Source and used as received.
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Toluene (Carlo Erba Reagents), ethanol (Carlo Erba Reagents), acetic acid (AcOH, Carlo Erba Reagents), Dimethylformamide (Carlo Erba Reagents), sodium azide (Carlo Erba Reagents), hydroxide (Sigma-Aldrich), methanesulfonyl chloride (Sigma-Aldrich), imidazole
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sodium
(Sigma-Aldrich), triphenylphosphine (Sigma-Aldrich), ammonium hydroxide (Sigma-Aldrich), monosodium phosphate (Sigma-Aldrich), disodium phosphate (Sigma-Aldrich), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC; Sigma-Aldrich), ethanolamine (ETA, N-hydroxysulfosuccinimide
(NHSS;
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Sigma-Aldrich),
Sigma-Aldrich),
N-(2-
hydroxyethyl)piperazine-N′-(2- ethanesulfonic acid) (HEPES; Euromedex), streptavidin (SigmaAldrich)
were
used
as
chloride
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hexaammineruthenium(III)
received.
Ferrocenemethanol (Ru(NH3)6Cl3,
(FcCH2OH;
Sigma-Aldrich),
Sigma-Aldrich),
potassium
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hexacyanoferrate(II) trihydrate (K4Fe(CN)6, Sigma-Aldrich) were of reagent grade quality or better and used without further purification. Heptakis (6-amino-6-deoxy)-beta-cyclodextrin was prepared by mesylation of the primary hydroxyle groups of β-CD followed a substitution with sodium azide and reduction according to the procedure reported in the literature [55,56].
2.2. Preparation of PS-b-PMMA Films.
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The gold substrate was cleaned by briefly rinsing with water and drying under a stream of nitrogen; this was followed by UV-ozone treatment for 5 minutes. After this period, the surface was treated with anhydrous ethanol for 10 minutes under agitation followed by drying with
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nitrogen. A thin film of PS-b-PMMA was prepared on the gold substrate via spin-coating (3000 rpm) from its toluene solution (2.0% w/w) and was then annealed at 170 °C in vacuum (ca. 0.3 Torr) for 24 h to form cylindrical PMMA domains in the film. The PMMA domains were
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degraded via UV irradiation using a UV lamp Fisher Scientific (Bioblock Scientific VL 6C/6W UV lamp, 254 nm; 12 W; ca. 2.5 mW cm-2) under an Argon atmosphere, which involves
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simultaneous cross-linking of the PS matrix and degradation of the cylindrical PMMA domains [52]. Subsequently, the degraded PMMA domains were removed by rinsing with AcOH for 2 min [40]. The thickness of a PS-b-PMMA film was measured using an imaging null-ellipsometer EP3 (Nanofilm, Germany). The ellipsometric thicknesses of annealed PS-b-PMMA films prior to
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the PMMA degradation were 87.2±1.2 nm; after UV/AcOH treatment there were a slight increase in the thickness of the films (up to 94.4±1.3 nm).
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2.3. Surface Modification of PS-b-PMMA-Derived Nanopores. The chemical modification of PS-b-PMMA-derived nanoporous was done according to the
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procedure of Li et al. [40]. PS-b-PMMA-derived nanoporous films on gold substrate were immersed in 1 mL phosphate buffer (0.1 mol L-1, pH 6) containing 400 mmol L-1 of EDC and 100 mmol L-1 of NHSS for 12 h with gentle shaking. Subsequently, the films were soaked in a 1 mL phosphate buffer solution containing 8 mmol L-1 of heptakis (6-amino-6-deoxy)-betacyclodextrin for 24 h with gentle shaking. Then the films were rinsed with phosphate buffer and immersed in an aqueous solution of ETA (1 mol L-1, pH 8.5) for 12 h with gentle shaking. The
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samples were rinsed with the phosphate buffer and then rinsed with milliQ water prior to the measurements.
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2.4. Attachment of linker and SA. The the functionalized NEAs were immersed in 150 µL of a 5 µM solution of tetravalent Fcbiotin linker (synthesized by Dubacheva et al. [54]) for 45 min. Then the films were rinsed with
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HEPES buffer (10 mmol L-1, 0.15 mol L-1 NaCl, pH 7.0).
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After this step, the films were soaked in 500 µL HEPES buffer containing 1 mg L-1 of streptavidin for 45 min. Subsequently, the films were rinsed and kept in a HEPES buffer for 5 min prior the measurements. 2.5. AFM Measurements.
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AFM images were obtained by tapping-mode imaging in air, using a Picoplus instrument (Molecular Imaging). A Picoscan controller and a Magnetic AC mode (MAC mode) control box were used to control the scanner. A small range scanner of 10x10 µm and a top-MAC nose were
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used for imaging. AFM tips with a magnetic coating (MAC Lever Type VI, Agilent Technologies) of a nominal spring constant 48 N m-1 were used. The measurement frequency
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was set to 15% below the resonant frequency. Scanning was carried out with the feedback adjusted to 20% amplitude reduction at a lateral scan frequency. All the images were analyzed using the Gwyddion 2.22 software. 2.6. SEM Measurements.
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SEM images were obtained by a secondary electron imaging mode with a Zeiss ultra 55 fieldemission gun scanning electron microscopy (FEG-SEM) (CMTC-INP, Grenoble, France) at an
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accelerating voltage of 3 kV, using an in-lens detector. 2.7. Electrochemistry Measurements.
Electrochemical experiments were performed with a conventional three-electrode potentiostatic
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system using a CHI 440 potentiostat (CH-Instruments, Inc., USA). Electrode potentials were referred to Ag/AgCl (3 mol L-1 KCl). The counter electrode was platinum and the working
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electrode was the functionalized gold sensor. Electrochemical detachment of the linker and its complex with streptavidin formed stepwise (linker/SA) was done in situ in the buffer by applying an oxidizing electric field (Eox=+0.55V, which was used to oxidize the Fc groups attached to the
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3. RESULTS AND DISCUSSION
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3.1. NEAs construction and characterization
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The PS-b-PMMA films deposited on gold substrates were characterized using AFM and SEM techniques. Figure 1 shows (a) tapping-mode AFM images of the PS-b-PMMA film before and (b) tapping-mode AFM image and (c) SEM image of the PS-b-PMMA film after PMMA etching. In Figure 1a, circular bumps were observed, suggesting that the PMMA domains were oriented perpendicular to the film surface [40]. After UV irradiation and subsequent acidic treatment with AcOH (Figure 1b), the circular bumps changed to circular depressions (pores) due to the removal of the PMMA domains. After the removal of the PMMA domains the presence of
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regularly spaced nanopores could also be observed by SEM imaging; circular domains having radius of 8 ± 1 nm were observed and, assuming a perfect orientation of cylindrical domains in
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the whole surface, the effective pore density is around 560 pores µm-2.
