Article pubs.acs.org/Langmuir

Atrazine-Based Self-Assembled Monolayers and Their Interaction with Anti-Atrazine Antibody: Building of an Immunosensor Magdaléna Hromadová,*,†,‡ Lubomír Pospíšil,† Romana Sokolová,† Jana Bulíčková,† Martin Hof,† Nathalie Fischer-Durand,§ and Michèle Salmain*,§ †

J. Heyrovský Institute of Physical Chemistry of ASCR, v.v.i., Dolejškova 3, 182 23 Prague, Czech Republic Faculty of Science, J. E. Purkyně University, 400 96 Ú stí nad Labem, Czech Republic § Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue P. et M. Curie, 75231 Paris cedex 05, France ‡

S Supporting Information *

ABSTRACT: As a part of our objective to build an immunosensor for the detection of the pesticide atrazine (ATZ) in environmental samples, we studied the self-assembling process of the disulfide derivative of the pesticide atrazine on a gold substrate. Atrazinebased self-assembled monolayers were characterized by ellipsometry, scanning tunneling microscopy, polarization−modulation infrared reflection−absorption spectroscopy (PM IRRAS), X-ray photoelectron spectroscopy and quartz crystal microbalance (QCM) measurements. Two different time constants for the adsorption process were observed, depending on the experimental method used. The QCM data reflect adsorption kinetics of the original disulfide compound, whereas ellipsometry and ex situ PM IRRAS refer to the formation of thiolate (ATZS) monolayers. In situ QCM data demonstrated the suitability of such monolayers for the detection of atrazine in aqueous samples. Exposure of the ATZS sensing surface to an anti−atrazine antibody (anti-ATZ IgG) resulted in complete coverage of the surface by antibody, whereas approximately half of the antibody molecules were displaced from the QCM sensor surface by further addition of atrazine into the solution.

1. INTRODUCTION Immunochemical detection revolutionized modern chemical analysis of pesticides due to simple mass fabrication of sensors and their low production cost, allowing analysis of a large number of samples at a reasonable time scale.1 Atrazine acts on plants by binding to the plastoquinone-binding protein that is part of photosystem II.2 It stops the emergence of broadleaf weeds and is mainly used for the protection of corn crops. Degradation of atrazine occurs via the action of microorganisms involving mostly hydrolysis reactions.3 A recent international study clearly demonstrated that atrazine is an endocrine disruptor acting across vertebrate classes by inducing estrogen synthesis and reducing androgen levels. Although it is classified as a persistent pollutant since its lifetime in soils is a matter of months, atrazine remains one of the most widely used pesticides in the U.S.4 In 2004, the European Union banned its use because of persistent water contamination.5 This ascertainment, together with toxicity concerns, compelled the European Union to set the maximum admissible concentration of any individual pesticide residue to 0.1 μg/L in surface and groud waters.6 Environmental issues caused by atrazine led to a design of an alternative pesticide terbutylazine, which differs only by one methyl group in the side chain. However, this small © 2013 American Chemical Society

variation has a profound effect on its adsorption and photodegradation properties.7,8 Large differences in the critical transition temperature of the compact film formation explain more efficient accumulation of atrazine in the groundwater compared to terbutylazine, without a possibility to efficiently undergo a photodegradation process. Ground water contamination by atrazine stimulated research interest in its detection in aqueous samples and development of highly sensitive immunosensors for atrazine monitoring.9−15 Among them, the piezoelectric immunosensors showed great promise since they operate in a label-free mode.16,17 Our previously reported carbonyl metallo immunoassay format for determination of atrazine in aqueous samples18 did not achieve the required sensitivity level 0.1 μg/L. In this work, we tried a different approach. We prepared and characterized atrazine-based SAMs that present the basis for an immunosensing device for indirect atrazine detection in environmental samples. An indirect immunosensor is based on the competitive binding of the Received: October 22, 2013 Revised: December 3, 2013 Published: December 6, 2013 16084

