ZnSe-Based Surface Enhanced Infrared Absorption Spectroscopy

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Au/ZnSe based surface enhanced infrared absorption spectroscopy as a universal platform for bioanalysis Wen-Jing Bao, Jian Li, Jin Li, Qian-Wen Zhang, Yang Liu, Cai-Feng Shi, and Xing-Hua Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04505 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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Analytical Chemistry

Au/ZnSe based surface enhanced infrared absorption spectroscopy as a universal platform for bioanalysis Wen-Jing Bao‡, Jian Li‡, Jin Li, Qian-Wen Zhang, Yang Liu, Cai-Feng Shi, and Xing-Hua Xia* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China Corresponding Author: [email protected], Tel: 0086-25-89687436 ABSTRACT: A versatile and sensitive platform for label-free bioanalysis has been proposed based on attenuated total reflectionsurface enhanced infrared absorption spectroscopy (ATR-SEIRAS) using Au/ZnSe as the enhancement substrate that allows wide spectral range down to 700 cm-1. Au nanoparticles are stably deposited on the surface of ZnSe prism due to the formation of Au-Se bond via electroless deposition, and the enhancement factor of the resultant Au/ZnSe substrate is about two times larger than that of the commonly used Au/Si substrate. As demonstration, the Au/ZnSe based SEIRAS has been applied to obtain abundant structural information in the fingerprint region and quantitative analysis of various biomolecular interactions such as DNA hybridization and immunoreaction without any labeling process.

KEYWORDS：ATR-SEIRAS, Au nanoparticles, ZnSe, DNA hybridization, bioanalysis

INTRODUCTION Surface enhanced infrared absorption spectroscopy (SEIRAS) is one of the essentially non-perturbing bioanalytical techniques for probing interfacial molecules and reactions with high sensitivity.1,2 As an extension of the conventional infrared spectroscopy, SEIRAS in attenuated total reflection (ATR) mode can significantly improve the detection sensitivity3,4 and effectively provide the structural and conformational information of biomolecules at the surface/interface,5-7 while the signal interference from the bulk solution and environmental variations can be markedly eliminated. Owing to these advantages, SEIRAS has been applied for the monitoring of interfacial protein conformation and function,6, 8-12 qualitative and label-free detection of various biomolecules,7,13 determination of specific recognition14 as well as structure-activity relationship of biomolecules.15 Recently, we demonstrated the distance dependent signal intensity in immunoassay based on the optical near-field effect, and showed the capability of SEIRAS as an in situ and real time method for sub-monolayer detection of biomolecular recognition events.16 The transparent range of prisms currently used for SEIRAS is a crucial limitation in the application. The cut-off wavenumber of the most widely used silicon prism is about 1500 cm1 in the mid-infrared region. Many important characteristics of biomolecules located in the fingerprint region (1350-400 cm-1), such as the phosphate backbone of nucleic acid, phospholipids in cell membrane and the coordination structure in protein between cofactor and peptide chain, are all hardly detected with the Si prism based SEIRAS. Thus, it is highly necessary to broaden the spectral scope of SEIRAS with prisms having wider transparent range. ZnSe is a commonly used prism in the application of multi-ATR-IR absorption spectroscopy due to its low refractive index and wide transparent range from 4000 cm1 to 650 cm-1 in the mid-IR region. Mizaikoff et al. reported the label-free detection of DNA hybridization by covalently attach-

