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Cite this: Chem. Commun., 2014, 50, 7787 Received 14th March 2014, Accepted 27th May 2014 DOI: 10.1039/c4cc01920b
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Distance-determined sensitivity in attenuated total reflection-surface enhanced infrared absorption spectroscopy: aptamer–antigen compared to antibody–antigen† Wen-Jing Bao,a Zhen-Dong Yan,b Min Wang,a Yun Zhao,a Jian Li,a Kang Wang,a Xing-Hua Xia*a and Zhen-Lin Wang*b
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Distance-dependent signal intensity in immunoassay by attenuated total reflection-surface enhanced infrared absorption spectroscopy is demonstrated by controlling the distance of target proteins away from the enhancement substrate. Based on this optical near-field effect, sensitive detection of protein molecules with a detection limit of 0.6 nM and investigation of the kinetics and thermodynamics of protein–aptamer/antibody interactions can be achieved.
Surface enhanced infrared absorption spectroscopy (SEIRAS) is a sensitive technique that exhibits great advantages for spectroelectrochemical1,2 and bioanalytical applications.3–6 As a complementary method to surface enhanced Raman scattering (SERS), SEIRAS in attenuated total reflection (ATR) mode selectively detects the vibrations with their dipole moment variation perpendicular to the surface,7–10 which is very useful in determining the orientation and conformation of biomolecules at the surface/interface.11,12 So far, SEIRAS has already been applied for qualitative analysis of biological species ranging from small biomolecules to genetic and proteomic targets,12–14 and furthermore to determine the specific recognition15 and the structure– activity relationship of biomolecules.16 Recently, we have successfully monitored the specific binding of an antibody and an antigen on a gold nanoparticle film by ATR-SEIRAS,17 demonstrating the quantitative applicability of this technique as a realtime sensing method for biomolecular recognition events. Despite these successes, the analytical sensitivity of SEIRAS is still too low to afford sub-nanomolar level detection, which greatly restricts its potential wide application. It is widely believed that the micro/nano-structured noble metal films on an optical prism exhibit a strong electromagnetic a
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China. E-mail:
[email protected]; Fax: +86-25-83685947; Tel: +86-25-83685947 b School of Physics, Nanjing University, Nanjing, 210093, China. E-mail:
[email protected]; Tel: +86-25-83592730 † Electronic supplementary information (ESI) available: Supplemental figures and tables, experimental details and an introduction to the modified twocompartment model. See DOI: 10.1039/c4cc01920b
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field, leading to the enhancement of vibrational absorption of bound molecules.5,18,19 One important method to enhance the absorption intensity is to fabricate metal nanoparticles and nanostructures with strong localized surface plasmon resonance (LSPR) located at the excitation frequency of molecular vibrational modes.20–24 Another key step to improve the detection sensitivity in SEIRAS is to make full use of the enhancement effect. According to the optical near-field effect,25,26 the enhanced electromagnetic field decays rapidly with the distance from the surface of metal nanostructures and the signal enhancement is restricted to the immediate vicinity of the surface. Here, we demonstrate experimentally the distancedependent signal intensity in the immunoassay by utilizing antibodies and aptamers as capture molecules. Aptamers are single stranded DNA or RNA molecules that can be designed to recognize a large number of proteins and other targets with excellent affinity and specificity.27,28 Compared with the conventional monoclonal antibodies, aptamers have a much smaller size and exhibit higher stability towards external changes.28 These unique properties make aptamers promising candidates as molecular recognition elements for the protein bioassays by ATR-SEIRAS. Herein, the affinity interaction of L-selectin with its antibody and aptamer, with a molecular weight of circa 60 kDa and 12 kDa, respectively, is selected as a model system for specific recognition investigation. An Au nanoparticle (NP) film is deposited on the surface of a silicon prism by galvanic displacement reaction29–31 and serves as a SEIRAS enhancement substrate (SI 1 and Fig. S1, ESI†). The antibody and the aptamer of L-selectin are separately attached to the Au NP film via covalent bonding (SI 1 and Fig. S2, ESI†). The SEIRA spectra of L-selectin are recorded in ATR mode using a homemade accessory. To obtain the sole SEIRA signal of target proteins, a background spectrum is recorded before L-selectin is added. The target proteins in the bulk solution are specifically captured to the interface, creating featured absorption bands of proteins (as illustrated in Fig. 1). As shown in Fig. 2a, the sharp peak at 1646 cm 1 is attributed to the amide I vibration of L-selectin, which is due
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Fig. 1 Illustration of the distance-dependent analysis of protein molecules on an Au nanoparticle film using ATR-SEIRAS.
