Nanostructured Disposable Impedimetric Sensors as Tools for Specific

ARTICLE pubs.acs.org/ac

Nanostructured Disposable Impedimetric Sensors as Tools for Specific Biomolecular Interactions: Sensitive Recognition of Concanavalin A Oscar A. Loaiza,*,† Pedro J. Lamas-Ardisana,† Elena Jubete,† Estibalitz Ochoteco,† Iraida Loinaz,† Germ
an Caba~nero,† Isabel García,‡,§ and Soledad Penad
es‡,§ †

Sensors and Photonics Unit, New Materials Department, CIDETEC-IK4, Parque Tecnol
ogico de San Sebasti
an, 20009 Donostia-San Sebasti
an, Spain ‡ Laboratory of GlycoNanotechnology, Biofunctional Nanomaterial Unit, CICbiomaGUNE and §CIBER-BBN, Parque Tecnol
ogico de San Sebasti
an, P° de Miram
on 182, 20009 San Sebasti
an, Spain

bS Supporting Information ABSTRACT: The development of sensors to detect specific weak biological interactions is still today a challenging topic. Characteristics of carbohydrate-protein (lectin) interactions include high specificity and low affinity. This work describes the development of nanostructured impedimetric sensors for the detection of concanavalin A (Con A) binding to immobilized thiolated carbohydrate derivatives (D-mannose or D-glucose) onto screen-printed carbon electrodes (SPCEs) modified with gold nanoparticles. Thiolated D-galactose derivative was employed as negative control to evaluate the selectivity of the proposed methodology. After binding the thiolated carbohydrate to the nanostructured SPCEs, different functionalized thiols were employed to form mixed self-assembled monolayers (SAM). Electrochemical impedance spectroscopy (EIS) was employed as a technique to evaluate the binding of Con A to selected carbohydrates through the increase of electron transfer resistance of the ferri/ferrocyanide redox probe at the differently SAM modified electrodes. Different variables of the assay protocol were studied in order to optimize the sensor performance. Selective Con A determinations were only achieved by the formation of mixed SAMs with adequate functionalized thiols. Important differences were obtained depending on the chain lengths and functional groups of these thiols. For the 3-mercapto-1-propanesulfonate mixed SAMs, the electron transfer resistance varied linearly with the Con A concentration in the 2.2
40.0 μg mL
1 range for D-mannose and D-glucose modified sensors. Low detection limits (0.099 and 0.078 pmol) and good reproducibility (6.9 and 6.1%, n = 10) were obtained for the D-glucose and D-mannose modified sensors, respectively, without any amplification strategy.

N

anoparticle architectures on electrodes attract substantial research efforts focused on the development of bioelectrochemistry.1
4 The general principle for their application lies in the use of bifunctional linkers where one end anchors to the nanoparticles while the other end anchors to metal5 or carbon electrodes.6
8 Other methods, such as nanoparticle adsorption at constant potential9 or electrodeposition,10 are also interesting alternatives since electrodes with gold nanoparticles could be advantageous substrates for biomolecule immobilization.11 Gold nanostructuring of screen printed carbon electrodes (SPCEs) is therefore an ambitious goal, since these electrodes present attractive characteristics such as ease of use, portability, or inexpensive production.12,13 Electrochemical impedance spectroscopy (EIS) is a sensitive and effective technique used to probe the interfacial properties of modified electrodes. This technique offers different advantages such as miniaturization and low-cost detection, serving also as an elegant way to interface biorecognition events and signal transduction.14,15 Recently, the use of nanomaterials in EIS sensors has received much attention.16 The most widely reported use of gold nanoparticles in EIS sensors involves their r 2011 American Chemical Society

incorporation into an ensemble of conductive substrate where a protein, oligonucleotide, or other probe molecule is immobilized.17
19 The advantages of these nanostructured interfaces include improved electrical connectivity through the nanoparticle network, chemical accessibility to the analyte through these networks, and an increased surface for sensing. The increase in surface area, which is related to the nanoparticle size, enhances the surface roughness at the nanometer scale. This way, the electrode modification with gold nanoparticles of adequate sizes has resulted in improved limits of detection and sensitivity.20 Molecular recognition between carbohydrates and specific proteins, by means of multivalent protein-carbohydrates interactions, mediates many important physiological and pathophysiological processes.21 These processes include cellular growth, adhesion, bacterial and viral infections, cancer metastasis, inflammation, and immune surveillance.22,23 Lectins (specific carbohydrate binding proteins) are a broad nonimmune proteins Received: November 25, 2010 Accepted: March 12, 2011 Published: March 21, 2011 2987