ing amino modified DNA to a surface of ZnSe via photochemical reaction.17 However, there are few researches based on ZnSe in the field of surface enhanced infrared spectroscopy due to the lack of simple and feasible methods to prepare the enhancement substrate. Cai et al. reported a modified ATR configuration by sandwiching an ultrathin water interlayer between a hemi-cylindrical ZnSe prism and a Si wafer as an integrated window. Thus, high quality spectral fingerprints down to 700 cm-1 can be obtained.18 However, the thin water layer will certainly absorb and in turn reduce the IR energy, especially the IR signals within low wavenumber region will be almost completely declined by the silicon layer. Therefore, direct fabrication of enhancement substrate on ZnSe becomes the key point to improve the ATR-SEIRAS technique. Physical deposition of metal nanofilm is a conventional method for preparing the enhancement material on prism.19, 20 However, the formed metal films have two major disadvantages: 1) the enhancement factor of metallic nanofilm is low because of the huge mismatch between metal particle size and IR wavelength, and 2) the performance is not stable due to the weak adhesion between metal film and prism. Another alternative and effective method is the nano-fabrication techniques enabling fine control of the structure and morphology of the resultant materials, thereby greatly improving the enhancement factor. Taubner et al. prepared gold nanotriangle array on ZnSe surface using nanosphere lithography and successfully tuned the localized surface plasmon resonance (LSPR) frequency to middleinfrared region by controlling the diameter of polystyrene nanospheres.21 Altug et al. designed a gold nanowire array with various aspect ratios using electron beam lithography and also achieved middle-infrared LSPR for nanomaterials on CaF2.22 However, nano-fabrication techniques are usually time consuming, area limited and require expensive equipment or complex process, which is difficult for widely application.

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Herein, we propose a simple approach to the electroless deposition of Au nanoparticles film on the surface of ZnSe hemispherical prism as the enhancement substrate of SEIRAS, which can significantly broaden the spectral range and improve the detection sensitivity of SEIRAS. After systematic characterizations of the reaction between HAuCl4 and ZnSe using XPS, XRD, ICP-AES, AFM, TEM and electrochemical techniques, a reaction mechanism is proposed. The adhesion between ZnSe and Au nanoparticles is enhanced due to the formation of AuSe bond. Fabrication conditions of the Au film deposition were optimized for obtaining higher enhancement. Finally, the specific interaction between biomolecules, i.e. DNA hybridization and immunoreaction was investigated to demonstrate the versatility of the present SEIRAS platform using Au/ZnSe as the enhancement substrate. EXPERIMENTAL SECTION Material and Apparatus. The hemispherical ZnSe ATR prism (20 mm in diameter) was purchased from Bosheng Quantum Technology (Changchun, China). The hemispherical Si (111) ATR prism (36 mm in diameter) was purchased from Alkor Technologies (Saint-Petersburg, Russia). Mercapto-hexanoic acid (MHA), 6-mercaptohexanol (MCH), N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) were obtained from SigmaAldrich. Bovine hemoglobin was from Biological Products Corp. (Shanghai, China). Rabbit IgG and goat anti-rabbit IgG were obtained from Boster Biological Technology, Ltd (Wuhan, China) and used without further purification. The ss-DNA sequences listed in Table S1 were custom synthesized from Sangon Biotechnology Co., Ltd. (Shanghai, China). Other reagents were of analytical grade and used as received. All aqueous solutions were prepared using ultrapure water (Millipore, USA). Infrared spectra were measured with a Nicolet 6700 Fourier transform spectrometer (Thermo Fisher, USA) equipped with a liquid-nitrogen-cooled MCT detector. Atomic force microscopy (AFM) imaging was performed on a Dimension Icon AFM system (Bruker, USA) using tapping mode under ambient conditions. Scanning electron microscopy (FE-SEM, S-4800, Hitachi, Japan) was used to characterize the morphology of the deposited gold films. X-ray photoelectron spectroscopy (XPS) analyses were carried out on a PHI 5000 VersaProbe X-ray photoelectron spectrometer system (ULVAC-PHI, Japan) equipped with Al Kα radiation as a probe under a chamber pressure of 5 × 10-10 mbar. The composition of plating solution after deposition was measured by inductively coupled plasma- atomic emission spectrometer (ICP-AES, Optima 5300DV, PerkinElmer, USA). An X-ray powder diffractometer (XRD, X-6000, Shimadzu, Japan) was used to determine the crystallization degree of gold nanoparticles. UV-vis spectra were collected with a UV3600 spectrometer (Shimadzu, Japan). All electrochemical measurements were performed on a CHI 660E electrochemical workstation (CH Instruments, USA). Preparation of the gold nanoparticle film on ZnSe. The hemispherical ZnSe ATR prism was polished with alumina powder (1 μm) and rinsed by sonication in anhydrous ethanol and ultrapure water, respectively. In a typical deposition, 100 μL 24 mM HAuCl4 was added on the surface of the ZnSe prism and reacted at 30 oC for 40 s. For kinetic and thermodynamic investigation of the deposition process as well as the morphology control of the Au nanoparticles, deposition conditions (i.e.,