Figure 1. Tapping-mode AFM images of the surfaces of a thin PS-b-PMMA film on a gold substrate (a) after the annealing at 170 °C in vacuum for 24 h and (b) after the removal of the
same film as in (b).
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PMMA domains via UV irradiation and subsequent AcOH treatment and (c) SEM image of the
Both imaging techniques show that the PMMA phase was removed and pores were created.
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However, it is not possible to infer if the PMMA domains were completely removed from the film, allowing the exposure of the bottom gold surface to form the NEA structure [40]. To
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address this issue, a series of cyclic voltammograms (CVs) were carried on the produced electrodes.
The suitability of the mesoporous insulating matrix to electrochemical applications relies on the availability of the inner volume of the pore to the species from the electrolyte solution and the accessibility of the electrode surface (the bottom of the NEA), step required for the initiation of the electron transport process through the mesoporous film [57]. Thus, the gold electrode coated
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with PS-b-PMMA thin film was exposed to solutions of redox probes and the electrochemical responses were compared to that of an uncoated electrode. The results are shown in Figure 2, where the CVs of (a) 0.5 mmol L-1 FcCH2OH and (b) 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1
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KNO3(aq) on naked gold substrates are compared to those coated with PS-b-PMMA films before
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and after UV irradiation.
Figure 2. Cyclic voltammograms (scan rate: 10 mV s-1) of (a) 0.5 mmol L-1 FcCH2OH and (b) 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) on a gold substrate (—) naked and coated with PS-b-PMMA film (···) before and (- - -) after UV irradiation. The absence of a peak-shaped CV in the electrodes with PS-b-PMMA film before UV radiation is due to the fact that the insulating non-conductive polymer layer covering the gold surface
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avoids the electrode reactions. The peak current observed in the covered electrode after UV is very similar to the current observed in the naked electrode, suggesting that the PMMA domains were indeed removed from the PS-b-PMMA films to form the PS-b-PMMA-based NEA
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structure, i.e, the gold surfaces at the bottom of the NEAs were exposed.
Another confirmation that NEAs were indeed obtained comes from the results shown in Figure 3, were CVs of FcCH2OH on the produced NEA electrodes (after UV radiation) are shown at
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increasing scan rates. The redox peaks were observed up to the scan rate of 4 V s-1, which is an
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indication that the reaction is controlled by the linear diffusion of the redox-active probe (FcCH2OH) under this condition [58,59]. At greater scan rates, the voltammograms become less peak-shaped and more sigmoidal, a fact that can be associated with the product of a mixture of radial and linear diffusion of the probe, due to overlapping of the individual diffusion layers, where radial diffusion becomes predominant as the scan rate is increased [60,61]. Therefore,
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when plotting the anodic peak current values as function of the square root of the scan rate, the behavior of a NEE is very distinct to the observed for a planar electrode. The profile transition of the voltammograms curves from peak-shaped to sigmoidal, is a typical behavior of micro and
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nano-electrodes [59].
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The insert plot in Figure 3 shows that the forward peak current for the naked electrode scales with the square root of the scan rate, v1/2, according to the Randles-Sevcik equation [62]. For the PS-b-PMMA based NEA, the peak current was found to be a linear function of the square root of the scan rate only at very low scan rates (5 to 100 mV s-1), a behavior similar to the observed in the naked macroelectrode, rather than what is expected from nanoelectrode ensembles [58,59]. The reason for this observation is likely due to fact that, at low scan rates, the diffusion layers originating at the individual and/or cluster of pores are heavily overlapped, resulting in the semi-
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infinite planar diffusion of bulk redox species – similar to what happens at a solid planar macroelectrode [63]. However, at higher scan rates (over 200 mV s-1) the porous nature starts to show its effect, when the data points deviate from the linear function [62]. It is also noted that, at
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such high scan rates, the capacitive background current increases much faster than the faradaic current and that the CV become more sigmoidal shaped, making it difficult to determine the peak
height.
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Figure 3. Cyclic voltammograms for 0.5 mmol L-1 FcCH2OH at the PS-b-PMMA-based NEA obtained with different potential scan rates: (a) 5, (b) 10, (c) 20, (d) 60, (e) 100, (f) 200, (g) 500, (h) 1000, (i) 2000, (j) 4000, (k) 6000, (l) 8000 and (m) 10000 mV s-1. The insert shows the
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dependence of the anodic peak current on the square root of the scan rate for the naked electrode (filled symbols) and PS-b-PMMA-based NEA (open symbols).
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3.2. Chemical Modification of the NEAs The remaining acidic groups at the PS matrix boundary in the pore’s walls were covalently bound to a β-CD derivative. Figure 4 shows (a) tapping-mode AFM and (b) SEM images of a
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PS-b-PMMA-derived nanoporous film on a gold substrate after the EDC-mediated amidation of the pores with heptakis(6-amino-6-deoxy)-beta-cyclodextrin. After the amidation reaction, both
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the thickness of the films and the density of the nanopores did not change, indicating that the reaction occurred only inside the pores. The change in effective pore radius was ca. 2 nm, which coincided with the outer diameter of β-CD molecule (ca. 1.54 nm) [64]. This suggests that heptakis(6-amino-6-deoxy)-beta-cyclodextrin was indeed attached to the nanopore surfaces,
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resulting in the shrinkage of the effective pore radius.
We also used CV to characterize PS-b-PMMA-derived nanoporous films whose surface -COOH groups were modified with heptakis(6-amino-6-deoxy)-beta-cyclodextrin via EDC-mediated
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amidation. The Figure 4 shows the CVs of (c) FcCH2OH and (d) Ru(NH3)6Cl3 before and after the amidation reaction. The peak current intensity ip of both redox-active probes decreased upon
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amidation. Since the FcCH2OH was used in large excess and we also used Ru(NH3)6+3 as a probe, we can discard the effect of possible host-guest interactions (with CD) and argue that the smaller ip values were probably due to the fact that the pore radii were smaller after amidation, because β-CD is massive enough to shrink the effective pore radius, reducing the mass transport of redox species to the bottom of each NEA [41].