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Chart 1. Chemical Structure of Atrazine (ATZ) and Disulfide (ATZSSATZ) Moleculesa

a

Schematic representation of the ATZ−based (ATZS) monolayer on the gold substrate. Atrazine moiety is highlighted in gray circle. IgG fraction was purified according to a published procedure.18 Rabbit IgG and donkey serum were purchased from Sigma. A phosphate buffer saline (PBS) of pH = 7.0 was prepared from 0.01 M phosphate buffer and 0.12 M NaCl, respectively. Gold(111) substrates were purchased from Arrandee, Werther, Germany. The thickness of gold layer was 250 ± 50 nm according to the manufacturer. Gold metal was deposited on a glass substrate (11 × 11 × 1.1 mm3) covered with an adhesion layer of 2.5 ± 1.5 nm of chromium. Each gold substrate was cleaned in the stream of argon and annealed with butane flame to achieve flat gold(111) regions before any surface characterization and deposition studies. After cooling down in an argon atmosphere, the substrates were immediately soaked for a specified amount of time in deaerated solutions of different compositions. The substrates were copiously rinsed with respective solvents, further rinsed with Milli-Q water and dried in a stream of nitrogen. Most of the time 1 × 10−3 M solution of ATZSSATZ in ethanol or in chloroform solvent was used for preparation of selfassembled monolayers (SAMs). In addition to purely ATZS-based SAMs, two mixed SAMs were prepared and characterized by XPS and IRRAS measurements. Sample 1 was prepared by incubation of the gold substrate in 1 × 10−3 M solution of ATZSSATZ in deaerated ethanol, sample 2 in a mixture of 5 × 10−4 M ATZSSATZ and 5 × 10−4 M MUD in ethanol, and sample 3 in a mixture of 2 × 10−4 M ATZSSATZ and 8 × 10−4 M MUD in ethanol. The incubation times varied from 2 to 22 h and are specified within the main text. 2.2. Ellipsometric Measurements. The thickness of adsorbed ATZS monomolecular films was measured in the air by Stokes Ellipsometer L116 S300 (Gaertner Scientific Corp., Chicago, U.S). The adsorption kinetics of ATZSSATZ compound and detection of protein binding to the sensor was measured by the ellipsometry technique using EL X-05 ellipsometer (DRE−Dr. Riss Ellipsometerbau GmbH, Germany). The technique is based on the reflection of the polarized light from a He−Ne laser (λ = 632.8 nm) and on the measurement of the two parameters Ψ (amplitude component) and Δ (phase difference) using the expression,

anti-atrazine antibody to either atrazine-based SAM or atrazine in the analyte solution. The main objective of the present work is to study the adsorption properties of a new disulfide compound (ATZSSATZ), which contains atrazine pesticide attached covalently at both ends of the molecule. Chart 1 shows the chemical structures of atrazine (ATZ) and ATZSSATZ molecules as well as the schematic representation of atrazine-based self-assembled monolayer on the gold substrate. The atrazine moiety is highlighted in gray circle and cartoon assumes the upright adsorption of thiolates (ATZS) on the gold substrate. Studies of self-assembled monolayers formed from disulfide compounds on metallic substrates are not numerous. The most studied couple is the cysteine/cystine system.19−21 In general, it is assumed that disulfides give thiolate SAMs on gold. In our previous work,22−24 we successfully functionalized gold surfaces with thiolate SAMs for immobilization of protein A, which is an affinity-capture protein for rabbit immunoglobulin G (IgG). Such a surface responded well to anti-rabbit IgG antigen. Present research is directed toward the development of a new label-free immunosensor for atrazine detection, where the sensing surface specifically interacts with anti-atrazine antibody, which is then competitively removed from the sensing surface by atrazine molecules in the environmental samples (see Scheme 1). Scheme 1. Indirect Immunosensor for Atrazine Detection Showing Four Stages: Addition of Antibody Y, Binding Of Antibody To ATZ-Based Monolayer, Addition of Atrazine Analyte (●) and Displacement of Antibody Y From the Surface

ρ=

rp rs

= tan Ψe(iΔ)

(1)

where ρ is the ratio of the Fresnel reflection coefficients for light polarized parallel (rp) and normal (rs) to the plane of incidence. The angle of incidence was 70°. Freshly annealed gold substrate was first mounted into the sample holder, which was placed from above into the cuvette containing a small stirring bar. The optical properties of gold substrate were measured first in air. Then 3 mL of 1 × 10−3 M solution of ATZSSATZ in absolute ethanol were added. The optical properties were measured subsequently every 5 s for rapidly stirred solution and the measurements continued, until constant values of Ψ and Δ were observed. From these readings, the refractive index of the interfacial structure was determined. The ethanolic solution of ATZSSATZ was replaced by 0.1 M KCl (flushing the cuvette with total of 50 mL solution) to remove any bulk and loosely adsorbed ATZSSATZ molecules from the system. Then 6 μL of atrazine-specific IgG (final concentration 50 mg/L) was added to the cuvette

