Sensitive Localized Surface Plasmon Resonance Multiplexing Protocols

Aug 15, 2012 - Sensitive Localized Surface Plasmon Resonance Multiplexing. Protocols. Kun Jia, Jean L. Bijeon, Pierre M. Adam, and Rodica E. Ionescu*...
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Sensitive Localized Surface Plasmon Resonance Multiplexing Protocols Kun Jia, Jean L. Bijeon, Pierre M. Adam, and Rodica E. Ionescu* Laboratoire de Nanotechnologie et d’Instrumentation Optique, Institute Charles Delaunay, Université de Technologie de Troyes, UMR-STMR CNRS 6279, 12 Rue Marie-Curie BP 2060, 10010 Troyes Cedex, France ABSTRACT: Herein are reported two new protocols to obtain different zones of localized surface plasmon resonance (LSPR) gold nanostructures on single glass substrate by using a vacuum evaporation technique followed by a high-temperature annealing (550 °C). The thickness of the gold film, considered as the essential parameter to determine specific LSPR properties, is successfully modulated. In the first protocol, a metal mask is integrated onto the glass substrate during vacuum evaporation to vary the gold film thickness by a “shadowing effect”, while in the second protocol several evaporation cycles (up to four cycles) at predefined areas onto the single substrate are performed. The resulting gold-modified samples are characterized using a transmission UV−vis extinction optical setup and scanning electron microscopy (SEM). The size distribution histograms of nanoparticles are also acquired. By employing the first protocol, thanks to the presence of different zones of gold nanoparticles on a single substrate, optimized LSPR responses to different (bio)functionalization zones are rapidly screened. Independently, the second protocol exhibited an excellent correlation between the nominative evaporated gold film thickness, gold nanoparticle sizes, and plasmonic properties (resonant wavelength and peak amplitude). Such substrates are further used in the construction of LSPR immunosensors for the detection of atrazine herbicide.

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of plasmonic properties, potential ability for miniaturization, and simple experimental setup, without the need of a prism to couple plasmons.21 LSPR technology has also been shown to be less sensitive to temperature fluctuations. The optical responses of LSPR biosensors are highly dependent on the local electromagnetic field distribution in the surroundings of nanoparticles. Considering the exponential decay of an evanescent plasmon field away from a nanoparticle surface, the distance-dependent optical response of the LSPR biosensor upon the formation of a functional layer at the metal surface can be described according to Jung et al.:22

ue to their optical properties, noble metal nanoparticles such as gold and silver have gained increasing research interests both in academic and industrial fields during recent years.1−5 The main optical property is the localized surface plasmonic resonance (LSPR), which occurs when the incident electromagnetic wave (light) is resonant with the intrinsic oscillation of free electrons of metal nanoparticles.6 As consequences, strong optic absorption with extremely large molar extinction coefficients,7 prominent Rayleigh scattering,8 and enhanced localized electromagnetic fields near the nanoparticles surface have been developed.9,10 The LSPR properties of metal nanoparticles, including resonant wavelength, peak amplitude, and shape of spectra, are determined by various parameters, such as nanoparticle composition, size, shape, and interparticle spacing.11 Due to the advancement of highly precise and controllable methodologies of synthesis like physical “top-down” protocols12−15 or chemical “bottom up” recipes,16−19 noble metal nanoparticles with diverse morphologies have been prepared with the purpose of tuning their LSPR properties from visible to near-infrared range. Beside the morphology of metal nanoparticles, the local dielectric environment also plays an important role in determining the plasmonic properties. The increasing refractive index of the surrounding medium results to a red-shift of resonant wavelength in the extinction measurement of metal nanoparticles, which is explored as the sensing mechanism of LSPR biosensors using metal nanoparticles.20 When compared to the already commercialized surface plasmon resonance (SPR) biosensor based on propagating plasmons, the recently emerging LSPR biosensor has some intrinsic advantages such as large tunability © 2012 American Chemical Society