Fig. 2 (a) The ATR-SEIRA spectra of 50 nM L-selectin (i) adsorbed onto Au NP film (black line); (ii) recognized by L-selectin aptamer modified Au NP film (red line); (iii) recognized by anti-L-selectin modified Au NP film (blue line) after 30 min in 50 mM PBS; (b) the intensity of amide II bands from 50 nM L-selectin (squares) and simulated local electrical field enhancement (normalized in intensity, black dots) with different distances for captured molecules from the Au NP film surface; (c) top view and (d) side view of the near-field intensity around the Au NP film at wavenumber of 1543 cm 1.
to the CQO stretching vibrations of the peptide groups.32 The amide I absorption region can be used to evaluate the secondary structure of protein layers,33 providing important structural information on the binding event. Here we note that 34 L-selectin belongs to the typical a-helix-rich proteins, and its secondary structure can be well maintained when binding to the antibody/aptamer at the modified Au NP film interface (Fig. S3, S4 and Table S1, ESI†). The amide II vibration, located at 1543 cm 1, which corresponds to the N–H bending vibration, also including a minor contribution from the C–N and C–C stretching vibrations,32 can be used for protein quantitation.35 The two bands remain unchanged after rinsing with PBS, indicating the strong interaction of L-selectin with its antibody/aptamer and that unbound L-selectin in the bulk solution contributes little to these signals. According to the optical near-field effect, the IR intensity of L-selectin on the surface of the Au NP-coated silicon prism in ATR mode is highly affected by the distance of protein molecules away from the surface. The intensity of amide II bands of L-selectin bound to its antibody/aptamer is compared with that of equivalent L-selectin directly adsorbed onto Au NPs. We hypothesize that the orientation of the assembly layer is perpendicular to the Au NP film surface and thus the distances
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can be estimated as 2.0 nm, 2.9 nm and 7.1 nm for the directly adsorbed, aptamer and antibody captured L-selectin according to molecular sizes, respectively. It is found that the intensity of absorbance originated from an equal amount of proteins decreases quasi-exponentially with the increasing distance of captured L-selectin to the surface of Au NP film (Fig. 2b). The local electrical field distribution of the Au NP film was simulated using the COMSOL Multiphysics software (COMSOL Inc.) to provide a description of the optical near-field effect (SI 6 and Fig. S5, ESI†). The thin film was modeled as a continuous monolayer consisting of hexagonal close-packed ellipsoids.18,36,37 As seen in Fig. 2c, the maximum local electrical field enhancement factor is about 4, which is obtained at numerous nanogaps due to the point effect. Since the vibrational signals go with the square of the near-field enhancement, the intensity of the near-field at a specific distance from the Au NP film is squared statistically, indicating that the average electrical field enhancement decays rapidly with increasing the distance (Fig. 2b), with a relatively good accordance to the experimental data. Therefore, we believe that utilizing an aptamer, instead of the corresponding antibody, will exploit the advantage of the enhancement effect and improve the absorption intensity. Using this strategy, sensitive detection of L-selectin using aptamers as molecular recognition elements can be achieved by ATR-SEIRAS, and the kinetics and thermodynamics of the aptamer–protein interaction are further investigated. The binding kinetics of L-selectin to its aptamer at the interface was monitored by real-time ATR-SEIRAS. As shown in Fig. 3a, the intensity of amide bands of L-selectin increases gradually with the process of surface recognition. The amide II vibration of a protein is usually applied for quantitative analysis, since it has been reported by Pitt et al.38 that the peak area of this band exhibited a linear relationship