2. EXPERIMENTAL SECTION 2.1. Material and Sample Preparation. Compound 11mercaptoundecanol (MUD) was obtained from Aldrich and used as received. Atrazine (ATZ) was purchased as a pesticide reference material from Dr. Ehrenstorfer, GmbH (Augsburg, Germany). Atrazine disulfide (ATZSSATZ) having chemical formula C32H50O6N12Cl2S2 (Chart 1) was synthesized according to a previously published procedure.25 The antiatrazine polyclonal antibody (anti-ATZ IgG) was obtained by immunization of rabbits and the 16085

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Figure 1. Ex situ STM topography image of area A (size 134 × 134 nm2, set−point current 2 nA, bias voltage 195 mV) and area B (size 48.6 × 48.6 nm2, set-point current 0.38 nA, bias voltage 100 mV) of the ATZS monolayer on the Au(111) substrate. Histograms of the step heights and holedepth values along the cross-sections in image B (black line) are shown on the right. containing 3 mL of 0.1 M KCl and Ψ and Δ parameters were recorded. The strength of the interaction between IgG and the interfacial layer was checked subsequently by monitoring the ellipsometric optical parameters after flushing the cuvette again with 50 mL of 0.1 M KCl. 2.3. Infrared Measurements. The FTIR instrument used for polarization−modulation infrared reflection−absorption spectroscopy (PM IRRAS) was a commercial Nicolet Nexus spectrometer. The external beam was focused on the sample with a mirror, at an optimal incidence angle of 75°. A ZnSe grid polarizer and a ZnSe photoelastic modulator, modulating the incident beam between p- and spolarizations (HINDS Instruments, PEM 90, modulation frequency = 37 kHz), were placed before the sample. The light reflected at the sample was then focused on the nitrogen-cooled MCT detector. The sum and difference interferograms were processed and Fourier transformed to yield the differential reflectivity ΔR/R = (Rp − Rs)/ (Rp + Rs), which is the PM IRRAS signal. A total of 128 scans were recorded at 8 cm−1 resolution for each spectrum. PM IRRAS signal from a blank was obtained using the gold substrate rinsed with pure solvent and dried before the measurement. The sample was then immersed for a specified amount of time in the ATZSSATZ solution, rinsed with the solvent, dried, and used for the PM IRRAS measurement. 2.4. Scanning Tunneling Microscopy Measurements. Scanning tunneling microscopy (STM) images of the gold surface topography and of the self-assembled monolayers were obtained in the air using a mechanically sharpened Pt/Ir (80:20%) tip and Nanoscope II Scanning Tunneling Microscope (Digital Instruments, Inc. Santa Barbara, U.S.). 2.5. XPS Measurements. The X-ray photoelectron spectra (XPS) were recorded using an ESCA Probe P spectrometer (Omicron Nanotechnology Ltd., Germany) with a monochromatic Al Kα X-ray source (hν = 1486.7 eV). The survey spectra were recorded with 50 eV pass energy at steps of 0.5 eV, and the high-resolution spectra were obtained by applying window pass energy of 20 and 0.1 eV energy steps. The analyzed circular spot size had diameter of 1.3 mm. The binding energy (BE) values were referenced to C 1s peak at 284.7 eV. Low energy electrons (1.2 eV) were used for sample charge compensation. The analysis was carried out under UHV conditions in the low 10−10 mbar range. The measured spectra were analyzed with CasaXPS program (Casa Software Ltd., Teignmouth, U.K.). The spectral lines were corrected using a Shirley-type of background, and their identification was based primarily on the database of the CasaXPS software with help of the additional spectral databases.26,27 2.6. Quartz Crystal Microbalance Measurements. Quartz Crystal Microbalance (QCM) technique is based on the measurements of the frequency changes Δf of an oscillating quartz crystal. Frequency change Δf is proportional to mass changes Δm at the gold

electrode surface of the quartz crystal according to the Sauerbrey equation, Δf = − Cf Δm