R = m(ηa − ηs)[1 − exp( −2d /Ld)]

(1)

where R is the plasmonic response (either the extinction wavelength shift or the peak amplitude changes), m is the refractive index sensitivity of nanoparticle, i.e., peak shift per refractive index unit (RIU), ηa and ηs are the refractive indexes of the adsorbed layer and the bulk medium, respectively, d is the thickness of the binding layer, and Ld is the decay length of the evanescent field, which is a characteristic parameter depending on the chemical compositions and physical dimensions of metal nanoparticles. From eq 1, it is reasonably expected that the plasmonic response of metal nanoparticles is decreased exponentially far away from the nanoparticle surface. Because an additional biorecognition layer is preimmobilized Received: July 1, 2012 Accepted: August 15, 2012 Published: August 15, 2012 8020

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liquid) was purchased from antibodies-online GmbH (Aachen, Germany), whereas monoclonal mouse anti-IgG human antibody was obtained from Sigma (Lyon, France). The PBS buffer was used to prepare antibody and antigen dilutions through all the experiments. All the chemicals are of analytical grade and used as received without further treatment. Preparation of Different Zones of Gold Nanoparticles on Single Glass Substrate. Classical microscope glass slides (Carl Roth GmbH & Co.KG, Germany) were cut to ∼25 × 8 mm2 and subsequently used as the substrates for gold film deposition step(s). Prior to the evaporation, all the glass substrates were washed with a mixture of detergent (Decon 90) and deionized water (2:8, v/v ratio) in an ultrasonic water bath (Elmasonic S30H) at 50 °C for 15 min. Further, the resulted samples were rinsed with excess amount of deionized water, drying under a N2 stream, and subjected to another ultrasonication washing in deionized water at 50 °C for 5 min. Finally, the glass substrates were rinsed three times with deionized water, dried in an oven at 100 °C for 10 min, and ready for the gold evaporation. The gold evaporation was conducted in an evaporator (MEB 400, PLASSYS, France) using the electron beam evaporation mode at ambient temperature under a high vacuum (pressure was around 1.0 × 10−6 Torr). The evaporating rate was adjusted to ∼0.01 nm/s by slowly changing the working current intensity. In order to obtain homogeneous layers of gold film on glass surfaces, the sample plate was slightly rotated during the evaporation process. The evaporated film thickness was monitored by a built-in quartz crystal sensor. Two different masks were used during the evaporation: in the first protocol, staples were fixed on top of glass substrate to create gold−glass interfacial areas where the gold evaporation process was modulated because of the “shadowing effects” of staples. In the second protocol, the gold evaporation cycle was repeated different times at different predefined areas onto the glass slide (scotch tape mask was used to separate different areas). After evaporation, the modified glass samples were transferred in a high-temperature oven (Nabertherm, Germany) to conduct the thermal annealing in the presence of oxygen at 550 °C for 8 h. Morphology Characterization and Spectroscopy of Gold Nanostructures. The morphology of annealed gold nanoparticles was characterized with field emission scanning electron microscopy (Raith, SEM FEG-eLine) using an accelerating voltage of 10 kV and a working distance of 10 mm. For SEM characterization, the samples were covered with an ultrathin layer (around 3 nm) of palladium by sputtercoating to suppress the charging effects. The annealed sample was also imaged by biological optical microscopy (DMS Didalab) using a 10× objective lens (SP10/0.25, 160/0.7) in transmission mode. For the LSPR measurements, the experimental configuration was constructed based on transmission UV−vis−NIR spectroscopy, which contains a white light source (DH-2000-BAC, Ocean Optics) and two optic fibers (one for illumination and another for the collection). A portable photospectrometer (QE65000, Ocean Optics) with wavelengths ranging from 200 to 1100 nm, resolution of 1 nm, and an integration time of 100 ms was used. Biofunctionalization of Gold Nanostructures. The annealed samples were washed with ethanol/acetone mixture (volume ratio of 1:1) in an ultrasonic bath at room temperature for 30 min and dried under a N2 stream, followed by immersion in 12MDA ethanolic solution (1 mM in 15 mL) for 12 h at room temperature. The gold nanoparticles (NPs) modified

on the nanoparticle surface to specifically detect the targeted analyte in a typical LSPR biosensor configuration, eq 1 should be adapted in the biosensing applications as follows:22 R = m(ηa − ηs)exp( −2d i /Ld)[1 − exp(− 2da /Ld)]