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Figure 1. Thiol-ending β-D-glucose (GlcC5SH), β-D-galactose (GalC5SH), and R-D-mannose (ManC5SH) conjugates.

family of animal or plant origin. One of them, concanavalin A (Con A), has been chosen as a protein model in the present work. Con A is a plant lectin which is a tetramer at physiological pH. Con A presents specific sites for Ca2þ and Mn2þ and a dependent carbohydrate binding domain for R-D-mannose/ glucose.24,25 Divalent cations must be present to permit the binding of Con A to carbohydrates because they are necessary in order to get an active Con A conformation.26 In this paper, we report the development of novel nanostructured impedimetric sensors for the detection of biomolecular specific interactions based on reactions between carbohydrates and proteins. The sensor configuration involves the immobilization, via self-assembled monolayer (SAM), of thiolated carbohydrate conjugates on homemade SPCEs modified with gold nanoparticles (AuNp/SPCE). Carbohydrate SAMs represent an optimal model to mimic the multivalent presentation of carbohydrate at the cell surface. D-mannose, D-glucose, and D-galactose were derivatized with an aliphatic linker (
C5SH) and attached to AuNp/SPCEs. Glucose and mannose were chosen due to their selective binding to Con A, while galactose was chosen as a negative control to evaluate the selectivity of the proposed methodology. After carbohydrate self-assembling at the AuNp/ SPCE surfaces, 11-mercapto-1-undecanol (MUA), 3-mercapto-1propanesulfonate (MPS), 6-mercapto-1-hexanol (MCH), or cysteamine (Cys) was used to form different mixed SAMs in order to evaluate the SAM composition effect on the Con A determination. To the best of our knowledge, it is the first time that selective binding of lectin to carbohydrate ligands has been detected by means of EIS using nanostructured SPCEs sensors.

’ EXPERIMENTAL SECTION Reagents and Solutions. 11-Mercapto-1-undecanol (MUA), sodium 3-mercapto-1-propanesulfonate (MPS), 6-mercapto-1hexanol (MCH), cysteamine (Cys), Tris
HCl, MgCl2, CaCl2, gold(III) chloride trihydrate, citric acid monosodium salt, potassium hexacianoferrate (III) and potassium hexacyanoferrate (II) trihydrate, and unconjugated and fluorescein isothiocyanate (FITC) conjugated concanavalin A (FITC-ConA), both Type IV from Canavalia ensiformis (Jack bean), were purchased from Sigma-Aldrich (Spain). KOH, KCl, and H2SO4 were purchased from Panreac. All chemicals were at least analytical grade and were used as received. Two buffer solutions were prepared daily and consisted of MgCl2 (1 mM), CaCl2 (1 mM), and Tris
HCl (10 mM solution, pH 7.4, TMC) and phosphate buffer (0.1 M solution, pH 7.2, PB). Also, KCl (10 mM) and H2SO4 (0.1 M) solution was used. Glycoconjugates of β-D-glucose, β-D-galactose, and R-Dmannose having a five mercapto-ending aliphatic carbon chain (Figure 1) were synthesized as already described.27
29 All chemicals used for the synthesis were purchased as reagent grade from Sigma Aldrich and used without further purification. T 9LC was performed on Silica Gel 60 F254 precoated on aluminum plates (E. Merck), and the glycoconjugates were