(2)

where Cf is the Sauerbrey constant equal to 56.6 Hz cm2 μg−1 for 5 MHz AT-cut quartz crystal. Quartz Crystal Microbalance (QCM) measurements were obtained using QCM200 controller (Stanford Research Systems, U.S.) employing one inch in diameter, AT-cut polished quartz crystals of nominal frequency 5 MHz in the arrangement quartz/chromium/gold. The sensing gold surface had the geometric area of 0.4 cm2 and the roughness factor of about 1.2. Before each experiment the sensing surface of QCM crystal was cleaned by piranha solution (1 part of 33% H2O2 and 3 parts of 98% H2SO4) for 5 min, washed with plenty of Milli-Q water and dried in a stream of argon. Use extreme caution when handling Piranha solution! It is a strong oxidizing agent and extremely corrosive. QCM crystal was immediately mounted into the holder and immersed in a beaker filled with 50 mL of stirred ethanol, which was placed in a water bath thermostatted at 25 ± 0.1 °C. After stabilization of the frequency, a known volume of a stock solution of 1 × 10−2 M ATZSSATZ in ethanol was added to the solvent, and the frequency was monitored. The experiment was repeated several times with increasing concentrations of ATZSSATZ. Selected atrazinefunctionalized QCM crystals were subsequently treated with 0.15% v/ v of donkey serum in PBS buffer of pH = 7 to block any nonspecific binding of proteins. These crystals were then mounted into the QCM holder and dipped into a mixture of 0.1 M KCl and ethanol (9:1 v/v ratio) thermostatted at 25 ± 0.1 °C. After stabilization of the frequency, a solution of anti-ATZ antibody (IgG fraction) was added (final concentration = 50 mg/L), and the frequency was monitored until it reached a plateau. Subsequently, a solution of atrazine was added (final concentration = 1 × 10−5 M), and the frequency was monitored again until stabilization.

3. RESULTS AND DISCUSSION 3.1. Immobilization of Atrazine on the Gold Substrate. Atrazine was immobilized on the gold substrate in one step by adsorption of ATZSSATZ molecules from ethanol or chloroform stock solutions in the absence of oxygen. Mixed monolayers were prepared also in one step using a mixture of ATZSSATZ and MUD at different ratios in the respective solvents. Atrazine-based (ATZS) monolayers are schematically depicted in Chart 1 with an atrazine moiety highlighted in gray. Several methods including ex situ STM, XPS, ellipsometry, and QCM were used for their characterization. 3.2. Ex Situ Characterization of Self-Assembled Monolayers and Film Formation Kinetics. Figure 1 16086

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Figure 2. PM IRRAS spectrum of gold substrate after 10 min deposition of ATZS monolayer from 1 × 10−3 M ATZSSATZ solution in chloroform. Graph on the right represents integrated peak area from 3200 to 2700 cm−1 as a function of the deposition time.

Table 1. Summary of the Atomic Concentrations (At%) of Carbon, Oxygen, Nitrogen and Chlorine Obtained from the XPS Spectra At% (C 1s) sample 1 sample 2 sample 3

At% (O 1s)

exp.

theor.

61.3 82.6 85.9

63.0 69.5 74.5

a

At% (N 1s)

exp.

theor.

15.3 15.7 14.1

11.1 10.5 10.0

a

At% (Cl 2p)

exp.

theor.

exp.

theor.a

18.3 1.7

22.2 17.1 13.3

5.1

3.7 2.9 2.2

b

a

b b

−3

On the basis of the solution composition in contact with gold substrate: sample 1, ethanolic solution of 1 × 10 M ATZSSATZ, deposition time 19 h; sample 2, ethanolic solution of 5 × 10−4 M ATZSSATZ and 5 × 10−4 M MUD, deposition time 22 h; sample 3, ethanolic solution of 2 × 10−4 M ATZSSATZ and 8 × 10−4 M MUD, deposition time 22 h. bDetection limit a