(2)

where di and da are the thicknesses of the recognition interface layer and adsorbed layer, respectively, assuming both layers have the same refractive index ηa. According to eq 2, in order to obtain the best sensitivity of an LSPR biosensor, attention should be dedicated (i) to increase the refractive sensitivity (m) of nanoparticles and (ii) to optimize the balance between decay length (Ld) and the biorecognition interface layer thickness (di and da). Therefore, by using nanoparticles of different shapes, sizes, and compositions it is possible to maximize the refractive index sensitivity (m). If the recognition biomolecules are located outside the sensing range of nanoparticles (di > Ld), the following binding events could not be detected anymore; on the other hand, if the decay length is much larger compared to size of the biomolecules (Ld≫ di), the biorecognition events would occupy a limited fraction of the sensing range of the nanoparticles. In both cases a poor sensitivity of the LSPR biosensor will be observed.23,24 In order to study the distance-dependent plasmonic properties of nanoparticles, various experiments have been conducted to obtain a controlled layer with known thickness and refractive index on the metal nanoparticle surface, by using different methods like self-assembled monolayer (SAM) of alkanethiol,25−27 electrostatic layer-by-layer (LBL) assembly of polyelectrolyte multilayers,28,29 and atomic layer deposition.30 By monitoring the LSPR response of nanoparticle versus different outer-layer thicknesses, the decay length of metal nanoparticles was observed to be of tens of nanometers (up to 40 nm) depending on the nanoparticles and assembling method used. Interestingly, contrary to these classical short decay lengths (up to 40 nm), some research groups also reported the interesting “long-range” plasmonic response, in which the sensing distance of these metal nanostructures could be extended to a few hundreds of nanometers.31−35 The present article is focused on the preparation of various LSPR structures on single glass substrate via modulation of the evaporated film thickness through two protocols. By monitoring the plasmonic response after each biomodification step to different zones of gold nanoparticles, the most sensitive plasmonic nanostructure was easily selected. Thus, a model plasmonic nanosensor was constructed to specifically detect atrazine in trace concentration of 50 pg/mL. The two designed protocols to simultaneously fabricate different zones of gold nanoparticles on single glass substrate open new possibilities to construct high-throughput and multiplexing LSPR biosensor.

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EXPERIMENTAL SECTION

Materials. 12-Mercaptododecanoic acid (12MDA), bovine serum albumin (BSA), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were acquired from Sigma-Aldrich (Schnelldorf, Germany). Atrazine, ethanol, and acetone were obtained from Fluka (Lyon, France). Phosphate-buffered saline (PBS, 10×, pH 7.4, 100 mL) was prepared in our lab using sodium chloride, sodium phosphate dibasic, and sodium phosphate monobasic received from Sigma (St. Quentin Fallavier, France) and deionized water (18.2 MΩ·cm) produced by a Millipore Milli-Q water purification system (Molsheim, France). Monoclonal mouse anti-IgG atrazine antibody (ABIN234335, purified 8021

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Figure 1. Photographs of samples obtained from the first protocol (a) and second protocol (b). First protocol: simultaneous fabrication of a controlled thickness gradient of gold nanoparticles on single glass substrate by using metal staples to produce an artificial glass substrate/ gold interface. Second protocol: four cycles of gold evaporation (2 nm film for each cycle) leading to four zones of 2, 4, 6, and 8 nm thickness. The area with the wanted thickness is protected by scotch tape.