detected by staining with 1:9 H2SO4
EtOH or with anisaldehyde solution (25 mL of anisaldehyde, 25 mL of H2SO4, 450 mL of EtOH, and 1 mL of CH3COOH) followed by heating at over 200 °C. Column chromatography was carried out on Silica Gel 60 (E. Merck; 0.2
0.5, 0.2
0.063, or 0.040
0.015 mm; Merck). Stock solutions of thiolated carbohydrate conjugates (1 mg mL
1) were prepared in a 75:25 EtOH/MetOH mixture and stored at 4 °C. Stock solutions of Con A (1 mg mL
1) and FITC-Con A (50 ng mL
1) were prepared in TMC buffer and stored frozen at
20 °C. Milli-Q ultrapure grade water was used throughout. Apparatus and Electrodes. All electrochemical measurements were carried out with an ECO Chemie Autolab PGSTAT302N potentiostat-galvanostat (KM Utrecht, The Netherlands) equipped with a FRA module, using the software package GPES 4.9 (General Purpose Electrochemical System). SPCEs were printed using a Thieme 110E screen printing machine, following a three electrode configuration, according to a previously described procedure.30 The configuration of these SPCEs includes a carbon disk working electrode (Ø = 4.6 mm), a silver/silver chloride pseudoreference electrode, and a carbon counter electrode. A conventional silver/silver chloride reference electrode in KCl (3 M) was used for some experiments. The potential values were referred to the screen printed silver/silver chloride pseudoreference electrode unless otherwise stated. Scanning electron microscopy (SEM) images were acquired from a JEOL JSM-5500LV. The diameters of the gold nanoparticles in the SEM images were determined with Digital Micrograph (TM) 3.7.0 (Gatan Inc.). 1H and 13C NMR spectra were acquired on Bruker DRX-500 spectrometers, and chemical shifts are given in parts per million (d) relative to tetramethylsilane as an internal reference or relative to D2O. FITC dyes were excited with a mercury lamp (Leica Microsystems CMS GmbH, Wetzlar, Germany). An inverted microscope (Leika DMI600B) in an epi-fluorescent configuration with an oil-immersion objective (numerical aperture = 4, 40 objective lens; Leica) was used for fluorescence imaging. Long and short wavelength emissions were spectrally separated by 505 or 595 nm long pass dichroic mirrors (Leica), respectively, and imaged onto a charge-coupled device camera (Hamamatsu, Sewickley, PA, USA). Sensor Preparation. Pretreatment of the SPCEs. A 60 μL drop of H2SO4 (0.1 M) and KCl (10 mM) solution was placed on each SPCE, and a potential of þ1.2 V was applied during 2 min. The electrodes were then rinsed with water and dried under an air flow. Gold Nanoparticle Electrodeposition onto SPCEs. A 100 μL drop of HAuCl4 (0.25 mM) and sodium citrate (7.5 mM) solution was placed on each pretreated SPCE, and a
100 μA current was applied during 120 s. After gold electrodeposition, the electrodes (AuNp/SPCEs) were copiously rinsed with water and dried again. Carbohydrate Immobilization. Five μL of a solution (300 μg mL
1) of thiolated D-mannose, D-glucose, or D-galactose derivative was deposited on the AuNp/SPCEs and left at room 2988

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Scheme 1. Steps for the Sensor Fabrication and Impedimetric Signals for Each Stage: AuNp/SPCE (a), AuNp/SPCE/Sugar (b), AuNp/SPCE/Sugar-MPS (c), and AuNp/SPCE/Sugar-MPS/Con A (d)