by approximately 45° with respect to the surface normal (see Figure S1 of the Supporting Information, SI). The kinetics of film formation was also studied ex situ by PM IRRAS technique. A representative spectrum for ATZS adsorbate on the gold substrate is shown in Figure 2 together with the integrated area of the bands assigned to the C−H stretching modes of the ATZSSATZ molecule between 2700 and 3200 cm−1. Data in Figure 2 also indicate that the deposition time of 2 h is sufficient to obtain maximum adsorbate coverage on the gold substrate. The peaks at 1739, 1662, 1620, and 1581 cm−1 have been assigned previously to ν(CO) of the ester moiety, to amide I band, two triazine ring stretching bands and to amide II band.25 XPS method was used for characterization of the samples prepared from solutions containing different mole fractions of ATZSSATZ and MUD in ethanol. MUD serves as a diluent for atrazine-containing species. In general, mixed monolayers allow control of the concentration of biological molecules (antigen or antibody), which has been shown to be essential in certain recognition applications.32 Assuming that each molecule of disulfide ATZSSATZ can give two ATZ-containing thiolates (ATZS) bound to the gold surface, a maximum of 100%, 60%, and 40% ATZS coverage (mole fraction in solution χ(ATZS)s = 1, 0.6, and 0.4) is expected for samples 1 to 3 within the ATZbased monolayers, the rest being MUD molecules. The XPS spectra of all three samples contained peaks for gold (Au 5d, Au 4f, Au 4d, Au 4p3/2, and Au 4p1/2) with approximately the same atomic concentration (35 ± 5 At%), which is typical of the selfassembled organic monolayers.33 The signal from sulfur atoms

shows the STM images of atrazine-based monolayer obtained from two different sample areas (images A and B). ATZS monolayer was prepared by incubation of the gold(111) substrate in 1 × 10−3 M solution of ATZSSATZ in ethanol during the deposition time of 17 h. Section analysis of the step heights leads to a value of 0.25 ± 0.05 nm, which corresponds to one gold atom thick steps. The pit depth analysis gives the value 0.20 ± 0.05 nm, which compares well with the thiolinduced gold substrate depressions observed within the selfassembled monolayers of organic thiols and dithiols on the gold(111) surface indicating the presence of a thiolate monolayer adsorption.28−30 STM images are consistent with the presence of a compact ATZS monolayer. The actual presence of SAM was confirmed by the ex situ ellipsometric determination of the ATZS film thickness. Ellipsometry was successfully used in the past for the determination of SAM thicknesses of various organic disulfides chemisorbed on the gold substrates.31 Two sets of samples were prepared for the ellipsometry measurement using 1 × 10−3 M solution of ATZSSATZ in ethanol and chloroform. The deposition time varied from 2 to 19 h. Taking the refractive index of ATZS film on the gold substrate as nATZS = 1.45, the film thickness was calculated to be 1.26 ± 0.11 nm for deposition from ethanol and 1.25 ± 0.01 nm for deposition from chloroform. These values are smaller than the maximum theoretical thickness of the ATZS monolayer equal to 1.81 nm and corresponding to the length of the molecular structure as obtained by SPARTAN’08 software package (Wave function, Inc., U.S.). Obtained thicknesses indicate that ATZS molecules are tilted 16087

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Table 2. Binding Energies BE (eV), Relative Peak Areas (% area) of the Individual Components of the C 1s Spectra and Calculated Mole Fraction χ(ATZS) of ATZS in the Monolayer (C−H, C−C)

a

(C−N,C−O)

(CN, CO)

χ(ATZS)

C1s

BE (eV)

% areaa

BE (eV)

% areaa

BE (eV)

% areaa

calc.

sample 1 sample 2 sample 3

284.7 284.7 284.6

46.9 (47.1) 74.9 (75.0) 71.9 (79.4)

286.0 285.9 285.9

26.4 (23.5) 15.5 (14.3) 22.4 (12.9)

288.2 288.2 288.1

26.7 (29.4) 9.5 (10.7) 5.7 (7.5)

1.0 0.27 0.19

In parentheses theoretical % area corresponding to χ(ATZS).

Figure 3. Optical parameters Δ and Ψ as a function of time for adsorption of 1.04 × 10−3 M solution of ATZSSATZ in ethanol in contact with gold(111) substrate at t = 25 °C (A). Surface coverage θ as a function of time for both ellipsometric parameters (B).

3.3. In Situ Characterization of Self-Assembled Monolayers and Film Formation Kinetics. Ellipsometry was used for determination of the progress of ATZSSATZ adsorption at the gold/ethanol interface. Figure 3A shows the change in optical parameters Δ and Ψ as a function of time during the adsorption of ATZSSATZ on the gold(111) substrate from 1.04 × 10−3 M stirred solution in ethanol at t = 25 °C. It is assumed that each ATZSSATZ molecule gives two chemisorbed ATZS moieties on the gold surface. The process may involve essentially two steps: adsorption of disulfide followed by the gold-thiolate bond formation and rearrangement. Details of a two-step process are not discernible from the ellipsometric data in Figure 3. The progress of adsorption shows single exponential decay for both optical parameters, which were transformed into the fractional surface coverage θ values,