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Figure 4. (a) Tapping-mode AFM and (b) SEM image of the surface of a thin PS-b-PMMA film
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on a gold substrate after the EDC-mediated amidation of the pores with heptakis(6-amino-6deoxy)-beta-cyclodextrin and the respective cyclic voltammograms (scan rate: 10 mV s-1) of (c) 0.5 mmol L-1 FcCH2OH and (d) 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) of the PS-bPMMA-based NEA (—) before and (···) after EDC-mediated amidation with heptakis(6-amino6-deoxy)-beta-cyclodextrin.
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As discussed above, the change in the pore radius produced by the amidation was ca. 2 nm. To be sure that the amidation reaction did occur, i.e., that the β-CD are really covalently bound to the pore’s walls, we carried out another experiment. For that, we used K4Fe(CN)6 as a redox-
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active probe. Since this molecule is an anion at pH 7.0 and any free -COOH groups will be deprotonated at this pH, the probe will be repelled off the nano pore in the presence of deprotonated carboxyl groups. However, if the acidic groups are converted to amides, this
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repulsion will be reduced.
Figure 5. Cyclic voltammograms (scan rate: 10 mV s-1) of 0.5 mmol L-1 [Fe(CN)6]−4 in 0.1 mol L-1 KNO3(aq) on a gold substrate (- - -) naked and the PS-b-PMMA-based NEA (—) before the amidation; (···) after amidation with heptakis(6-amino-6-deoxy)-beta-cyclodextrin; (―x―) after amidation with heptakis(6-amino-6-deoxy)-beta-cyclodextrin and ETA.
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Figure 5 shows CVs from three different steps: the PS-b-PMMA-based NEA (i) without any βCD (before the amidation); (ii) with β-CD (after amidation) and (iii) with β-CD amidation followed by another amidation reaction, with ETA. The later step was to ensure that most of the
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acidic groups were converted to amides; we used a small molecule, ETA, because it doesn’t have the same steric hindrance as β-CD. As expected, the first curve (before any amidation) did not show redox peaks due to the electrostatic repulsion between the anionic Fe(CN)6-4 and the
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deprotonated -COOH groups on the nanopore surface at neutral pH [40]. However, a small redox peak can be observed after β-CD introduction — indicating that some carboxyl groups were
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amidated with β-CD. When a second amidation (with ETA) reaction was carried on, the peak was even larger, because the number of negatively charged carboxyl groups were smaller and more probe molecules could access the bottom of the nanopores. The summarized conclusions arising from the Figure 5 experiments are that (i) the β-CD was indeed covalently bonded to the
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nano pore walls and that (ii) due to the large volume of β-CD molecule not all the carboxyl
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groups in the nanopore surfaces are converted to amides.
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3.3. Supramolecular functionalization and biomolecule detection Using the produced electrodes, we carried out experiments to study the ability of our NEA to detect biomolecules by doing specific host guest interactions. Whereas non specific interaction of the Fc-biotin linker and the unfunctionalized NEAs (PS-b-PMMA UV radiated film without the β-CD) were not observed, the linker was indeed attached to the β-CD moieties of the functionalized NEAs, as is represented in the schema of Figure 6(a). The presence of the linker decreased the effective radius of the pores, as demonstrated by the smaller peak current at the
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voltammogram shown in Figure 6(b). The linker occupies an important volume inside the pore causing its partial obstruction and, therefore, decreasing the flow of the electrochemical probe to the bottom of the pores. The reversibility of the supramolecular Fc/β-CD complex was shown by
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inducing the electrochemical detachment of the Fc-biotin linker. This was achieved by applying an oxidizing electric field in situ, producing the Fc oxidation peak current at Figure 6(c). The electrochemical detachment was done in KNO3(aq) where an oxidizing electric field (Eox= +0.55
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V) was applied under gently stirring conditions. Successive scans were made until this peak disappeared completely. These experiment also allowed us to quantify the amount of Fc-linker
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previously connected to the NEAs: by integrating the value of the total current under the Fc peak (7.811x10-6 C) and dividing it by the Faraday constant and the area of the gold electrode, we found that 8.10x10−11 mol cm-2 of Fc were present in the electrode, which corresponds to 2.02x10−11 mol cm-2 of the Fc-linker (as each linker has 4 Fc groups) [54]. Taking into account
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the effective pore density of 560 pores µm-2, each nanopore was estimated to contain on average 217 Fc-linker. Assuming a pore radius of 6 nm and a film thickness of 94.4 nm on average, a surface density of the Fc-linker on the pore’s wall was estimated around 0.98 ×10-11 mol cm-2.
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The surface density is roughly half of that found for a similar Fc-linker bound to a planar surface, i.e. 2.00 ×10-11 mol cm-2 [65]. Sterical constraints due to pore curvature and confinement could
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be at the origin of this decreased surface density.
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Figure 6. (a) Scheme illustrating the attachment and detachment of the Fc-biotin linker to the β-
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CD bound to the nanopores walls; (b) cyclic voltammograms (scan rate: 10 mV s-1) of 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) on the β-CD decorated NEA (—) before the attachment ; (···) after the attachment and (- - -) after the detachment of the Fc-biotin linker; (c) cyclic voltammogram of 0.1 mol L-1 KNO3(aq) showing the oxidation of Fc at E > +0.3 V. Another observation that is worth to mentioning: it has been shown that streptavidin can be physically adsorbed on hydrophobic surfaces such as polystyrene [66,67]. Keeping that in mind, we also exploited the possibilities of non-specific interactions between the Fc-Linker and the
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organic part of the electrodes (PS-matrix) by adding it to the non-functionalized NEAs. All the electrochemical measurements (not shown here) indicated that non-specific interactions, such as
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adsorption, did not happen in our system. According to the experiment shown on Figure 7, the NEAs with biotin were able to detect the presence of the target molecule, the protein SA. The current peak was decreased when SA was
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present in the solution, as a consequence of a further blockage of the pore due to the presence of SA molecules, taking in account that the diameter of a SA is ∼4 nm, less than one third of the
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pore’s diameter [68]. As the peak current is proportional on the numbers of pores blocked by SA, this effect can have a linear dependence with the SA concentration in the medium, allowing also its quantitative detection. However, even in the presence of concentrated SA solution we were still able to observe a redox peak of the probe; this suggests that not all the pores were blocked,
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meaning that some pores didn’t have the Fc-biotin linker attached or that the protein moiety of the linker was hanging outside the pore, where the interaction streptavidin and biotin took place. The mean effective pore diameter after the amidation reaction of 12 nm is almost three times the
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mean diameter of the target molecule. However, after introduction of the linker, the pore diameter becomes smaller (Fc-biotin inker, 0.7 nm [54]). Assuming two Fc-biotin linker units in
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the same pore, the diameter would shrink to about 10 nm. Thus, the PS-b-PMMA selected for this system seems to be right, since one single molecule of SA is able to block at least half of the entrance of a nanochannel and restrict the approaching of a second SA unit. If the pore radius was higher or much smaller, this effect could be missed.