with 12MDA were extensively rinsed with ethanol to remove the excess thiol and dried with N2. Further, the terminal carboxyl groups of the thiolated surface were activated with mixed EDC/NHS (0.4 mM/0.1 mM) aqueous solution at room temperature for 50 min. Next, monoclonal antibodies (anti-IgG atrazine or anti-IgG human) diluted in PBS buffer (100 μL of 100 μg/mL) were incubated with the activated gold nanostructures at 4 °C for 12 h. After PBS rinsing, a BSA solution (0.1 mg/mL) was used to block the nonspecific binding sites of antibody-modified NPs. Finally, various atrazine dilutions with concentrations ranging from 50 pg/mL to 5 ng/mL diluted in PBS were incubated with antibody-modified nanoparticles for 3 h at 4 °C to complete the antibody−antigen immunoreactions. The LSPR responses were recorded after each (bio)functionalization step. It should be noted that, for steps involving PBS buffer, a rinsing step with ddH2O before spectroscopy measurements was necessary, otherwise the salt crystals from buffer were deposited on the substrate surface, which could produce interferences in the LSPR measurements.

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RESULTS AND DISCUSSION Independent Preparation of Various Zones of Gold Nanoparticles on Single Glass Substrate: Two Protocols. Among the tremendous methods to fabricate gold nanoparticles on solid substrate, the vacuum evaporation followed by thermal annealing of thin gold film on appropriate substrate was considered as a promising option, mainly due to its cost-effectiveness

Figure 2. LSPR spectra of (a) different zones of NPs in the glass−gold transitions (1−4) by using protocol 1 and their corresponding SEM morphology (b).

and large plasmonic tunability.36 It has been reported that the nominative thickness of evaporated film plays a decisive role in 8022

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Figure 3. Gold nanoparticles size distribution histograms for different zones within the glass−gold interfacial area, when protocol 1 is used.

gold evaporation (2 nm film for each cycle). The scotch tape was used as a mask to prevent certain glass areas from gold deposition. Consequently, “barcode-like” substrates containing four different nominative thicknesses (2, 4, 6, and 8 nm) of gold film were prepared. Thus, different nanoparticle sizes and shapes (i.e., different colors) on a single substrate were developed after high-temperature annealing step at 550 °C (Figure 1b). Spectroscopy and SEM Morphology Characterization of Different Gold Nanoparticles on Single Substrate. For the samples prepared with the first protocol, the authors have focused on the gold−glass interfacial area. In order to better understand the plasmonic spectra and SEM morphology, different gold nanoparticles in the vicinity of the gold−glass interfacial area were numbered from 1 to 4, corresponding to the position moving from the glass side to gold side, as indicated with a black arrow in Figure 2a. The LSPR spectra for the four different zones were recorded, while the SEM images corresponding to different zones of gold nanoparticles are exhibited in Figure 2b. Experimentally, when moving from area 1 to area 4 within the interfacial zones, the maximum extinction wavelength red-shifted from 568 nm for curve 1 to 677 nm for curve 4, with a concomitant increasing of peak amplitude from 0.2 to 0.475, respectively. From the size distribution histograms corresponding to different areas shown in Figure 3, it is obvious that the average size of gold nanoparticle was increasing from 15 nm for area 1 to 45 nm for area 4. Thus, the red-shift of resonant extinction wavelength and increasing of optical density recorded in Figure 2a were mainly attributed to the increasing of nanoparticle size. For the sample obtained with the second protocol, the measured LSPR spectra for four independent zones were correlated with their initial evaporated film thicknesses (Figure 4a). It should be noted that, in the beginning, as the nominative gold film is increasing, the peak amplitude is obviously increased while the resonant wavelength is slightly shifted. However, the wavelength shift becomes dominant as further increasing of film thickness. For example, when the nominative evaporated film thickness increased from 2 to 4 nm, the maximum optical density was increased from 0.23 to 0.45