temperature overnight. Once the electrodes had dried out (AuNp/SPCE/sugar), they were rinsed with water to remove any unbound thiolated sugar and dried carefully. A 5 μL drop of MPS 40 mM in PB solution was then placed onto the AuNp/ SPCE/sugar electrodes and left to dry under a N2 stream for 30 min. Subsequently, the modified electrodes (AuNp/SPCE/ sugar-MPS) were soaked in PB solution for 20 s. This procedure was also used to build the other mixed SAMs. Con A-Carbohydrate Interaction. Interaction of immobilized sugars with Con A was obtained by dropping 10 μL of lectin solution (prepared in TMC), onto the AuNp/SPCE/sugar-MPS. Once the electrodes had dried out for at least 1 h (AuNp/SPCE/ sugar-MPS/Con A), they were rinsed with water to remove any unbound Con A. Finally, the electrodes were dried by passing through an air flow. This procedure was also used for the studies carried out with the FITC-Con A. Electrochemical Detection of Con A-Carbohydrate Interaction. Impedimetric and cyclic voltammetry measurements were carried out after depositing 80 μL of K3Fe(CN)6/K4Fe(CN)6 (2.5 mM; for each component) in KCl (0.1 M) solution. Impedance measurements were performed at the equilibrium potential of the Fe(CN)64
/Fe(CN)63
couple with a 0.01 V (rms) sinusoidal excitation amplitude. Measurements were made at 40 steps per decade in the appropriate frequency range, five times at each frequency and averaged during each run. The impedance Z is expressed in term of a real (Z0 ) and an imaginary (
Z00 ) component. The changes in resistance were calculated according to ΔRct ¼ Rct ðsugar-MPS-ConAÞ
Rct ðsugar-MPSÞ where Rct (sugar-MPS) is the value of the charge transfer resistance when the thiolated sugar and MPS are immobilized on the electrode and Rct (sugar-MPS-Con A) is the resistance value after the interaction between Con A and the corresponding sugar. Scheme 1 shows the steps for the fabrication of the sensor and the impedimetric signals for each stage. Cyclic voltammetry was used to monitor the fabrication process of the carbohydrate nanostructured sensor and the Con A detection. Voltammograms were recorded from
0.1 to þ0.4 V at a scan rate of 0.1 V s
1 in K3Fe(CN)6/K4Fe(CN)6 (2.5 mM). Fluorescence Microscopy. The FITC dye in the functionalized (AuNp/SPCE/sugar-MPS/FITC-Con A) electrodes was excited with a mercury lamp (leica Microsystems CMS GmbH, Wetzlar, Germany). A 480/40 nm band-pass excitation filter and a 527/30 nm band-pass emission filter were used for FITC detection.

Electrochemical Characterization of SAMs. Cyclic voltammetry was used to determine the gold surface in the AuNp/SPCE and the self-assembled molecules in different SAM or mixed SAM modified AuNp/SPCE. The total gold surface area were estimated from the cyclic voltammograms, recorded from 0 to þ1.6 at 0.1 V s
1 in H2SO4 0.5 M, by integrating the cathodic peaks for the reduction of the gold oxide monolayers on the AuNp surfaces. The theoretical value of 482 μC 3 cm
2 and the reduction charges previously obtained were used for this calculation.31 The amount of self-assembled molecules was estimated in every case from the cyclic voltammograms, recorded from
0.5 to
1.4 at 0.1 V s
1 in deaerated KOH (0.5 M). The integration of the cathodic peaks for the reductive desorption of the SAMs was related to the self-assembled moles by Faraday’s law.32,33 A conventional Ag/AgCl (KCl, 3 M) reference electrode was used in these experiments.

’ RESULTS AND DISCUSSION Characterization of Electrodeposited Gold Nanoparticles. Figure 2 shows the SEM images of SPCEs before (panel a) and after gold electrodeposition at different cathodic currents (panels b
f). As can be seen in the figure, spherical gold nanoparticles are directly electrodeposited, proving the effectiveness of the method for the nanostructuration of the SPCE surface. Images of the AuNp/SPCEs at different points were analyzed (five electrodes were studied for each electrodeposition current). The results indicate that the diameter and the distribution of the gold nanopaticles were dependent on the current employed for the electrodeposition. For example, big particles (Ø = 1540 ( 240 nm) and low homogeneous distributions were observed when reductions at 10 μA were carried out (Figure 2b). Both characteristics are typical for low current electrodepositions over nonuniform surfaces, where the ratio between the growth and nucleation rates for the nanoparticles is high and the electrodeposition occurs at preferential places, i.e., uncovered edge sites of the graphite particles at the SPCE surface.34 When the reduction currents were increased, the mean diameters of the nanoparticles diminished and the superficial distribution was more homogeneous. Nevertheless, the relation between mean diameter and anodic current changed upon 50 μA (Figure 2g). Hydrogen evolution was observed from this current value by an evident surface bubbling. It can be expected that this hydrogen is also involved in the gold reduction near the electrode surface, through a homogeneous redox reaction, which could explain the 2989