was below the detection limit for the experimental conditions used. Therefore, we focused on the experimentally obtained atomic concentrations of carbon (C 1s), oxygen (O 1s), nitrogen (N 1s), and chlorine (Cl 2p) atoms, which are for all three samples summarized in Table 1 together with theoretical values expected for the SAM composition based on the solution mole fraction values. A very good agreement between theoretical and experimental atomic composition is achieved for sample 1 (ATZS monolayer). In the case of samples 2 and 3, it is obvious that the monolayer contains more MUD and less ATZS than expected from the solution composition used for the SAM preparation. Since ATZS chemical structure contains C 1s atoms corresponding to CO and CN moieties that are not present in MUD molecule, the highresolution C 1s spectra convey information on the mole fraction of ATZS within the mixed SAMs. Figure S2 of the SI shows the high resolution C 1s spectra of all three samples together with the best fit of the spectrum to three individual components, which are assigned to the sum of C−C and C−H carbon atoms (peak centered at 284.7 eV), the sum of the C−N and C−O carbons (peak at 285.9 eV) and the sum of the C O and CN atoms (peak at 288.2 eV), respectively. Table 2 lists the relative peak areas for each component of the C 1s spectrum obtained experimentally. Last column gives the mole fraction of ATZS within the SAM that best fits the data for all three samples. During the fitting procedure, attention was focused on the peak centered at 288.2 eV, as it reflects solely the presence of ATZS molecules. We observed that a one step procedure used for the preparation of mixed monolayers leads to lower mole fraction of ATZS in a monolayer containing MUD. Values of χ(ATZS) obtained from XPS are 0.27 and 0.19, which are much smaller than those based on the solution composition χ(ATZS)s. This finding is also supported by the IRRAS characterization of the samples (Figure S3 of the SI).

θ=

Δ0 − Δ Ψ −Ψ = 0 Δ0 − Δ∞ Ψ0 − Ψ∞

(3)

where Δ0 and Ψ0 are the initial optical parameter values at time equal to zero, and Δ∞ and Ψ∞ are the final optical parameters corresponding to θ equal to 1. Figure 3B represents time dependence of thus obtained fractional surface coverage θ for both parameters in one graph. Adsorption kinetics parameters were obtained by fitting θ values in Figure 3B to kinetic equations derived for Langmuir adsorption isotherm model, which assumes that adsorption is limited to one monolayer and all surface sites are mutually independent and equivalent.34−36 The rate of the monolayer formation can be expressed as the following: dθ = ka(1 − θ )C − kdθ dt

(4)

where C is the concentration of adsorbate, ka and kd are the adsorption and desorption constants, respectively. Time 16088

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Table 3. Kinetic Parameters and Mass Uptake from QCM Analysis C (mol L−1) 1.8 1 8.7 5 1

× × × × ×

−4

10 10−4 10−5 10−5 10−5

kobs (s−1) 0.0293 0.0193 0.0192 0.022 0.018

± ± ± ± ±

0.0007 0.0003 0.0007 0.001 0.001

Δm (ng cm−2)

−Δ feq (Hz) 3.1 2.4 2.0 1.5 0.7

± ± ± ± ±

0.3 0.2 0.2 0.3 0.2

55 42 35 27 12

± ± ± ± ±

5 4 4 5 4

ΓATZS (molecules cm−2) (7.7 (5.9 (4.9 (3.8 (1.7

± ± ± ± ±

0.7) 0.6) 0.6) 0.7) 0.6)

× × × × ×

1013 1013 1013 1013 1013

Figure 4. Response (ellipsometric parameters Δ and Ψ) of ATZS-coated gold sensor toward rabbit anti-ATZ IgG. Addition of anti-ATZ IgG (6 μL, final concentration 50 mg/L) to 3 mL of 0.1 M KCl (a) and subsequent flushing with 50 mL of 0.1 M KCl (b).

dependence of the fractional surface coverage θ is given by the following: θ (t ) =

Δf (t ) =

= Δfeq [1 − exp(−kobst )]

C [1 − exp(− (kaC + kd)t )] = θ(∞) C + k d / ka

[1 − exp(−kobst )]