the final morphology of gold nanoparticles after annealing, which in turn determines their plasmonic properties.37 Most of published works have evaporated one fixed thickness of gold film on single substrate; thus, a specific plasmon resonance has been developed for each annealed sample. In the present work, the authors have fabricated different zones of nanoparticles on single glass substrate, which has remarkable advantages in the multiplexing LSPR nanobiosensor applications. Given the importance of evaporated film thickness as mentioned above, the two proposed protocols both focus on the modulation of evaporated gold film thickness in a controlled manner before annealing. Specifically, the first protocol is based on the simultaneous fabrication of different zones of gold nanoparticles from a single evaporation step, whereas the second one is based on a multicycles gold evaporation procedure. A single glass substrate was used in both protocols. Protocol 1: Simultaneous Fabrication of Different Zones of Gold Nanoparticles on Single Glass Substrate. In this protocol, metal staples were fixed on the top of a glass substrate, leaving a gap between the mask (staples) and the substrate (glass). Due to the “shadowing effects” of used staples, an artificial gold−glass interfacial area was created, where the thickness of evaporated gold film in transversal directions was gradually modulated, leading to different gold nanoparticles after high-temperature annealing (550 °C). Indeed, as shown in Figure 1a, two transition zones located exactly at the gold−glass interface (e.g., area underneath the staples) were observed for the annealed sample. When these special areas were projected using a conventional optical microscope, a multicolor transition zone was recorded, and the color changed from violet (glass side) to blue (gold nanoparticle side). According to a systematic study of thermal annealed gold nanostructures, the sample with varying evaporated film thickness exhibited different colors after annealing. Thus, a violet color corresponded to thinner evaporated film, whereas the blue color was attributed to the thicker film, which corroborates our assumption of “shadowing effect” of staples mask. Protocol 2: Several Gold Evaporation Cycles Induced the Appearance of Different Zones of Nanoparticles on Single Glass Substrate. The second protocol is based on four cycles of 8023

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while the resonant wavelength only slightly red-shifted from 561 to 566 nm. However, when the evaporated film thickness further increased from 4 to 6 nm, a relative large red-shift of resonant wavelength (25 nm) was observed, while the maximum optical density was only increased 0.08. Finally, when the film thickness further increased to 8 nm, a large wavelength red-shift to 742 nm was observed, and a much wider peak with maximum optical density declined to 0.4 was recorded. Extinction of smaller particles is dominated by absorption, and thus there is a small size-induced red-shift. For bigger particles when extinction is dominated by scattering a strong red-shift is observed. From the corresponding SEM images shown in Figure 4b and the size distribution histogram in Figure 5, as the nominative evaporated film thickness increased, the interparticle distance was increased, which can imply a blue-shift of the resonant peak; however, if the interparticle gaps are larger than particle diameters this effect will be negligible. Size increasing becomes the dominant factor for the thicker film. It should be noted that the size distribution for the 8 nm film sample (Figure 5) was less uniform than that of samples with lower film thicknesses, which is partly responsible for the observed spectra broadening effects in this case. Additionally, the broadening effect is attributed to radiation damping induced by the nanoparticle’s size increases. Moreover, by comparing the spectroscopic data with the nanoparticles morphology obtained from the two protocols, the plasmonic peaks for the sample of the first protocol were generally wider than those recorded for the sample of the second protocol. Indeed, a narrower size distribution was observed for the sample of the second protocol (see Figures 3 and 5), which explains the sharper peaks observed with protocol 2. More homogeneous nanoparticles lead to a sharper plasmonic extinction peak.38 In short, it is clear that the plasmonic properties (resonant wavelength, peak width, and amplitude) are determined by the morphological features of gold nanoparticles, which can be readily modulated by using different nominative evaporated film thicknesses. As a brief summary, a correlation between initial film thickness and plasmonic properties is plotted in Figure 6 and analyzed in Table 1. The most intense plasmonic peaks are obtained for particles with an average size of 57.5 nm, which is in agreement with Mie theory calculations.39 LSPR Responses after (Bio)functionalization of Gold Nanoparticles. As discussed in the introduction, the sensitivity optimization of an LSPR nanosensor is quite complicated due to various parameters that have their individual influences through plasmonic properties. However, by using our multiplex LSPR structures on single substrate, one can easily select the optimized LSPR structure by monitoring the plasmonic responses upon simultaneous modification of different gold nanoparticles. As an example, the plasmonic spectra corresponding to various successive biomodification steps for a sample of protocol 1 are shown in Figure 7. It is noticed that after the thiol functionalization step, the resonant wavelength was slightly red-shifted. Further, when the larger biomolecules (anti-IgG human antibody) were immobilized in these interfacial areas, more obvious wavelength shifts were observed. Specifically, after binding of 100 μg/mL of anti-IgG human antibody on gold nanoparticles, 25 and 15 nm red-shifts of resonant wavelength were measured for curves 4 and 3, respectively. However, only 6 nm of shift was observed for curve 2 and no obvious plasmonic responses were recorded for