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Figure 7. (a) Scheme illustrating the complex linker/SA formation and the detachment of this complex linker to the β-CD bound to the nanopore walls; (b) cyclic voltammograms (scan rate: 10 mV s-1) of 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) on the β-CD decorated NEA (—) before the attachment and (―x―) after the attachment of the Fc-biotin linker; (- - -) after complex linker/SA formation and (···) after the detachment of complex linker/SA.
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4. CONCLUSION
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We have presented a method for obtaining a very versatile NEA that can be adapted to specifically detect the targeted molecule. After the introduction of β-CD groups at the nanopore inner surfaces, the NEA can be programmed by choosing the right Fc-linker; in our case, the linker contained a biotin unit, thus allowing the detection of streptavidin. By changing the linker,
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other molecules can be also detected. The concept opens a window to many possibilities,
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including the development of systems for fast and versatile detection. Relying on the reversibility of the Fc/β-CD interaction, the linker could be changed in situ in flow systems, allowing instantaneous selection and detection of new target molecules. This method can be further exploited to develop equipments able to detect the presence of a specific protein or other biomacromolecule in a complex mixture; such devices can find many applications, ranging from
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clinical diagnosis to the detection of circulating antibodies specific to a single protein. AUTHOR INFORMATION
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Corresponding Author
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* R. Borsali. E-mail: [email protected] Funding Sources
CNRS, CARNOT POLYNAT, CNPq and CAPES-COFECUB (project 620/08).
ACKNOWLEDGMENT The authors acknowledge the financial support from CNRS, Grenoble Institute Carnot Polynat, Greenanofilms, CNPq and CAPES-COFECUB (project 620/08). H. Bonnet is acknowledged for
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his assistance in the AFM technique and S. Pradeau is acknowledged for her collaboration in the
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By means of self-assembly of Block Copolymer a versatile nanoarray electrode for protein detection is proposed
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Nanopores from UV-exposed block copolymer thin film were modified by adding the host molecule β-CD
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The guest - protein-specific binding agents attached to Ferrocene - were connected to the pores
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The electrode was able to specifically detect and quantify the target protein
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The device is very versatile: new targets can be selected by changing the guest molecule
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S0032-3861(17)30674-2
DOI:
10.1016/j.polymer.2017.07.015
Reference:
JPOL 19827
To appear in:
Polymer
Received Date: 30 May 2017 Revised Date:
27 June 2017
Accepted Date: 4 July 2017
Please cite this article as: Fayad SJ, Minatti E, Soldi V, Fort Sé, Labbé P, Borsali R, A versatile nanoarray electrode produced from block copolymer thin films for specific detection of proteins, Polymer (2017), doi: 10.1016/j.polymer.2017.07.015. 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.
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A Versatile NanoArray Electrode produced from Block Copolymer Thin Films for specific detection
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of proteins
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Samira J. Fayad,a,b Edson Minatti,b Valdir Soldi,b Sébastien Fort,a Pierre Labbé,c and Redouane Borsali*,a
Grenoble Alpes University, CNRS, CERMAV UPR 5301, 38000 Grenoble, France
b
Department of Chemistry, Universidade Federal de Santa Catarina, 88040-900 Florianópolis,
Grenoble Alpes University, CNRS, DCM UMR 5250, 38000 Grenoble, France
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c
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Brazil
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a
Keywords: Self-Assembly, Thin Film, Single Molecule, Block Copolymer, Nanoelectrode Arrays, Cyclodextrin, electrochemistry
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ABSTRACT
This work describes how nanostructured thin films obtained from the self-assembly of block
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copolymers (BCPs) systems can be used as a nanoelectrode array (NEA) that can be programmed to specifically detect targeted molecules. Namely, poly(styrene-b-methacrylate) (PS-b-PMMA) thin films, after removal of PMMA phase, produced regular spaced 560 pores
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µm-2 with 16 nm of diameter. The nanopores were then chemically modified by the introduction of β-cyclodextrin (β-CD) molecules. By using the supramolecular interaction of β-CD and
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ferrocene (Fc), the pores could be programed by the introduction of molecules linked to Fc able to interact with target species. In the model system shown here, the linker had a biotin unit, aiming the detection of streptavidin. By changing the linker, other molecules can also be detected. This concept opens a window to many possibilities, including the development of
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1. INTRODUCTION
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devices for fast and versatile molecule detection.
The use of BCPs to obtain well-ordered nanostructures in the solid state is widely described in
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the current literature [1-6]. Distinct morphologies with different size and shapes can be easily tailored by changing the molecular characteristics of the BCP, such as molar mass, block volume fractions and block incompatibility [6-13]. The nano domains created by the self-assembly of BCPs can be transferred to solid substrates that can act as templates or scaffolds to the fabrication of highly ordered nanomaterials [14-18]. One of the fields taking advantages of such possibilities is electrochemistry, where BCPs have
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been used to obtain nanostructures for several applications, such as sensor materials and electrodes [19,20]. The fabrication and behavior of macrosized nanoelectrode ensembles (NEEs, where the nanoelectrodes are randomly spaced) and NEAs (where there is an order in the
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spacing between the individual nanoelectrodes) are very attractive [21].
It is well known that both the sensitivity and the selectivity of electrodes can be increased by
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using nanoelectrodes arrays, when compared to regular electrodes with planar surfaces [22-29]. They improve the signal-to-background current ratios and allow extremely low detection limits
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[30,31]. In a NEA, each single electrode is at the bottom of a nanopore; the size of the pore can be finely tuned, allowing single molecule detection. The mobility and detection of the target molecule are limited by the size and chemical characteristics of the nanopores [32]. In order to produce NEEs and NEAs, several strategies have been described in the literature and
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can be summarized as two general methods: the physical or chemical scratching of a previously regular surface in order to obtain the nano sized holes (by means of X-ray, electron nanolithography, plasma, and others) [33-35] or by using an already nano-organized surface, such as
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the use of nano-templates made from the self-assembly of block copolymers in thin films deposited onto the metallic surface of the electrode and followed by the selective removal of one
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of the phases (blocks) from the polymeric matrix [36-38]. The latter case is much more interesting, since the size and spacing of the pores can be easily adjusted by changing the molecular weight and block volume fraction of the polymers [39,40]. By playing with the variables in the equilibrium phase diagram of the block copolymer [10,41,42] one can choose the morphology of the self-assembly and obtain the desired pattern and, consequently, produce the required NEAs.