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the composition and concentration of the plating solution, deposition time and temperature) were controlled as shown in Table S2 in detail. Electrochemical characterization of the soluble species formed during the deposition. 10.0 mL 1.0 mM HAuCl4 was added on the surface of ZnSe and deposited at 30 oC for 1 min, 2 min, 3 min and 5 min, respectively. Then, cyclic voltammograms of the plating solutions after the reaction were measured with a glass carbon electrode at a scan rate of 100 mV/s. The supporting electrolyte was 0.1 M K2SO4 (pH = 5.0). The infrared enhancement effect of Au nanofilm on ZnSe. Au nanoparticles were first deposited on a surface of ZnSe at 30 oC for 3 min with different additives. The deposition solution contains 24 mM HAuCl4 added with no additive, 10 mM KCl, 10 mM KBr, 10 mM KI and 1% PVP, respectively. Then, 1.0 μL 1.0 mM KSCN was dropped on the surface of Au/ZnSe prism with different morphologies described as above and dried with an IR lamp. IR extinction spectra of the five Au/ZnSe substrates were collected with bare ZnSe as the background spectrum. Au nanoparticles were further prepared on the surface of ZnSe at 20 oC for 1 min with 24 mM HAuCl4 as the deposition solution. The Au nanoparticles on silicon substrate were deposited at 60 oC for 1 min in a solution of 0.01 M HAuCl4 + 0.1 M Na2SO3 + 0.033 M Na2S2O3 + 0.033 M NH4Cl + 0.5M HF. To make a direct comparison of the enhancement effect for the two substrates, An/ZnSe and Au/Si were incubated in 0.2 μM MCH overnight separately and then rinsed with water to form a MCH SAM on the surface of Au nanoparticles. ATR-IR absorption spectra of MCH on the two substrates were collected with bare ZnSe and Si as the background spectra, respectively. The incidence angle was 75o. Optimization of deposition condition for Au/ZnSe as the substrate for bioanalysis. Au nanoparticles were prepared on the surface of ZnSe at 30 oC with 10 mM HAuCl4 as the deposition solution. The deposition time was set as 20 s, 30 s, 40 s and 60 s, respectively. Afterwards, 500 μL 0.5 mg/mL BHb prepared in 50 mM PBS (pH=7.4) was added to the surface of Au/ZnSe with different deposition time for 30 min. Then, SEIRA spectra of BHb on different substrate were collected with PBS solution as the background spectrum. Detection of DNA hybridization. First, 400 μL 0.25 μM probe DNA was added to the Au/ZnSe surface and allowed to assemble overnight to form a homogeneous DNA layer. In order to eliminate the electrostatic repulsion interaction among DNA strands, the solution of ss-DNA was prepared in 1 M NaCl. The modified Au nanoparticles film was subsequently dipped in 100 μL 40 μM MCH for 30 min to remove DNA molecules physically adsorbed on the surface and acted as passivation layer. Afterwards, the surface was rinsed with 50 mM PBS (pH=7.4) solution and was treated as the background spectrum. IR spectra of target DNA were obtained after adding 50 μL 1.0 μM target DNA. Detection of immunoreaction. Au/ZnSe was immersed in 400 μL 5 mM 6-Mercaptohexanoic acid (MHA) overnight to form a self-assembled layer terminated with carboxyl groups. A 400 μL mixed solution of 25 mM EDC/NHS (1:1, in 10 mM PBS, pH=5.8) was added to the detection cell and reacted for 30 min and then the activated surface was immediately incubated in 200 μL 20 μg/mL rabbit IgG (in 50 mM PBS, pH=7.4) for 1 h after rinsing with 50 mM PBS. To minimize the nonspecific interactions, the surface was soaked in 300 μL 1% BSA