Figure 4. LSPR spectra (a) and corresponding SEM morphology of gold nanoparticles (b) for 550 °C annealed samples using protocol 2 with nominative evaporated film thickness of 2 nm (b1), 4 nm (b2), 6 nm (b3), and 8 nm (b4). The numbers 1, 2, 3, and 4 are corresponding to the different evaporated thicknesses of 2, 4, 6, and 8 nm before high-temperature annealing. 8024

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Figure 5. Gold nanoparticle size distribution histograms for annealed samples (550 °C, 8 h) with different initial film thicknesses of 2 nm (zone 1), 4 nm (zone 2), 6 nm (zone 3), and 8 nm (zone 4), when protocol 2 is used.

Figure 6. Correlation between evaporated film thickness before annealing and its plasmonic features of resonant wavelength and maximum extinction after annealing at 550 °C for 8 h.

Figure 7. LSPR responses for different areas upon simultaneous biomodification of gold nanoparticles within the gold−glass interfacial zone (12MDA: 12-mercaptododecanoic acid).

Table 1. Physical Dimensions of Gold Nanoparticles and Corresponding LSPR Features for Samples with Different Initial Evaporated Film Thicknesses from 1 to 8 nma physical dimensions nominative evaporated film thickness [nm] 1 2 3 4 5 6 7 8 a

average size [nm] 8.2 10.2 17.5 21.6 42.8 57.5 75.4 110.4

± ± ± ± ± ± ± ±

2.4 2.8 3.9 5.4 8.3 10.8 15.3 23.7

aspect ratio

resonant wavelength [nm]

max extinction

± ± ± ± ± ± ± ±

544.65 555.15 560.82 566.34 570.11 594.14 618.76 722.53

0.108 0.229 0.339 0.447 0.509 0.547 0.509 0.408

1.12 1.14 1.08 1.13 1.06 1.11 1.23 1.35

It has been reported that the decay length of metal nanoparticles is increased with the raising of metal nanoparticle size,40 and as discussed previously, the match between decay length of a nanoparticle and physical size of a biomolecule was important to obtain better plasmonic sensitivity in LSPR biosensor.28 Moreover, according to the work of Nath and Chilkoti,41 the sensitivity of the LSPR nanosensor using larger gold nanoparticles (39 nm) was much better than that of smaller nanoparticles (13 nm). Interestingly, the average gold nanoparticles size corresponding to the most sensitive LSPR area in our work was also around 40−50 nm (Figure 3). On the basis of these results, the different LSPR sensitivities observed for different gold nanoparticle zones (Figure 7) could be attributed to a better match between the nanoparticle decay length and physical dimension of absorbed biomolecules. LSPR Immunosensor for Specific Detection of Atrazine. Even though the specific mechanism responsible for the best LSPR sensitivity is still unclear, the corresponding

optical spectra

0.06 0.08 0.09 0.11 0.08 0.07 0.12 0.16

All the samples were annealed at 550 °C for 8 h.