with the total amount of the protein at the interface. Here, we found that the peak intensity of amide II, besides its area, could also be used to calculate the quantity of the protein (Fig. S6, ESI†). Therefore, the intensity of an amide II band located at 1647 cm 1 in Fig. 3a is plotted as a function of the binding time (Fig. 3b). The binding kinetics of L-selectin to its aptamer shows a typical character of the interfacial reaction, i.e., first a rapid increase in the early five minutes, then a slowing down process, and eventually reaching the equilibrium state after 20 min. To gain a deep insight into the kinetics of this binding event, a modified two-compartment
Fig. 3 (a) A series of SEIRA spectra as a function of binding time in a solution of 50 nM L-selectin + 50 mM PBS (pH = 7.4) on the surface of aptamermodified Au NP film; (b) the intensity of amide II bands (black squares) of the spectra in (a) and a fitting curve (red line) as a function of binding time.
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Science Foundation of China (21035002, 21275070, 51271092, and 21121091) and the Innovation Project for College Graduates of Jiangsu Province (CXZZ13_0039).
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Notes and references
Fig. 4 (a) The ATR-SEIRA spectra of a protein recognized by an immobilized aptamer from different concentrations of L-selectin in the bulk solution. The concentrations (nM) from top to bottom are: 33.7, 25.3, 20.8, 16.0, 11.0, 8.3, 5.6, 2.9, 1.8, and 0.6. All the spectra were recorded after 30 min of binding so as to represent the equilibrium state; (b) the intensity of amide II bands (black square) of the spectra in (a) and the fitting curve (red line) as a function of the concentration of L-selectin in solution.
model39 is applied here to simulate the specific recognition of proteins in the solution to captured aptamers at an interface (SI 8, ESI†). Accordingly, the fitting result in Fig. 2b indicates that the association rate constant is (7.2 0.5) 104 M 1s 1, implying a fast kinetics for the aptamer–protein interaction. The study of the thermodynamics of the aptamer–protein interaction is valuable to the improvement of the design of an aptamer structure. Here we further recorded a series of SEIRA spectra of captured proteins at the interface with different concentrations of L-selectin in the bulk solution (Fig. 4a). Every spectrum was recorded at 30 min after the addition of L-selectin to ensure the attainment of the equilibrium state (Fig. S7, ESI†). As can be seen in Fig. 4b, attributed to the character of the surface reaction essentially, the binding capacity of L-selectin on the aptamer modified Au NP film shows a nonlinear increase along with gradual addition of the protein to the bulk solution. It is worth mentioning that the detection limit of target L-selectin is 0.6 nM and it has been improved by one order of magnitude in contrast to immunoassay by ATR-SEIRAS (Fig. S8, ESI†). Similarly, the result was simulated using a modified two-compartment model. The association equilibrium constant is calculated to be (5.5 0.6) 107 M 1, indicating high affinity for the aptamer–protein interaction.40 In summary, the optical near-field effect in ATR-SEIRAS was demonstrated experimentally by a model system of specific interaction of protein–aptamer/antibody binding. Highly sensitive, in situ and label-free monitoring of the immunoreaction can be achieved using ATR-SEIRAS and aptamers as the capturing agents at the Au NP film/solution interface. From the featured IR absorption of target proteins, a detection limit of sub-nanomolar level can be readily obtained using the present technique. Due to the fast kinetics and high affinity of the specific binding of the aptamer–protein, this new approach based on ATR-SEIRAS is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies. This work was financially supported by the National 973 Basic Research Program (2012CB933800), the National Natural
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