For instance, if the thin film is obtained using the conditions that favor
cylindrical domains of one block perpendicularly oriented to the electrode surface, the removal
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of this phase will produce NEAs [19]. Another advantage of the use of BCPs templates is the larger density of pores compared to traditional lithography. In addition, by using BCPs the pores
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can be regularly spaced and have long range order. Usually, when removing one of the phases of a thin film made from a self-assembled BCP, the remaining phase is left with some residual and reactive groups at the boundary of the now extinct
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phase [43]. This behavior can be exploited to add new chemical functionalities to the pore walls — a way to input detection selectivity to the obtained NEA [44-46]. For instance, the inclusion
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of biomolecules, such as proteins or antibodies, can lead to specific interactions with target molecules; the specific interactions in enzyme-substrate or antibody-antigen coupling are then transferred to the NEAs, making the detection very specific and selective. The PS-b-PMMA is one of the BCPs most often used to produce nano-templates [47-50]. The
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segregation phase diagram is well known and the influence of variables such as temperature, molar mass and volume fractions are thoroughly described in the literature [38-40,51]. One of the most important characteristics of this copolymer is its behavior when irradiated with UV
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light: the PS block is cross-linked and the PMMA block is degraded [44, 52]. Thus, in a selfassembled PS-b-PMMA thin film, the PMMA phase can be removed by UV radiation. If the film
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is made by perpendicularly arranging the cylindrical PMMA domains in a PS matrix, nano-pores will then be obtained in which the size and spacing of the pores can be controlled by the molar mass and block composition of the BCP. In addition, the PS boundary is left with residuals and reactive carboxyl acid groups as previously observed by Li et al. [43]. The introduction of supramolecular chemistry in NEAs is particularly attractive, because it allows controllable molecular recognition and structural modifications at specific areas of the
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nanopores. In this work, we used PS-b-PMMA to obtain thin films with PMMA hexagonally packed cylinders on a PS matrix deposited on gold surfaces. After the removal of the PMMA phase, the nanopores walls were chemically modified by the introduction of β-CD molecules.
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The redox behavior of Fc is well-known for its reversibility in host–guest supramolecular systems with β-CD [53,54]. By taking advantage of these interactions, the pores could be easily changed by introduction of other molecules covalently bound to Fc that are able to interact with
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target molecules. In this way, we demonstrate a versatile way to obtain NEAs that have specific interactions with analytes, allowing single molecule detection. To prove the method, we chose a
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protein-ligand model system, namely an orthogonal bifunctional tetravalent Fc-biotin linker, in which a cyclodecapeptide scaffold is coupled to one biotin molecule at one end and four Fc functionalities at the other end [54]. The supramolecular attachment of Fc to β-CD leaves the biotin anchored at the surface of the nanopores walls. The biotin has a very strong and specific
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interaction with streptavidin (SA), therefore allowing the NEA to detect the presence of this
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protein in the aqueous medium.
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2. EXPERIMENTAL SECTION
The proposed method can be summarized as: (1) casting of nano-organized diblock copolymer thin film onto a conductive gold subtract; (2) removal of one of the copolymer phases by means of UV radiation to obtain regularly spaced nanopores; (3) chemical modification of the nanopores to include selective binding molecules; (4) detection of target molecule due to the reduction of pore entrance.
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2.1. Chemicals and Materials.
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All solutions were prepared with water having a resistivity of 18 MΩ cm or higher (Millipore system, France). PS-b-PMMA (Mn 53 000 g mol-1 for PS and 20 500 g mol-1 for PMMA and Mw/Mn 1.08) was purchased from Polymer Source and used as received.
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Toluene (Carlo Erba Reagents), ethanol (Carlo Erba Reagents), acetic acid (AcOH, Carlo Erba Reagents), Dimethylformamide (Carlo Erba Reagents), sodium azide (Carlo Erba Reagents), hydroxide (Sigma-Aldrich), methanesulfonyl chloride (Sigma-Aldrich), imidazole
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sodium
(Sigma-Aldrich), triphenylphosphine (Sigma-Aldrich), ammonium hydroxide (Sigma-Aldrich), monosodium phosphate (Sigma-Aldrich), disodium phosphate (Sigma-Aldrich), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC; Sigma-Aldrich), ethanolamine (ETA, N-hydroxysulfosuccinimide
(NHSS;
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Sigma-Aldrich),
Sigma-Aldrich),
N-(2-
hydroxyethyl)piperazine-N′-(2- ethanesulfonic acid) (HEPES; Euromedex), streptavidin (SigmaAldrich)
were
used
as
chloride
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hexaammineruthenium(III)
received.
Ferrocenemethanol (Ru(NH3)6Cl3,
(FcCH2OH;
Sigma-Aldrich),
Sigma-Aldrich),
potassium
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hexacyanoferrate(II) trihydrate (K4Fe(CN)6, Sigma-Aldrich) were of reagent grade quality or better and used without further purification. Heptakis (6-amino-6-deoxy)-beta-cyclodextrin was prepared by mesylation of the primary hydroxyle groups of β-CD followed a substitution with sodium azide and reduction according to the procedure reported in the literature [55,56].
2.2. Preparation of PS-b-PMMA Films.
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The gold substrate was cleaned by briefly rinsing with water and drying under a stream of nitrogen; this was followed by UV-ozone treatment for 5 minutes. After this period, the surface was treated with anhydrous ethanol for 10 minutes under agitation followed by drying with
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nitrogen. A thin film of PS-b-PMMA was prepared on the gold substrate via spin-coating (3000 rpm) from its toluene solution (2.0% w/w) and was then annealed at 170 °C in vacuum (ca. 0.3 Torr) for 24 h to form cylindrical PMMA domains in the film. The PMMA domains were
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degraded via UV irradiation using a UV lamp Fisher Scientific (Bioblock Scientific VL 6C/6W UV lamp, 254 nm; 12 W; ca. 2.5 mW cm-2) under an Argon atmosphere, which involves
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simultaneous cross-linking of the PS matrix and degradation of the cylindrical PMMA domains [52]. Subsequently, the degraded PMMA domains were removed by rinsing with AcOH for 2 min [40]. The thickness of a PS-b-PMMA film was measured using an imaging null-ellipsometer EP3 (Nanofilm, Germany). The ellipsometric thicknesses of annealed PS-b-PMMA films prior to
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the PMMA degradation were 87.2±1.2 nm; after UV/AcOH treatment there were a slight increase in the thickness of the films (up to 94.4±1.3 nm).