area 1. In short, the LSPR sensitivity was increased when the nanoparticle size increases from zone 1 to zone 4 within the gold−glass interfacial areas. 8025

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peak intensity modulations become more important while the wavelength shift is independent of the concentration of the atrazine antigen concentration. Specifically, the optical density increased from 0.547 for binding of 50 pg/mL atrazine to 0.576 for 0.5 ng/mL atrazine and was further increased to 0.608 after binding of higher atrazine content of 5 ng/mL. From the obtained results, where the small molecules of atrazine (about 220 Da) were specifically detected through an LSPR immunosensor configuration, the main plasmonic response was first considered as wavelength shift due to large antibody immobilization, after which the optical intensity changes became predominant once the different antigen concentrations have been tested. To ensure that the LSPR responses result from specific immunoreactions between anti-IgG atrazine and its antigen (atrazine), control experiments were conducted using the same LSPR nanobiosensor setup when an atrazine nonspecific antibody (anti-IgG human) has been used instead of the specific one. The plasmonic spectra corresponding to different biomodification steps are shown in Figure 8b. It is clear that the plasmonic wavelength is red-shifted after modification of thiol and anti-IgG human antibody. However, after exposure to 5 ng/mL atrazine, any plasmonic shifts were not observed. Therefore, the LSPR responses shown in Figure 8a do correspond to the specific binding of by atrazine antigen to its antibody. All the plasmonic responses (resonant wavelength and peak intensity) corresponding to different biomodification steps and antigen bindings are summarized in Table 2. Even though there are many chemical procedures for the biofunctionalization, the authors emphasize the large applicability of their gold nanoparticle preparation through extremely sensitive plasmonic biosensors.

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CONCLUSIONS To summarize, in this work, two new protocols are proposed for the preparation of different zones of gold nanoparticles onto a single glass substrate. Both protocols stem from the idea to modulate the evaporated film thickness at a predefined area on the same glass slide. The first protocol using staple masks was explored to readily select the optimized plasmonic structures after biofunctionalization steps, while the second protocol was used to explore the excellent correlation between evaporated film thickness, nanoparticle morphology, and plasmonic properties. On the basis of these results, we have constructed an LSPR immunosensor to specifically detect trace content of atrazine as 50 pg/mL.

Figure 8. LSPR biosensor for specific detection of atrazine (a) and experimental control using atrazine nonspecific IgG antibody under the same conditions (b). The initial evaporated film thickness was 5 nm followed by thermal annealing at 550 °C for 8 h (12MDA: 12mercaptododecanoic acid).

optimized nanoparticles should have the average size of 40 nm, based on the obtained results (Figures 7 and 3) and previously published work.40 Because the gold nanoparticle sizes were highly dependent on initial evaporated film thickness (results in Table 1), gold nanoparticles with average size of 42.8 nm could be obtained by annealing the sample with 5 nm evaporated gold film thickness at 550 °C for 8 h. Taking into consideration these parameters, we have constructed an immunosensor using samples with 5 nm gold film thickness to specifically detect atrazine herbicide. As shown in Figure 8a, the immobilization of MDA alkanethiol layer and anti-IgG atrazine antibodies leads to a wavelength red-shift of 4 and 13 nm, respectively. Next, after binding of antigen (atrazine), the wavelength shift was rather small as 5 nm for the highest content atrazine. Interestingly, the

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AUTHOR INFORMATION

Corresponding Author

*Phone: (33) 3 25 75 97 28. Fax: (33) 3 25 71 84 56. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