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Figure 2. SEM images of the working electrode of SPCEs before (a) and after electrochemical gold deposition at different cathodic currents (μA): 10 (b), 40 (c), 60 (d), 80 (e), and 100 (f). Graph of the mean diameter of gold nanoparticles vs cathodic current (g). Solution: HAuCl4 (0.25 mM) and sodium citrate (7.5 mM). Electrodeposition time: 2 min.

changes regarding nanoparticle size and distribution. The best results were obtained by gold electrodeposition at 100 μA, i.e., smallest diameters with the lowest dispersion (Ø = 89 ( 7 nm) and the most homogeneous distribution of the gold nanoparticles over the SPCE surface (Figure 2f). Reduction at higher currents was discarded, since nanoparticle aggregates were observed. Therefore, subsequent studies were carried out with AuNp/SPCEs obtained by 250 s gold electrodeposition at 100 μA. Characterization by Fluorescence Microscopy. It has been reported that the interaction of Con A with D-mannose is highly specific.35
37 Additionally, it is also known that Con A presents a low biological affinity for D-galactose.38 In order to verify both these facts, a simple fluorescent labeling assay was performed. Galactose (AuNp/SPCEs/galactose-MPS) and mannose (AuNp/SPCEs/mannose-MPS) sensors were labeled with FITC-Con A and visualized by fluorescence imaging. From the obtained images, it could be seen that AuNp/SPCEs/mannoseMPS/FITC-Con A was successfully formed, showing numerous fluorescent signals along the sensor surface (Figure S-1b, Supporting Information), such emission was not observed on the galactose coated electrodes (Figure S-1a, Supporting Information). The detection of fluorescence, after the FITC-Con A interaction, confirmed the effectiveness of the method for the carbohydrate immobilization at the sensor surface and, as mentioned above, the different Con A affinities for the mannose and galactose. It was also observed that the size of the fluorescence spots was not as homogeneous as the gold nanoparticles at the SPCE surface (Figure 2f). This fact could be related to the uneven distribution of the fluorescein isothiocyanate labels (3
6 FITC mol per protein mol) and the formation of small Con A aggregates at pH 7.2.39

EIS Detection of the Carbohydrate-Con A Binding. The immobilization process was monitored by electrochemical impedance spectroscopy (EIS). This is a powerful tool to probe the features of surface modified electrodes. Figure 3a displays the Nyquist plots obtained for AuNp/SPCEs, AuNp/SPCEs/ mannose, and the AuNp/SPE/mannose-MPS before and after their interaction with Con A, in the presence of equimolar [Fe(CN)6]4
/3
. The most adequate fit for the experimental data was given by the equivalent circuit, showed in the inset of Figure 3a, previously proposed by Shobha Jeykumari et al.40 The model equivalent circuit contains the solution resistance (Rs), the bulk electronic capacitor of the thiol layer (Cb), the finite length Warburg diffusion impedance (ZO) of the immobilized Con A, and the charge transfer resistance of the electrochemical reaction (Rct). This parallel combination of Rct and Cb gives rise to a semicircle in the complex plane plot of
Z00 against Z0 (Nyquist plot); the semicircle diameter is equal to the charge transfer resistance (Rct) which exhibits the electron transfer kinetics of the redox-probe at the electrode interface. ZQ is the diffusion impedance of the redox couple of the mediator in the solution. ZO and ZQ can be expressed as: qffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffi ð1Þ Z0 ¼ Y0
1 , 0 ðjωÞtan h 2 BðjωÞ

ZQ ¼ Y0
1 Q ðjωÞ
n

ð2Þ

where Y0 is the admittance parameter, expressed at S cm
2, independent of frequency, j = (
1)1/2, ω = angular frequency = 2πf, B is the diffusion factor, and ZQ = ZCPE corresponding to the constant phase angle element (CPE). The dimensionless exponent n reflects the roughness of the electrode surface (the higher 2990

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Figure 3. Nyquist plots from AuNp/SPCE (blue, 1), nAuNp/SPCEs/carbohydrate (black, b), and nAu/SPCE/carbohydrate-MPS before (red, 9) and after its interaction with Con A (green, 2). Thiolated D-mannose (a) and D-galactose (b) sensors. [Fe(CN)6]3
/4
(1:1), 2.5 mM, in KCl, 0.1 M. Mannose, 300 μg mL
1; MPS, 1 mM; Con A, 10 μg mL
1.