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for 10 min and carefully rinsed by PBS buffer. Finally, goat anti-rabbit IgG at a desired concentration was added and corresponding IR spectra were collected with rabbit IgG/Au /ZnSe as the background spectrum. SEIRAS measurement. Detection was processed with a homemade ATR accessory and the diameter of the detection cell was 6 mm. Unpolarized IR radiation was totally reflected at the ZnSe prism/solution interface with an incident angle θ=75o and was detected with a liquid-nitrogen-cooled MCT detector. All the spectra were plotted in absorbance unit relative to a baseline which was recorded by immersing the as-prepared surface in buffer solution for 30 min before further measurement. The spectral range was 4000-650 cm−1 with a resolution of 4 cm−1. RESULTS AND DISCUSSIONS The experimental design is shown in Scheme 1. The morphology and structure of the Au nanoparticles film on ZnSe obtained by electroless deposition were investigated with SEM (Figure 1a) and AFM (Figure 1b). As shown in Figure 1a, the thin film is composed of nano-islands with an average size of 120 nm. Large numbers of nano-gaps are formed to provide the enhanced local electrical field due to the point effect, which can produce large infrared signal enhancement. The AFM image shows that the thickness of the film is about 30 nm. The characteristic diffraction peaks at 38.4o, 44.5o, 64.8o and 77.9o from the X-ray diffraction pattern (Figure 1c) correspond to the (111), (200), (220) and (311) planes of Au crystal respectively, indicating that the deposited nanoparticles are gold nano-structures with typical face-centered cubic lattice. The substrate, ZnSe crystal, also has a face-centered cubic structure with the lattice planes marked in Figure 1c.

Scheme 1. Schematic illustration of the Au/ZnSe based ATRSEIRAS detection of DNA hybridization. Au nanoparticles film is deposited on top of a ZnSe hemispheical prism as IR enhancement substrate. DNA hybridization is monitored by ATRSEIRAS. Wide range enhanced IR spectrum includes fingerprint region information can be recorded with the help of this platform.

Figure 1. (a) SEM image, (b) AFM image, and (c) X-ray diffraction pattern of the Au nanoparticles deposited on a surface of ZnSe at 20 oC for 1 min. The concentration of HAuCl4 was 5 mM. The inset in panel (a) shows the size distribution of Au nanoparticles. The scale bar in panel (a) is 400 nm. (d) Scheme of the band structure of bulk ZnSe crystal in aqueous solution and the standard redox potential for AuCl4-/Au. The conditional redox potential for AuCl4-/Au with a concentration of 5 mM is 0.96V. ZnSe is an intrinsic semiconductor material and its band structure in aqueous solution23 is shown in Figure 1d. The potentials correspond to the valence band and conduct band of bulk ZnSe are 0.79 eV and -1.91 eV, respectively. The electrochemical potential of AuCl4-/Au, which is 1.002 V versus standard hydrogen electrode (SHE), is more positive than the valence band of ZnSe. Thus, AuCl4- can be reduced via injecting holes into the valence band of ZnSe, leading to the electroless deposition of Au nanoparticles on the surface of ZnSe and the oxidation of ZnSe at the same time. To gain a deep insight into the mechanism of electroless deposition, high resolution XPS was applied to analyze the composition of superficial nanomaterials. For ZnSe, the peak at 54.0 eV is assigned to Se2- 3d, which splits into two peaks (Se2- 3d5 and Se2- 3d3) in high resolution spectrum (Figure 2a).24 After deposition, the peak of Se2- 3d red-shifts slightly, indicating the probable interaction between Se and other atoms. In addition, two small shoulder peaks appear after electroless deposition, which are featured as low intensity and high binding energy that are close to Se(IV), showing the formation of SeO2 during the deposition. The spectrum of Au 4f gives more information about this reaction. The two characteristic peaks located at 82.7 eV and 86.4 eV are related to Au 4f7 and Au 4f5 features respectively (Figure 2b), further confirming that the nanofilm is made of Au nanoparticles.25 Moreover, a new pair of peaks at 84.0 eV and 87.6 eV arises with the same energy gap and intensity ratio as metallic Au after deposition, implying the characteristic binding energy related to Aun+ 4f7 and Aun+ 4f5, respectively. Although the bonding nature of Au is not very clear, the binding energy values are consistent with that found for Au (I) complexes. These new features can be ascribed to the formation of gold selenide (AuxSe) that shows quite similar XPS signals with previously reported gold silicide.25 Additional analysis by XPS, ICP-AES and HR-TEM further support the conclusion (Table S3, Figure S1, Table S4). The formation of AuxSe could