C Δf [1 − exp( −(kaC + kd)t )] C + kd /ka max (6)

where Δf max represents maximum change of frequency that can be achieved to form a fully compact self-assembled monolayer or the binding capacity of the QCM crystal, Δfeq is the steadystate or equilibrium frequency. All other parameters are the same as in eq 5. The experimental parameters kobs and Δfeq obtained by fitting data in Figure S4 of the SI to eq 6 are summarized in Table 3 together with corresponding mass changes Δm of the crystal according to eq 2. The observed rate constants can be plotted against the ATZSSATZ concentration C (see Figure S5 of the SI) to obtain ka, kd, and Keq values of the adsorption process. Assuming that fast kinetic response of the QCM crystal reflects the adsorption of disulfides rather than the formation of chemisorbed thiolate species the following values were obtained ka = 59 ± 20 M−1 s−1, kd = (17 ± 2) × 10−3 s−1, and Keq = 3412 ± 1243, respectively. The steady-state surface concentration of ATZS species forming ATZ-based SAMs on the gold substrate was calculated from Δm values and is given in the last column of Table 3. It corresponds to ΓATZS = 2 ΓATZSSATZ, i.e., twice the surface concentration of the original disulfide. A reasonable estimate of the maximum surface concentration of ATZS in SAM is (9 ± 1) × 1013 molecules cm−2 assuming that ATZS molecules are tilted by approximately 45° with respect to the surface normal. In such a case, the steady-state fractional surface coverage θ(∞) is 0.86 ± 0.12 at bulk ATZSSATZ concentration 1.8 × 10−4 M and approaches unity for higher concentrations (for example see Figure 1).

(5)

where kobs = ka C + kd and θ(∞) = C/(C + kd/ka) = C/(C + 1/ Keq). The θ(∞) value expresses the steady−state fractional surface coverage, whereas Keq represents the equilibrium constant for the film formation process. The θ(∞) value approaches unity for strongly adsorbing species (high Keq) and at high adsorbate concentrations. Best fit of the experimental surface coverage θ(t) to eq 5 yields the observed rate constant kobs = (1.84 ± 0.04) × 10−3 s−1 at ATZSSATZ concentration 1.04 × 10−3 M. Kinetics of the SAM formation was also followed by QCM technique, where the frequency of the oscillating quartz crystal was monitored as a function of time. Frequency change Δf was converted to mass change Δm at the gold electrode surface of the quartz crystal according to Sauerbrey equation (see eq 2), which is strictly valid only for uniform, thin, and rigid films.37,38 Our system conforms to these properties (see Figure 1). The frequency shifts of QCM crystals, dipped into stirred ethanol solution and recorded after addition of various amounts of ATZSSATZ at 25 °C as a function of time, are shown in Figure S4 of the SI. As expected, modest but significant changes of the frequency were measured resulting from the adsorption of ATZSSATZ. The adsorption kinetic curves were fitted to kinetic eq 6, which is the same as eq 5, following a simple substitution for θ(t) = Δf/Δf max and θ(∞) = Δfeq /Δf max, 16089