n is, the less rough is the electrode). When n = 0, CPE is reduced to a resistor, when n = 0.5, the system is equal to a Warbur impedance, and when n = 1, Y0Q = Cdl. The impedance spectra follow the theoretical shape and include a semicircle portion, observed at higher frequencies, which corresponds to the electron transfer limited process, followed by a linear part, characteristic of the lower frequencies which could be attributable to a diffusion limited electron transfer. As can be seen in Figure 3a, significant differences in the impedance spectra were observed during stepwise modification of the electrode surface. The AuNp/SPE exhibited an expected fast electron transfer process with a diffusion limited step characteristic of an electrochemical process. The consecutive attachments of the thiolated D-mannose and MPS gave rise to subsequent increases in the electron transfer resistance, resulting in increases in the semicircular section of the Nyquist plot. Interaction of Con A with the D-mannose produced an additional barrier for the redox probe access to the AuNp/SPCE modified electrode, resulting in a further increase in the electron transfer resistance, which is reflected by another substantial increase in the semicircular part of the spectrum. This last increase could be related to the concentration of Con A, which interacts with the immobilized thiolated carbohydrate on the nanostructured SPCE. On the other hand, Figure 3b presents the impedance signals registered in the same way but with a thiolated D-galactose modified sensor. It could be seen that the signals are equal before and after Con A interaction which corroborates the above-mentioned low affinity between Con A and D-galactose. It should also be mentioned that similar results were obtained by cyclic voltammetry. In this case, subsequent decreases in the electron transfer resistance can be detected by the peak-to-peak potential separation increases in the cyclic voltammograms for [Fe(CN)6]4
/3
(2.5 mM) in KCl (0.1 M) solution (Figure S-2, Supporting Information). Although both techniques are adequate for the detection of the AuNp/SPCE/mannose-MPS and Con A interaction, the magnitude of the signal change and the data acquisition facility by EIS are greater than those obtained by cyclic voltammetry. As a result of this, the detection of Con A interaction in subsequent studies was carried out exclusively by means of the electron transference resistance changes (ΔRct) from EIS measurements.

Effect of Different Mixed SAMs on the Sensors Selectivity and Sensitivity. Working variables were optimized taking the

ΔRct between the modified electrode before and after its interaction with Con A as criterion of selection. The assays were carried out using thiolated D-mannose as recognition element for Con A. The influence of four thiols, 11-mercapto-1-undecanol (MUA), 3-mercapto-1-propanesulfonate (MPS), 6-mercapto-1hexanol (MCH), and cysteamine (Cys), forming mixed monolayers with thiolated D-mannose on ΔRct was evaluated. As it can be seen in Figure 4, the sensor response was strongly dependent on the thiol used for the mixed monolayer. The Con A detection was only successful at MCH or MPS mixed monolayer electrodes while the sensitivity was too low in the case of Cys or MUA. Cys is a short chain alkanethiol with an amine functional group. It has been reported that the self-assembling of short chain alkanethiols results in low ordered structures, full of local defects, where the monolayer is not a physical barrier for the electron exchange in diffusion redox systems.41
43 Cys monolayers can introduce high positive charge densities at the surfacesolution interfaces through the protonation of the amine groups. These ammonium moieties can facilitate the electron exchange processes with anionic redox systems, like ferro/ferricyanide, because electrostatic attraction forces are generated between the electrode surface and the present redox molecules.44 This fact could explain why the formation of the Cys mixed monolayer results in a Rct decrease with respect to the AuNp/SPCE/ mannose value. Furthermore, there were no changes in the Nyquist plots after the Con A interaction, which is reasonable since the easier electron exchange pathway, across Cys selfassembled molecules, is not remarkably hindered by the mannose-Con A interaction. The effect of MUA was completely different to that of Cys. MUA is a moderate long chain alkanethiol, and therefore, it tends to build compact and ordered monolayers where the electron tunneling should be the main mechanism for electron exchange. This implies an extra Rct which can be clearly observed if the AuNp/SPCE/mannose and AuNp/SPCE/mannose-MUA impedance diagrams are compared. Nevertheless, very small ΔRct were registered after the interaction of AuNp/SPCE/ mannose-MUA with Con A. This could be interpreted as a noneffective Con A binding at the electrode surface. It is feasible considering that the access to the D-mannose molecules could 2991