significantly enhance the adhesion between Au nanoparticles and ZnSe, which will maintain long-term stability of the Au nanofilm during the application.

Figure 2. High resolution X-ray photoelectron spectra for (a) Se 3d and (b) Au 4f features of Au nanoparticles film on ZnSe surface (Black line: the raw data; blue line: the baseline for data fitting; red line: the fitted curve). All spectra are calibrated using C 1s peak as the standard. Electrochemical cyclic voltammetry (CV) was then used to characterize the soluble species formed during the deposition. The reduction potential of residual AuCl4- and oxidation potential of Au are located at 0.73 V and 1.11 V, respectively, while the signal at 0.82 V is related to the adsorption of Cl-. Moreover, there is another reduction signal appeared at about 0.14 V whose intensity grows with the increase of deposition time (Figure S2). By calculating the conditional redox potential of H2SeO3/Se: E = Eo −

α Se 59 .2 lg 4 α H SeO 2

− 59 .2 pH = 0 .168 V

(1)

3

it can be inferred that the peak at 0.14 V is attributed to the reduction of H2SeO3 to Se. To sum up, the mechanism of electroless deposition can be described as following equations: AuCl4- + 3e = Au + 4Cl(2) + 2+ + (3) ZnSe + 2H2O + 6h = Zn + SeO2 + 4H AuCl4- is reduced via injecting holes into the valence band of ZnSe, leading to the oxidation of ZnSe to SeO2. Partially reduced Au binds to Se2- at the initial period of deposition, and the resultant AuxSe enhances the interaction of small Au particles with the ZnSe substrate. Then, the as-generated SeO2 is partially dissolved in the weak acidic media, which results in the continuous reaction between ZnSe and HAuCl4. Au nanoparticles grow with the deposition time and interconnected nanofilm will be finally formed. The sensitivity of SEIRAS is highly related to the morphology and structure of Au nanofilm on ZnSe prism. Thus, it is essential to understand the kinetics and thermodynamics of the reaction between ZnSe and HAuCl4, which are both important factors that determine the morphology of the Au nanoparticles. The interaction between ZnSe and HAuCl4 can be modeled as a standard first-order reaction. Hence, the reaction rate constant and activation energy are calculated simply based on the reaction rate equation (4) and Arrhenius equation (5), respectively: ln C(HAuCl4) = -kt +ln C(HAuCl4)0 (4) (5) ln k = ln A-Ea / RT where C(HAuCl4) is the concentration of HAuCl4 after deposition time of t, C(HAuCl4)0 is the initial concentration of HAuCl4, k is the reaction rate constant, Ea is the activation energy of the