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2.3. Surface Modification of PS-b-PMMA-Derived Nanopores. The chemical modification of PS-b-PMMA-derived nanoporous was done according to the
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procedure of Li et al. [40]. PS-b-PMMA-derived nanoporous films on gold substrate were immersed in 1 mL phosphate buffer (0.1 mol L-1, pH 6) containing 400 mmol L-1 of EDC and 100 mmol L-1 of NHSS for 12 h with gentle shaking. Subsequently, the films were soaked in a 1 mL phosphate buffer solution containing 8 mmol L-1 of heptakis (6-amino-6-deoxy)-betacyclodextrin for 24 h with gentle shaking. Then the films were rinsed with phosphate buffer and immersed in an aqueous solution of ETA (1 mol L-1, pH 8.5) for 12 h with gentle shaking. The
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samples were rinsed with the phosphate buffer and then rinsed with milliQ water prior to the measurements.
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2.4. Attachment of linker and SA. The the functionalized NEAs were immersed in 150 µL of a 5 µM solution of tetravalent Fcbiotin linker (synthesized by Dubacheva et al. [54]) for 45 min. Then the films were rinsed with
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HEPES buffer (10 mmol L-1, 0.15 mol L-1 NaCl, pH 7.0).
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After this step, the films were soaked in 500 µL HEPES buffer containing 1 mg L-1 of streptavidin for 45 min. Subsequently, the films were rinsed and kept in a HEPES buffer for 5 min prior the measurements. 2.5. AFM Measurements.
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AFM images were obtained by tapping-mode imaging in air, using a Picoplus instrument (Molecular Imaging). A Picoscan controller and a Magnetic AC mode (MAC mode) control box were used to control the scanner. A small range scanner of 10x10 µm and a top-MAC nose were
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used for imaging. AFM tips with a magnetic coating (MAC Lever Type VI, Agilent Technologies) of a nominal spring constant 48 N m-1 were used. The measurement frequency
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was set to 15% below the resonant frequency. Scanning was carried out with the feedback adjusted to 20% amplitude reduction at a lateral scan frequency. All the images were analyzed using the Gwyddion 2.22 software. 2.6. SEM Measurements.
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SEM images were obtained by a secondary electron imaging mode with a Zeiss ultra 55 fieldemission gun scanning electron microscopy (FEG-SEM) (CMTC-INP, Grenoble, France) at an
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accelerating voltage of 3 kV, using an in-lens detector. 2.7. Electrochemistry Measurements.
Electrochemical experiments were performed with a conventional three-electrode potentiostatic
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system using a CHI 440 potentiostat (CH-Instruments, Inc., USA). Electrode potentials were referred to Ag/AgCl (3 mol L-1 KCl). The counter electrode was platinum and the working
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electrode was the functionalized gold sensor. Electrochemical detachment of the linker and its complex with streptavidin formed stepwise (linker/SA) was done in situ in the buffer by applying an oxidizing electric field (Eox=+0.55V, which was used to oxidize the Fc groups attached to the
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surface).
3. RESULTS AND DISCUSSION
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3.1. NEAs construction and characterization
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The PS-b-PMMA films deposited on gold substrates were characterized using AFM and SEM techniques. Figure 1 shows (a) tapping-mode AFM images of the PS-b-PMMA film before and (b) tapping-mode AFM image and (c) SEM image of the PS-b-PMMA film after PMMA etching. In Figure 1a, circular bumps were observed, suggesting that the PMMA domains were oriented perpendicular to the film surface [40]. After UV irradiation and subsequent acidic treatment with AcOH (Figure 1b), the circular bumps changed to circular depressions (pores) due to the removal of the PMMA domains. After the removal of the PMMA domains the presence of
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regularly spaced nanopores could also be observed by SEM imaging; circular domains having radius of 8 ± 1 nm were observed and, assuming a perfect orientation of cylindrical domains in
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the whole surface, the effective pore density is around 560 pores µm-2.
Figure 1. Tapping-mode AFM images of the surfaces of a thin PS-b-PMMA film on a gold substrate (a) after the annealing at 170 °C in vacuum for 24 h and (b) after the removal of the
same film as in (b).
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PMMA domains via UV irradiation and subsequent AcOH treatment and (c) SEM image of the
Both imaging techniques show that the PMMA phase was removed and pores were created.
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However, it is not possible to infer if the PMMA domains were completely removed from the film, allowing the exposure of the bottom gold surface to form the NEA structure [40]. To
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address this issue, a series of cyclic voltammograms (CVs) were carried on the produced electrodes.
The suitability of the mesoporous insulating matrix to electrochemical applications relies on the availability of the inner volume of the pore to the species from the electrolyte solution and the accessibility of the electrode surface (the bottom of the NEA), step required for the initiation of the electron transport process through the mesoporous film [57]. Thus, the gold electrode coated
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with PS-b-PMMA thin film was exposed to solutions of redox probes and the electrochemical responses were compared to that of an uncoated electrode. The results are shown in Figure 2, where the CVs of (a) 0.5 mmol L-1 FcCH2OH and (b) 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1
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KNO3(aq) on naked gold substrates are compared to those coated with PS-b-PMMA films before
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and after UV irradiation.
Figure 2. Cyclic voltammograms (scan rate: 10 mV s-1) of (a) 0.5 mmol L-1 FcCH2OH and (b) 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) on a gold substrate (—) naked and coated with PS-b-PMMA film (···) before and (- - -) after UV irradiation. The absence of a peak-shaped CV in the electrodes with PS-b-PMMA film before UV radiation is due to the fact that the insulating non-conductive polymer layer covering the gold surface
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avoids the electrode reactions. The peak current observed in the covered electrode after UV is very similar to the current observed in the naked electrode, suggesting that the PMMA domains were indeed removed from the PS-b-PMMA films to form the PS-b-PMMA-based NEA
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structure, i.e, the gold surfaces at the bottom of the NEAs were exposed.