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Figure 4. Nyquist diagrams employing different mixed monolayers: AuNp/SPCE/mannose (black), AuNp/SPCE/mannose-thiol’ (red), and AuNp/ SPCE/mannose-thiol’/Con A (blue). [Fe(CN)6]3
/4
(1:1), 2.5 mM, in KCl, 0.1 M. Thiolated D-mannose, 300 μg mL
1; thiol, 1 mM; Con A, 10 μg mL
1.

be hindered by the surrounding longer MUA self-assembled molecules. Alternatively, MCH presents a shorter chain than MUA and the mixed monolayer is expected to be less ordered. This fact was confirmed by the impedance diagrams where the signals for the AuNp/SPCE/mannose and AuNp/SPCE/mannose-MCH present a quite similar behavior, showing that the MCH modification did not suppose an additional Rct. Nevertheless, Con A detection was possible because significant ΔRct increases were registered after lectin binding. In this case, the MCH selfassembling into the D-mannose SAM did not facilitate or hinder the electron transfer, so small changes at the electrode interface, such as the Con A binding, could result in large ΔRct. Finally, MPS is a short chain alkanethiol with a sulfonate functional group. Although MPS mixed monolayers should present low packed structures, the increase in Rct with respect to that magnitude at AuNp/SPCE/mannose is evident at the impedance signals. Unlike the case where Cys was employed, the use of MPS introduces anionic structural charges at the electrode interface which generates electrostatic repulsion forces with the ferro/ferricyanide system. Thus, a decrease in the electron exchange rate is obtained.45 Moreover, the comparison between

the AuNp/SPCE/mannose-MPS and AuNp/SPCE/mannoseMCH responses allows us to conclude that the Rct with the ferro/ ferricyanide system is more affected by the electrostatic repulsion than by the change length, at least in the case of short chain thiols. The AuNp/SPCE/mannose-MPS permits the analyte detection since the Con A binding blocked the easiest pathway for the redox system to access the electrode (i.e., the D-mannose self-assembled molecules at the electrode interface), hampering the electron exchange. As a result, only MPS and MCH mixed monolayers were chosen to carry out the subsequent optimization studies. The concentrations of MPS and MCH forming mixed monolayers were also studied. AuNp/SPCE/mannose and AuNp/ SPCE/galactose were used as recognition (signal) and control (background) elements for Con A, respectively. For the nonmixed monolayer sensors, the signal and background responses were similar, indicating high nonspecific Con A adsorption over the sensor surfaces. The formation of mixed monolayers avoided these adsorptions and increased the difference between signals and background, allowing the specific Con A detection. In both cases, increasing thiol concentrations resulted in an enhancement of specificity for Con A detection. Nevertheless, for each tested concentration, MPS provided greater signal-background ratios than 2992

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MCH (Figure 5). The greatest difference was obtained for MPS 40 mM. This MPS concentration was chosen for further studies. A feasible explanation of these observations could be deduced from the coverage values of the different SAMs. A molecular density of 35.6 ( 3.2 μC 3 cm
2 (n = 5) was estimated for the AuNp/SPCE/mannose. This value could be related to a loosely packed monolayer where the adsorption of the Con A would happen by the direct linking of the protein to the gold AuNp. The packing densities for AuNp/SPCE/MPS, AuNp/SPCE/MCH, AuNp/SPCE/mannose-MPS, and AuNp/SPCE/mannoseMCH were close to 70 μC 3 cm
2 (Table S-I, Supporting Information) which indicates tightly packed SAMs.33 In these cases, the SAMs could act as an effective blocking agent, avoiding the nonspecific Con A adsorption and improving the selectivity of the sensors. Furthermore, the peaks for the reductive desorption of both mixed SAMs clearly suggest homogeneously mixed phases since these processes occur at a potential value comprised in the interval between desorption potentials of each singlecomponent SAM32 (Figure S-3, Supporting Information). Analytical Characteristics. The repeatability of the measurements was calculated from the ΔRct obtained from the interaction of the sensor with 10 μL of Con A, 2.14 μM. Relative