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reaction, T is the reaction temperature, R is the molar gas constant and A is the pre-exponential factor. HAuCl4 has an intrinsic UV-vis absorption located at 312 nm in 0.5 M H3PO4-NaH2PO4 (pH=2.5) solution (Figure S3), which can be used to quantitatively measure the concentrations of HAuCl4 at different stages of the reaction with ZnSe.26 The absorption intensity of HAuCl4 significantly decreases with the increase of deposition time and shows good accordance with the equation for the first-order reaction (Figure S4). The reaction rate constants at various temperatures are therefore calculated to be 1.05 × 10-2 s-1 (20 oC), 3.56 × 10-2 s-1 (40 oC ), 5.16 × 10-2 s-1 (60 oC) and 1.24 × 10-1 s-1 (80 oC). According to the Arrhenius equation, the activation energy for the electroless deposition between HAuCl4 and ZnSe is calculated as 27.6 kJ/mol (Figure S5), which is quite low and close to that of reaction between HAuCl4 and Si,27 implying that both of the reactions are diffusion-controlled. As a result, the diffusion of reactants in the solution and the concentration of HAuCl4, as well as the reaction temperature, will effectively affect the kinetics of the electroless deposition, and finally control the morphology of Au nanofilm. The morphology of Au nanofilm was further investigated under various deposition conditions (i.e., the composition and concentration of the plating solution, deposition time and temperature). It is proved that the concentration of HAuCl4 plays a key role in morphological control of Au nanoparticles on ZnSe. Polyhedral nanoparticles are obtained at low concentration of HAuCl4 (Figure S6), while nanosheet-like structures are formed at higher concentrations (Figure 3). However, the structures of Au nanoparticles show no significant difference at elevated deposition temperatures because of low activation energy (Figure S7). It is worth mentioning that the surface coverage of Au nanoparticles can be determined monotonically by both the reaction temperature and deposition time, in other words, similar coverage (for example, Figure 3d and Figure S7d) can be obtained simply via maintaining the product of reaction rate (i.e. temperature) and deposition time, which gives a robust approach to get the optimized morphology. Another effective way to tune the morphology of Au nanofilm is the introduction of additives that favor the anisotropic growth of Au nanoparticles or alter the diffusion of reactants. As shown in Figure S8a-c, nano-flower, nano-square and nano-sheet structures are obtained after the addition of Cl-, Br- and I- ion into the HAuCl4 solution, respectively, as a result of the selective adsorption of halogen ions to the specific planes of Au nanoparticles as reported previously.28-31 The introduction of polyvinylpyrrolidone (PVP) or ethanol can affect the diffusion of HAuCl4, so as to change the shape of Au nanoparticles (Figure S8d,e).27, 31 In addition, the adsorption of PVP on Au (100) and (111) crystal plane leads to the formation of truncated polyhedron Au nanoparticles.32-34


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Figure 4. (a) ATR-IR absorption spectra of MCH monolayer (prepared from 0.2 μM MCH) self-assembled on the surface of (I) bare Si; (II) bare ZnSe; (III) Au/Si and (IV) Au/ZnSe, respectively. Au/ZnSe was prepared with 1 min deposition using 24 mM HAuCl4 at 20 oC; (b) ATR-IR absorption spectra of 500 μL 0.5 mg/mL BHb absorbed on Au/ZnSe fabricated with different deposition time. All the incidence angle was 75o. The Au/Si was deposited at 60 oC for 1 min in a solution of 0.01 M HAuCl4 + 0.1 M Na2SO3 + 0.033 M Na2S2O3 + 0.033 M NH4Cl + 0.5M HF.