Table 2. LSPR Resonant Wavelength and Maximum Extinction for Stepwise Preparation of a Biosensor for Atrazine Detection experiment specific nonspecific specific nonspecific

resonant wavelength [nm] max extinction

annealed gold substrate

thiol

anti-IgG atrazine antibody

antigen 50 pg/mL

antigen 0.5 g/mL

antigen 5 ng/mL

550.07 552.4 0.48 0.444

553.95 555.27 0.503 0.457

563.24 560.1 0.517 0.465

565.56 560.1 0.547 0.465

565.56 560.1 0.576 0.465

567.11 560.6 0.608 0.479

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(27) Haes, A. J.; Zou, S. L.; Schatz, G. C.; Van Duyne, R. P. J. Phys. Chem. B 2004, 108, 6961−6968. (28) Kedem, O.; Tesler, A. B.; Vaskevich, A.; Rubinstein, I. ACS Nano 2011, 5, 748−760. (29) Kiel, M.; Klotzer, M.; Mitzscherling, S.; Bargheer, M. Langmuir 2012, 28, 4800−4804. (30) Whitney, A. V.; Elam, J. W.; Zou, S. L.; Zinovev, A. V.; Stair, P. C.; Schatz, G. C.; Van Duyne, R. P. J. Phys. Chem. B 2005, 109, 20522−20528. (31) Rindzevicius, T.; Alaverdyan, Y.; Kall, M.; Murray, W. A.; Barnes, W. L. J. Phys. Chem. C 2007, 111, 11806−11810. (32) Szunerits, S.; Das, M. R.; Boukherroub, R. J. Phys. Chem. C 2008, 112, 8239−8243. (33) Galopin, E.; Noual, A.; Niedziolka-Jonsson, J.; JonssonNiedziolka, M.; Akjouj, A.; Pennec, Y.; Djafari-Rouhani, B.; Boukherroub, R.; Szunerits, S. J. Phys. Chem. C 2009, 113, 15921− 15927. (34) Galopin, E.; Niedziolka-Jonsson, J.; Akjouj, A.; Pennec, Y.; Djafari-Rouhani, B.; Noual, A.; Boukherroub, R.; Szunerits, S. J. Phys. Chem. C 2010, 114, 11769−11775. (35) Szunerits, S.; Ghodbane, S.; Niedziolka-Jonsson, J.; Galopin, E.; Klauser, F.; Akjouj, A.; Pennec, Y.; Djafari-Rouhani, B.; Boukherroub, R.; Steinmuller-Nethl, D. J. Phys. Chem. C 2010, 114, 3346−3353. (36) Karakouz, T.; Tesler, A. B.; Bendikov, T. A.; Vaskevich, A.; Rubinstein, I. Adv. Mater. 2008, 20, 3893−3899. (37) Tesler, A. B.; Chuntonov, L.; Karakouz, T.; Bendikov, T.; Haran, G.; Vaskevich, A.; Rubinstein, I. J Phys. Chem. C 2011, 115, 24642−24652. (38) Zhang, X. M.; Zhang, J. H.; Wang, H.; Hao, Y. D.; Zhang, X.; Wang, T. Q.; Wang, Y. N.; Zhao, R.; Zhang, H.; Yang, B. Nanotechnology 2010, 21, 465702. (39) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J Phys. Chem. B 2006, 110, 7238−7248. (40) Okamoto, T.; Yamaguchi, I.; Kobayashi, T. Opt. Lett. 2000, 25, 372−374. (41) Nath, N.; Chilkoti, A. Anal. Chem. 2004, 76, 5370−5378.

ACKNOWLEDGMENTS The authors are grateful for the financial support of the Stratégique Program 2009−2012 from the University of Technology of Troyes (UTT) and to the France−Israel Bilateral Research Network Programme 2009−2011. The ANR program ANR-07-Nano-032 “NP/CL” is also acknowledged for supplement of the optical setup. The authors thank François Weil (LASMIS-UTT team) for providing the thermal processing equipment and Wang Huan and Rafael SalasMontiel (UTT) for the fruitful discussions about the evolution of metal nanoparticles plasmonics and for their experimental assistance. Kun Jia thanks the Chinese Scholarship Council for funding his Ph.D. scholarship in France.

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dx.doi.org/10.1021/ac301825a | Anal. Chem. 2012, 84, 8020−8027