Figure 5. Influence of the thiol concentration forming mixed monolayer in the Con A detection with Au/Np/SPCE/mannose (300 μg mL
1) and Au/Np/SPCE/galactose (300 μg mL
1). Ten μL Con A, 10 μg mL
1. Solution of [Fe(CN)6]3
/4
(1:1), 2.5 mM, in KCl, 0.1 M.

standard deviations (RSD) calculated from ten consecutive measurements were 3.5 and 2.2% for AuNp/SPCE/glucose-MPS and AuNp/SPCE/mannose-MPS sensors, respectively. These results indicate the excellent stability of the developed sensors. The reproducibility of the sensor preparation was also tested. Results from the ΔRct at 10 different AuNp/SPCE/glucose-MPS and AuNp/SPCE/mannose-MPS constructed in the same manner yielded RSDs of 6.9 and 6.1%, respectively. These results demonstrate that the fabrication procedure of these sensors is reliable, allowing reproducible impedimetric responses for different sensors. Con A calibrations with D-mannose and D-glucose AuNp/ SPCE/sugar-MPS sensors were made under the optimized working conditions for five successive calibration plots stated at the 95% confidence level. Linear calibration graphs were obtained between 0.21 and 3.85 pmol ranges for D-mannose and D-glucose modified sensors, with calibration slopes of 681 ( 29 (r = 0.998) and 299 ( 17 (r = 0.997) Hz pmol
1, respectively. These different slopes can be explained because the bound is stronger between D-mannose and Con A than D-glucose and ConA.35,36 It is also worth noticing that the AuNp/SPCE/ galactose-MPS presented insignificant ΔRct for increasing Con A concentrations. The nonspecific Con A adsorption on the electrode is the most feasible explanation for this data, since the Con A and galactose biological affinity is negligible. The limits of detection and determination were calculated according to the 3s/m and 10s/m criteria, respectively, where m is the slope of the respective calibration plot and s was estimated as the standard deviation (n = 10) of ΔRct from Con A 0.214 pmol solutions. These values were, respectively, 0.099 and 0.331 pmol for AuNp/SPCE/glucose-MPS and 0.078 and 0.26 pmol for AuNp/SPCE/mannose-MPS. Several factors could be involved in the achievement of such sensitive values without signal amplification. First, the high EIS sensitivity toward surface changes which converts this technique into a powerful tool for the screening of carbohydrate-Con A interactions. Second, the nanostructuration of the SPCEs with gold nanoparticles results in a discontinuous high surface area where the recognition elements can be easily immobilized and the accessibility for the Con A interaction is facilitated.46 Third, is an adequate choice of the second thiol forming the mixed monolayer, which provides sensitivity and selectivity to the Con A detection. In this particular case, the MPS incorporation could play an important role in the arrangement of the biomolecular recognition elements. The selfassembling process with MPS drives a stretch conformation for the alkane chains of the thiolated carbohydrates,47,48 maximizing the space between the nanoparticle surface and the sugar residues and minimizing the steric hindrances for the recognition of Con A. Finally, Table 1 summarizes the analytical performance from different detection methods previously reported. Duncan et al.

Table 1. Analytical Performance of Different Detection Approaches for the Detection of ConA detection method

immobilization method

optic (SPR)

sugar immobilization using gold coated glass prism modified with APTS

electrochemical (voltamperometric)

immobilization of alkanethiol/glycolipid vesicles onto gold electrodes

optic (fluorescence)

immobilization of trityl-derivatized mannose on a polystyrene microplate

optic (spectrophotometric) optic (spectrophotometric)

glycolipid molecules self-assembled onto gold nanoparticles interaction of Con A with mannose-stabilized gold nanoparticles

EIS

immobilization of mixed SAMs on gold nanostructured SPCE 2993

lineal range

D.L.

(μg 3 mL-1)

(μg 3 mL-1)

reference

0.5

50

0.25 (2.4 nM)

52

0.12

51

10
100 0.25
10 0
30 2.5
7.5 20
40 2.23
40

0.01 (0.1 nM)