Figure 3. The SEM images of the Au nanoparticles deposited on the surface of ZnSe at 20 oC for (a) 30 s; (b) 1 min; (c) 2 min; (d) 5 min. The concentration of HAuCl4 was 24 mM. Scale bar: 0.5 μm. The signal enhancement is directly correlated with the electromagnetic field enhancement of gold nanostructures on the surface of ZnSe prism, which is intrinsically determined by the morphology and structure of Au nanoparticles. Based on these requirements, Au nanoparticles films with different morphologies in the presence of various additives were selected for further optical investigation (Figure S8, 9). It can be found from the extinction spectra that the Au nanoparticles with several typical morphologies only show broad and continuous absorption in the middle infrared range as a result of the coupling effect among the Au nanoparticles, which provides comparable enhancement with that of Au NPs prepared without additives for the probe molecules of SCN-1 on its surface (Figure S9). The enhancement of Au/ZnSe substrate was then investigated with mercaptohexanol (MCH) self-assembly monolayer as the signal molecule to compare with that of widely used Au/Si substrate in the ATR mode (Figure 4a). Herein, we focus on the continuous Au nanofilm on ZnSe due to the comparable surface coverage of nanoparticles with Au/Si. The asymmetric and symmetric stretching vibrations of methylene group, located at 2920 cm-1 and 2850 cm-1, respectively, are clearly seen on the surface of Au/Si and Au/ZnSe. We hypothesize that the orientation and density of the self-assembly layer are the same on both surfaces, thus, it can be estimated that the enhancement factor of Au/ZnSe is about two times larger than that of Au/Si based on the band intensity and the real surface area of Au nanoparticles measured with an electrochemical method (Figure S10). The higher enhancement can be attributed to the low refractive index of ZnSe, which leads to a stronger evanescent electromagnetic field in the ATR mode.22

According to the above results, the surface coverage of Au nanoparticles on ZnSe, which depends on the deposition time and temperature, can effectively affect the absorption intensity of target molecules. An optimized fabrication temperature is set as 30 oC, which is moderate for experimental control and minimizes the thermal impact to precise ZnSe prism. The morphology of Au films fabricated at 30 oC with different time exhibits a faster kinetics and similar shape evaluation with time compared with the fabrication at 20 oC (Figure S7a, S11), confirming the appropriateness for the time setting. The obtained Au/ZnSe substrate prepared at 30 oC was then used for enhanced IR analysis of biomolecules. Taking consideration of the complex interaction between biomolecules and Au/ZnSe, the deposition condition was further optimized using bovine hemoglobin (BHb) as the signal biomolecule. As shown in Figure 4b, the maximum intensity of BHb is acquired on the surface of Au/ZnSe prepared at 30 oC and electroless deposition time of 40 s, which is about three times of that on the optimized Au/Si surface mentioned above. Compared with Si prism, ZnSe has a much lower cut-off frequency in the middle infrared range and higher enhancement factor. Therefore, it is firmly believed that SEIRAS using Au/ZnSe as the enhancement substrate will provide more abundant structural information and higher sensitivity for bioanalysis. Herein, the first label free SEIRAS investigation of DNA hybridization and a measurement of immunoreaction were applied as demonstration to show the versatility of this platform in bioanalysis. In our previous research, we reported for the first time an approach to the in situ study of DNA hybridization using the Au/Si based ATR-SEIRAS.13 Owing to the optical nature of Si prism, it is difficult to achieve quantitative analysis based on the absorption signal of the target DNA molecule itself, instead, a sandwich assay structure with 4-mercaptobenzoic acid modified Au nanoparticles as IR probe was applied to monitor the kinetics of DNA hybridization. Herein, the interfacial DNA hybridization reaction between the immobilized and free state DNA can be real time monitored directly without labeling when the Au/ZnSe based SEIRAS is used. Probe ss-DNA was selfassembled on the surface of Au nanofilm via coordination interaction between terminal thiol group and gold substrate, and the corresponding ATR-SEIRAS spectrum, with a spectral range down to 700 cm-1, shows a typical characteristics for ssDNA (Figure S12, curve a). The broaden peak around 1677 cm1 is attributed to the stretching vibration of C=C and C=N bonds from all the four kinds of bases (Figure S13). Another two signals located at 1208 cm-1 and 1088 cm-1 are assigned to the stretching vibration of P=O bands and asymmetric stretching vibration of P-O bonds from phosphate backbone groups35, respectively. Afterwards, the surface was passivated with mercaptohexanol, which causes obvious negative peaks since physically adsorbed probe DNA is removed from the surface (Figure