Another confirmation that NEAs were indeed obtained comes from the results shown in Figure 3, were CVs of FcCH2OH on the produced NEA electrodes (after UV radiation) are shown at
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increasing scan rates. The redox peaks were observed up to the scan rate of 4 V s-1, which is an
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indication that the reaction is controlled by the linear diffusion of the redox-active probe (FcCH2OH) under this condition [58,59]. At greater scan rates, the voltammograms become less peak-shaped and more sigmoidal, a fact that can be associated with the product of a mixture of radial and linear diffusion of the probe, due to overlapping of the individual diffusion layers, where radial diffusion becomes predominant as the scan rate is increased [60,61]. Therefore,
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when plotting the anodic peak current values as function of the square root of the scan rate, the behavior of a NEE is very distinct to the observed for a planar electrode. The profile transition of the voltammograms curves from peak-shaped to sigmoidal, is a typical behavior of micro and
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nano-electrodes [59].
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The insert plot in Figure 3 shows that the forward peak current for the naked electrode scales with the square root of the scan rate, v1/2, according to the Randles-Sevcik equation [62]. For the PS-b-PMMA based NEA, the peak current was found to be a linear function of the square root of the scan rate only at very low scan rates (5 to 100 mV s-1), a behavior similar to the observed in the naked macroelectrode, rather than what is expected from nanoelectrode ensembles [58,59]. The reason for this observation is likely due to fact that, at low scan rates, the diffusion layers originating at the individual and/or cluster of pores are heavily overlapped, resulting in the semi-
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infinite planar diffusion of bulk redox species – similar to what happens at a solid planar macroelectrode [63]. However, at higher scan rates (over 200 mV s-1) the porous nature starts to show its effect, when the data points deviate from the linear function [62]. It is also noted that, at
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such high scan rates, the capacitive background current increases much faster than the faradaic current and that the CV become more sigmoidal shaped, making it difficult to determine the peak
height.
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exact
Figure 3. Cyclic voltammograms for 0.5 mmol L-1 FcCH2OH at the PS-b-PMMA-based NEA obtained with different potential scan rates: (a) 5, (b) 10, (c) 20, (d) 60, (e) 100, (f) 200, (g) 500, (h) 1000, (i) 2000, (j) 4000, (k) 6000, (l) 8000 and (m) 10000 mV s-1. The insert shows the
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dependence of the anodic peak current on the square root of the scan rate for the naked electrode (filled symbols) and PS-b-PMMA-based NEA (open symbols).
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3.2. Chemical Modification of the NEAs The remaining acidic groups at the PS matrix boundary in the pore’s walls were covalently bound to a β-CD derivative. Figure 4 shows (a) tapping-mode AFM and (b) SEM images of a
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PS-b-PMMA-derived nanoporous film on a gold substrate after the EDC-mediated amidation of the pores with heptakis(6-amino-6-deoxy)-beta-cyclodextrin. After the amidation reaction, both
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the thickness of the films and the density of the nanopores did not change, indicating that the reaction occurred only inside the pores. The change in effective pore radius was ca. 2 nm, which coincided with the outer diameter of β-CD molecule (ca. 1.54 nm) [64]. This suggests that heptakis(6-amino-6-deoxy)-beta-cyclodextrin was indeed attached to the nanopore surfaces,
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resulting in the shrinkage of the effective pore radius.
We also used CV to characterize PS-b-PMMA-derived nanoporous films whose surface -COOH groups were modified with heptakis(6-amino-6-deoxy)-beta-cyclodextrin via EDC-mediated
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amidation. The Figure 4 shows the CVs of (c) FcCH2OH and (d) Ru(NH3)6Cl3 before and after the amidation reaction. The peak current intensity ip of both redox-active probes decreased upon
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amidation. Since the FcCH2OH was used in large excess and we also used Ru(NH3)6+3 as a probe, we can discard the effect of possible host-guest interactions (with CD) and argue that the smaller ip values were probably due to the fact that the pore radii were smaller after amidation, because β-CD is massive enough to shrink the effective pore radius, reducing the mass transport of redox species to the bottom of each NEA [41].
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Figure 4. (a) Tapping-mode AFM and (b) SEM image of the surface of a thin PS-b-PMMA film
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on a gold substrate after the EDC-mediated amidation of the pores with heptakis(6-amino-6deoxy)-beta-cyclodextrin and the respective cyclic voltammograms (scan rate: 10 mV s-1) of (c) 0.5 mmol L-1 FcCH2OH and (d) 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) of the PS-bPMMA-based NEA (—) before and (···) after EDC-mediated amidation with heptakis(6-amino6-deoxy)-beta-cyclodextrin.
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As discussed above, the change in the pore radius produced by the amidation was ca. 2 nm. To be sure that the amidation reaction did occur, i.e., that the β-CD are really covalently bound to the pore’s walls, we carried out another experiment. For that, we used K4Fe(CN)6 as a redox-
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active probe. Since this molecule is an anion at pH 7.0 and any free -COOH groups will be deprotonated at this pH, the probe will be repelled off the nano pore in the presence of deprotonated carboxyl groups. However, if the acidic groups are converted to amides, this
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repulsion will be reduced.
Figure 5. Cyclic voltammograms (scan rate: 10 mV s-1) of 0.5 mmol L-1 [Fe(CN)6]−4 in 0.1 mol L-1 KNO3(aq) on a gold substrate (- - -) naked and the PS-b-PMMA-based NEA (—) before the amidation; (···) after amidation with heptakis(6-amino-6-deoxy)-beta-cyclodextrin; (―x―) after amidation with heptakis(6-amino-6-deoxy)-beta-cyclodextrin and ETA.
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Figure 5 shows CVs from three different steps: the PS-b-PMMA-based NEA (i) without any βCD (before the amidation); (ii) with β-CD (after amidation) and (iii) with β-CD amidation followed by another amidation reaction, with ETA. The later step was to ensure that most of the
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acidic groups were converted to amides; we used a small molecule, ETA, because it doesn’t have the same steric hindrance as β-CD. As expected, the first curve (before any amidation) did not show redox peaks due to the electrostatic repulsion between the anionic Fe(CN)6-4 and the
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deprotonated -COOH groups on the nanopore surface at neutral pH [40]. However, a small redox peak can be observed after β-CD introduction — indicating that some carboxyl groups were
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amidated with β-CD. When a second amidation (with ETA) reaction was carried on, the peak was even larger, because the number of negatively charged carboxyl groups were smaller and more probe molecules could access the bottom of the nanopores. The summarized conclusions arising from the Figure 5 experiments are that (i) the β-CD was indeed covalently bonded to the
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nano pore walls and that (ii) due to the large volume of β-CD molecule not all the carboxyl
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groups in the nanopore surfaces are converted to amides.