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The existence of multiple time-scales for SAM formation has been confirmed by many authors.39−44 For alkanethiol SAMs Bain et al. described a two-step process, where the film thickness reached almost 90% of its final value within the first minute, whereas the final film properties were reached only after several hours.40 Han and Uosaki reported these two steps to be diffusion-limited physisorption followed by a chemisorption process.41 Our results do not grant the use of two rate constants for SAM formation within one experimental QCM run, but the existence of two different kinetic processes is possible to discern by selection of the experimental method. For example, our in situ ellipsometric data provide kobs values, which are 1 order of magnitude slower than the kobs values obtained from QCM measurements. This is undoubtedly due to the fact that mass change parameter Δm does not distinguish between one physisorbed ATZSSATZ disulfide molecule and two chemisorbed ATZS thiolate species. 3.4. Interaction of Antiatrazine Antibody with Atrazine-Based SAM. Interaction of atrazine-based SAM with antiatrazine antibody was monitored by both ellipsometry and QCM measurements. QCM technique is well suited for the host−guest complex formation45 and antigen−antibody association46,47 studies. For example, QCM technique confirmed suitability of alkanethiol SAMs for immobilization of biomolecules.48 Figure 4 shows the ellipsometric parameters Δ and Ψ of the atrazine-based sensing surface ATZS-monolayer (see Chart 1) in contact with 3 mL of 0.1 M aqueous KCl solution. At time t = 0 s (indicated by arrow a in Figure 4), the atrazine-specific IgG was added to the cuvette and changes in Δ and Ψ were monitored. The interaction between atrazine moiety and atrazine-specific IgG leads to a decrease of Δ and small increase of Ψ parameter. Steady-state is achieved within several hundreds of seconds and signal does not change after the replacement of anti-ATZ IgG solution by pure KCl solution and monitoring of the signal (indicated by arrow b in Figure 4). At this stage, all steps of the immunosensing device as shown in Scheme 1 have been followed by the QCM technique. At first, selected atrazine-functionalized QCM crystals were treated with 0.15% v/v of donkey serum in PBS buffer of pH = 7 to block any non-specific binding of anti-ATZ IgG. After rinsing with buffer, the sensor was immersed in 0.1 M KCl solution (1:9 v/v ratio of ethanol/water) and thermostatted at 25 ± 0.1 °C. Figure 5 shows changes of the frequency response Δf of the gold QCM sensor upon addition of anti-ATZ antibody (IgG fraction) to the solution indicated by arrow a. After stabilization of the signal, frequency change of Δf = −9.6 Hz was observed. Assuming that each molecule of IgG occupies an area of 1.8 × 10−12 cm2, such a decrease in frequency corresponds effectively to a complete coverage of the available surface by antibody molecules. At this point (indicated by arrow b in Figure 5), the solution was spiked with atrazine (final concentration 1 × 10−5 M ATZ). QCM crystal responded by a positive frequency shift of Δf = +4.8 Hz, which means that approximately half of the antibody molecules were displaced from the sensing surface by ATZ present in solution. Thus, we demonstrated that the functional material was able to recognize and bind specific antibodies, and this binding was reversed by addition of atrazine pesticide into the solution as depicted in Scheme 1.

Figure 5. QCM frequency change Δf of the ATZS-based SAM on the gold substrate in contact with 0.1 M KCl ethanol/water solution (1:9 v/v ratio) after addition of atrazine-specific IgG (final concentration 50 mg/L, arrow a), followed by an addition of ATZ (final concentration 1 × 10−5 M, arrow b) at constant temperature of 25 ± 0.1 °C.

were characterized by ellipsometry, scanning tunneling microscopy, PM IRRAS, XPS, and QCM techniques. Two different time scales for the ATZS-based SAM formation were observed depending on the experimental method used. QCM analysis provided kobs values at least 1 order of magnitude faster than in situ ellipsometry and ex situ PM IRRAS measurements. This observation most likely reflects the fact that QCM response is directly related to the adsorption (physisorption) of the original disulfide ATZSSATZ species, whereas slower kinetics relates to the formation of thiolates (chemisorption). Ellipsometry reflects this slower process. Finally, we have demonstrated that such ATZ-based monolayers are suitable for indirect immunoassay detection of pesticide atrazine in the aqueous samples. For practical analytical purposes, our study recommends millimolar concentrations of ATZSSATZ and a deposition time of 2 h as sufficient for the functional ATZbased SAM formation.



ASSOCIATED CONTENT

S Supporting Information *

Orientation of the ATZS molecules on the gold substrate; XPS of mixed SAMs; infrared reflection−absorption spectroscopy of mixed SAMs; QCM study of the SAM formation kinetics and concentration dependence of the experimental kobs values. This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*Tel.: +420 266 053 197; fax: +420 286 582 307; e-mail: [email protected]−inst.cas.cz. *Tel.: +33 1 43 26 00 61; e-mail: [email protected] Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Grant Agency of the Czech Republic (GACR 13-19213S), the Academy of Sciences of the Czech Republic (M200401202), and the Czech-French exchange program Barrande. The Centre National de la

4. CONCLUSIONS We studied the self-assembly process of atrazine derivative ATZSSATZ on the gold substrate. Self-assembled monolayers 16090

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Recherche Scientifique and the French Ministry of Research are gratefully acknowledged. The authors would like to express thanks to Dr. P. Dubot and RNDr. Petr Sajdl, CSc. for their help with PM IRRAS and XPS measurements. Finally, we would like to thank A. Miszta for performing time-dependent ellipsometric experiments.



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Atrazine-based self-assembled monolayers and their interaction with anti-atrazine antibody: building of an immunosensor.

As a part of our objective to build an immunosensor for the detection of the pesticide atrazine (ATZ) in environmental samples, we studied the self-as...
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