S12, curve b). Upon the addition of target DNA, featured peaks related to DNA bases (located around 1713 cm-1 and 1635 cm1 ) and phosphate groups arise. Their intensities remain unchanged after rinsing with NaCl solution, indicating the strong and specific interaction of probe ss-DNA with its complementary DNA (Figure S12, curves c and d). The sensitive detection of DNA molecules via its bases and phosphate groups enables quantitative and label-free analysis of DNA hybridization with real time ATR-SEIRAS. As shown in Figure 5a, the intensities of the bases, as well as the phosphate groups of target DNA, increase with the reaction time. It takes about 1 h to reach the equilibrium state in a solution of 1.0 M NaCl (Figure 5b), much faster than that of previous results13 due to the feasibility of high ionic strength buffer applied. The association rate constant of DNA hybridization is calculated to be (1.52 ± 0.06) ×104 M-1∙s-1 using a modified two-compartment model.36 The signal intensity of phosphate backbone is twice larger than that of bases (Figure 5b), leading to a more obvious evolution of reaction kinetics, which also shows the superiority of acquiring a wide scope spectrum with our platform.

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new enhancement substrate, quantitative and label-free analysis of DNA hybridization and immunoreaction have been achieved with ATR-SEIRAS, demonstrating the versatility of this platform for various bioanalytical analysis, and also extremely improves the performance and expand the application scope of ATR-SEIRAS.

ASSOCIATED CONTENT Supporting Information. Additional details of experimental conditions; XPS analysis, HR-TEM images, cyclic voltammogram analysis, ICP-AES analysis of Au/ZnSe substrate; UV-vis absorption spectra, SEM images of different substrate fabrication conditions; IR extinction spectra for enhancement factor comparison and SEIRAS spectra in bioanalysis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (X.H. Xia)

Author Contributions ‡

W.J. Bao and J. Li contributed equally to this work.

ACKNOWLEDGMENT Figure 5. (a) Time-dependent SEIRA spectra of 300 μL 0.2 μM target DNA added to the probe DNA modified Au/ZnSe surface; (b) The absorption intensity of P-O bonds (black squares) and bases (red circles) from target DNA as a function of hybridization time and the corresponding fitting curve. The present Au/ZnSe enhancement substrate can also be used to monitor other biorecognition reactions, such as immunoreaction. As demonstration, the affinity interaction between Rabbit IgG and Goat anti-Rabbit IgG was investigated. After the immersion of solution containing Goat anti-rabbit IgG on the Au/ZnSe surface modified with Rabbit IgG, typical IR signal of protein arises. The signal intensities of amide I and amide II bands located at 1635 cm-1 and 1543 cm-1 show a typical character of the interfacial reaction, i.e., first a rapid increase in the early stage, then a slowing down process, and eventually reaching the equilibrium state after 40 min (Figure S14). The association rate constant of antigen-antibody interaction is calculated from the intensity evolution of amide II band with the modified two-compartment model29 as 4.94 ×105 M-1∙s-1.

CONCLUSIONS In summary, stable gold nanostructures with significant IR enhancement factor can be chemically deposited on the surface of ZnSe, which improves the sensitivity and broaden the spectral range of ATR-SEIRAS. The deposition reaction is revealed as diffusion controlled process, thus, the morphology of Au structures on ZnSe can be well controlled by adjusting the concentration of HAuCl4 and adding additives. Under optimal deposition conditions, the obtained Au/ZnSe substrate shows twice larger enhancement factor compared to the reported Au/Si substrate. With the extremely extended spectral range and signal enhancement, fingerprint region information of trace level biological analytes can be successfully monitored. Based on this

This work was supported by grants from the National Natural Science Foundation of China (21327902, 21635004).

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ZnSe-Based Surface Enhanced Infrared Absorption Spectroscopy

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