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3.3. Supramolecular functionalization and biomolecule detection Using the produced electrodes, we carried out experiments to study the ability of our NEA to detect biomolecules by doing specific host guest interactions. Whereas non specific interaction of the Fc-biotin linker and the unfunctionalized NEAs (PS-b-PMMA UV radiated film without the β-CD) were not observed, the linker was indeed attached to the β-CD moieties of the functionalized NEAs, as is represented in the schema of Figure 6(a). The presence of the linker decreased the effective radius of the pores, as demonstrated by the smaller peak current at the
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voltammogram shown in Figure 6(b). The linker occupies an important volume inside the pore causing its partial obstruction and, therefore, decreasing the flow of the electrochemical probe to the bottom of the pores. The reversibility of the supramolecular Fc/β-CD complex was shown by
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inducing the electrochemical detachment of the Fc-biotin linker. This was achieved by applying an oxidizing electric field in situ, producing the Fc oxidation peak current at Figure 6(c). The electrochemical detachment was done in KNO3(aq) where an oxidizing electric field (Eox= +0.55
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V) was applied under gently stirring conditions. Successive scans were made until this peak disappeared completely. These experiment also allowed us to quantify the amount of Fc-linker
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previously connected to the NEAs: by integrating the value of the total current under the Fc peak (7.811x10-6 C) and dividing it by the Faraday constant and the area of the gold electrode, we found that 8.10x10−11 mol cm-2 of Fc were present in the electrode, which corresponds to 2.02x10−11 mol cm-2 of the Fc-linker (as each linker has 4 Fc groups) [54]. Taking into account
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the effective pore density of 560 pores µm-2, each nanopore was estimated to contain on average 217 Fc-linker. Assuming a pore radius of 6 nm and a film thickness of 94.4 nm on average, a surface density of the Fc-linker on the pore’s wall was estimated around 0.98 ×10-11 mol cm-2.
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The surface density is roughly half of that found for a similar Fc-linker bound to a planar surface, i.e. 2.00 ×10-11 mol cm-2 [65]. Sterical constraints due to pore curvature and confinement could
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be at the origin of this decreased surface density.
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Figure 6. (a) Scheme illustrating the attachment and detachment of the Fc-biotin linker to the β-
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CD bound to the nanopores walls; (b) cyclic voltammograms (scan rate: 10 mV s-1) of 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) on the β-CD decorated NEA (—) before the attachment ; (···) after the attachment and (- - -) after the detachment of the Fc-biotin linker; (c) cyclic voltammogram of 0.1 mol L-1 KNO3(aq) showing the oxidation of Fc at E > +0.3 V. Another observation that is worth to mentioning: it has been shown that streptavidin can be physically adsorbed on hydrophobic surfaces such as polystyrene [66,67]. Keeping that in mind, we also exploited the possibilities of non-specific interactions between the Fc-Linker and the
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organic part of the electrodes (PS-matrix) by adding it to the non-functionalized NEAs. All the electrochemical measurements (not shown here) indicated that non-specific interactions, such as
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adsorption, did not happen in our system. According to the experiment shown on Figure 7, the NEAs with biotin were able to detect the presence of the target molecule, the protein SA. The current peak was decreased when SA was
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present in the solution, as a consequence of a further blockage of the pore due to the presence of SA molecules, taking in account that the diameter of a SA is ∼4 nm, less than one third of the
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pore’s diameter [68]. As the peak current is proportional on the numbers of pores blocked by SA, this effect can have a linear dependence with the SA concentration in the medium, allowing also its quantitative detection. However, even in the presence of concentrated SA solution we were still able to observe a redox peak of the probe; this suggests that not all the pores were blocked,
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meaning that some pores didn’t have the Fc-biotin linker attached or that the protein moiety of the linker was hanging outside the pore, where the interaction streptavidin and biotin took place. The mean effective pore diameter after the amidation reaction of 12 nm is almost three times the
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mean diameter of the target molecule. However, after introduction of the linker, the pore diameter becomes smaller (Fc-biotin inker, 0.7 nm [54]). Assuming two Fc-biotin linker units in
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the same pore, the diameter would shrink to about 10 nm. Thus, the PS-b-PMMA selected for this system seems to be right, since one single molecule of SA is able to block at least half of the entrance of a nanochannel and restrict the approaching of a second SA unit. If the pore radius was higher or much smaller, this effect could be missed.
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Figure 7. (a) Scheme illustrating the complex linker/SA formation and the detachment of this complex linker to the β-CD bound to the nanopore walls; (b) cyclic voltammograms (scan rate: 10 mV s-1) of 0.5 mmol L-1 Ru(NH3)6Cl3 in 0.1 mol L-1 KNO3(aq) on the β-CD decorated NEA (—) before the attachment and (―x―) after the attachment of the Fc-biotin linker; (- - -) after complex linker/SA formation and (···) after the detachment of complex linker/SA.
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4. CONCLUSION
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We have presented a method for obtaining a very versatile NEA that can be adapted to specifically detect the targeted molecule. After the introduction of β-CD groups at the nanopore inner surfaces, the NEA can be programmed by choosing the right Fc-linker; in our case, the linker contained a biotin unit, thus allowing the detection of streptavidin. By changing the linker,
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other molecules can be also detected. The concept opens a window to many possibilities,
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including the development of systems for fast and versatile detection. Relying on the reversibility of the Fc/β-CD interaction, the linker could be changed in situ in flow systems, allowing instantaneous selection and detection of new target molecules. This method can be further exploited to develop equipments able to detect the presence of a specific protein or other biomacromolecule in a complex mixture; such devices can find many applications, ranging from
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clinical diagnosis to the detection of circulating antibodies specific to a single protein. AUTHOR INFORMATION
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Corresponding Author
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* R. Borsali. E-mail: [email protected] Funding Sources
CNRS, CARNOT POLYNAT, CNPq and CAPES-COFECUB (project 620/08).
ACKNOWLEDGMENT The authors acknowledge the financial support from CNRS, Grenoble Institute Carnot Polynat, Greenanofilms, CNPq and CAPES-COFECUB (project 620/08). H. Bonnet is acknowledged for
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his assistance in the AFM technique and S. Pradeau is acknowledged for her collaboration in the
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By means of self-assembly of Block Copolymer a versatile nanoarray electrode for protein detection is proposed
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Nanopores from UV-exposed block copolymer thin film were modified by adding the host molecule β-CD
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The guest - protein-specific binding agents attached to Ferrocene - were connected to the pores
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The electrode was able to specifically detect and quantify the target protein
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The device is very versatile: new targets can be selected by changing the guest molecule
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