SPR Detection of Low-Molecular-Weight Species Using Molecular Imprinting of Gold Nanoparticles Matrix

Analytical Approaches for Detection of Small Molecules

It is known that upon the association of low-molecular-weight substrates, the dielectric changes at the Au surface are too small to allow detectable SPR spectral changes. Different methods to enhance the dielectric changes upon binding the analyte to the surface were reported by implementing latex particles [66], liposomes [67], or proteins [68] as amplifying labels for the recognition events. One effective method to amplify the recognition process has involved the conjugation of Au NPs to the SPR chip surface. The coupling between the localized plasmon of the NPs and the surface plasmon wave was found to significantly affect the SPR spectrum [69, 70]. Due to the proximity of gold nanoparticles to the binding sites, even small dielectric changes caused by a small amount of analyte close to the sites change its equilibrium. Thus, changes in the dielectric properties of the imprinted matrix as a result of the binding of the analyte could be amplified by the conjugation of metallic NPs ensemble to the sensing complex.

Molecular Imprinting in Polymer-Gold Nanoparticles Composite Matrix

As the carboxylic acid residues exhibit similar dimensions and structural features to the nitro groups in the explosives, and since ionic and/or H-bonds between the carboxylate and the anilinium residues could provide affinity interactions between the carboxylic acids and the electrogenerated bis(aniline)-crosslinked Au NP composite, it was argued that the structurally related carboxylic acids could act as templates for the generation of imprinted sites for structurally analogous nitrosubstituted explosives [71]. The scheme in Fig. 5.10 outlines the method to synthesize an imprinted Au NP composite for the detection of PETN (1) or NG (2].

Figure 5.10 Schematic presentation for the electropolymerization of a composite of bis(aniline)-crosslinked Au NPs for the sensing of PETN or NG using citric acid as an imprinting template. Reprinted with permission from Ref. [71]. Copyright (2011) American Chemical Society.

The thioaniline-functionalized Au NPs were electropolymerized on a thioaniline-modified Au-coated glass surface in the presence of citric acid (3) as an imprinting template. The resulting bis(aniline)- crosslinked matrix was then rinsed to remove the imprinting template molecule. The electropolymerization of the Au NPs and the elimination of the imprinting template were followed by SPR spectroscopy.

A nonimprinted Au NP composite was similarly prepared by the electropolymerization of the modified Au NPs in the absence of the imprinting template. Figure 5.11 A exemplifies the SPR spectra observed upon the treatment of the citrate-imprinted Au NP matrix (panel I) and the nonimprinted Au NP composite (panel II) with 2 pM PETN. Whereas a noticeable shift in the SPR curve is observed for this low PETN concentration on the citrate-imprinted composite, no shift is evident for the nonimprinted matrix. Figure 5.11B, curve I, shows the reflectance changes of the citric-acid-imprinted Au NP composite upon interaction with different concentrations of PETN. As the concentration of PETN increases, the reflectance changes are intensified, consistent with a higher content of PETN that binds to the composite. For comparison, Fig. 5.1 IB, curve II, depicts the reflectance values observed upon the treatment of the nonimprinted Au NP composite within the same PETN concentration range. Evidently, only minute reflectance changes are observed, implying that PETN does not bind to the nonimprinted Au NP matrix. The calibration curve corresponding to the reflectance changes of the citrate-imprinted composite upon treatment with different concentrations of PETN is shown in Fig. 5.11C.

Figure 5.11 (A) SPR curves corresponding to (I) the citrate-imprinted

bis(aniline)-crosslinked Au NP composite (a) before and (b) after the addition of PETN, 2 pM, and (II) the nonimprinted bis(aniline)-crosslinked Au NP composite (a) before and (b) after the addition of PETN, 2 pM. (B) Sensograms corresponding to the changes in the reflectance intensities, at a constant angle i? = 65.0°, upon addition of variable concentrations of PETN: (a) 200 and (b) 400 fM and (c) 2, (d) 8, (e) 20, (f) 80, (g) 200, and (h) 400 pM to (I) the citrate-imprinted Au NP matrix and (II) the nonimprinted matrix. (C) Calibration curve relating the reflectance changes to the concentrations of PETN on the citrate-imprinted matrix. The inset shows the lower concentration region of the calibration curve. Error bars correspond to a set of N = 5 measurements. All measurements were performed in ethanol. Reprinted with permission from Ref. [71]. Copyright (2011) American Chemical Society.

The detection limit achieved for analyzing PETN by the citrate- imprinted matrix is 200 fM. The matrix interface can be used repeatedly, regenerating it after an analysis by rinsing, although a slight degradation of the matrix with each subsequent analysis has been observed.

Peculiarities of SPR Detection of Explosives Using LSPR Nanoantenna

Upon the application of dicarboxylic acids, such as succinic acid or fumaric acid, as imprinting templates, sensing abilities toward PETN decreased (not shown). These results emphasize the significance in the selection of an appropriate template analogue for PETN detection. Nitroglycerin exhibits, however, structural similarity to the citric acid imprinting template, and thus it was anticipated that the citrate-imprinted matrix would also sense the NG explosive. Figure 5.12 A, curve I, shows the reflectance changes of the citrate- imprinted matrix upon interaction with different concentrations of NG. For comparison, Fig. 5.12A, curve II, depicts the reflectance changes in the nonimprinted Au NP composite upon interaction with different concentrations of NG. Figure 5.12B shows the calibration curves for both citrate-imprinted and nonimprinted matrixes.

The detection limit for analyzing NG by the imprinted composite corresponds to 20 pM, while reflectance changes level off to a saturation value at a concentration of ca. 2 nM, consistent with the saturation of the imprinted sites. Evidently, the detection limit for analyzing PETN by the citrate-imprinted Au NP matrix is ca. 100- fold lower as compared to the analysis of NG on the same matrix. Calibration curve in Fig. 5.12B relates the reflectance changes to the concentrations of NG and allows deriving the association constant of NG to the citrate-imprinted sites (К_а = 2.5xl0¹⁰ M'¹). In comparison, the enhanced sensitivity to PETN was reflected by the significantly higher association constant (ca. 40-fold higher) of PETN to the citrate-imprinted sites. The enhanced association of PETN to the citric-acid-imprinted sites, as compared to the NG binding to these sites, may be attributed to the fact that, in addition to the three carboxylic acid residues that mimic the -0N0₂ functionalities of PETN, the additional OH group present in the imprint molecule (citric acid) acts cooperatively in generating a binding domain for the fourth -0N0₂ group present in PETN. That is, the imprinted sites are structurally optimized to accommodate the substrate with four

-0N0₂ groups. Figure 5.12B, curve b, shows the calibration curve for analyzing NG by the nonimprinted matrix. The reflectance changes are minute, and they do not allow the detection of NG. It was also found that the imprint of other carboxylic acids, such as isocitric, succinic, or maleic acids, does not lead to Au NP matrixes that allow effective sensing of NG at the low-concentration range.

Figure 5.12 (A) SPR sensograms corresponding to the changes in the

reflectance intensities, at a constant angle 9 = 65.0°, upon addition of variable concentrations of NG: (a) 20, (b) 80, and (c) 400 pM and (d) 2, (e) 8, and (f) 40 nM to (I) the citrate-imprinted Au NP matrix and (II) the nonimprinted matrix. (B) Calibration curves relating the reflectance changes to the concentrations of NG on (a) the citrate-imprinted and (b) the nonimprinted matrixes. Error bars correspond to a set of N = 5 measurements. All measurements were performed in ethanol. Reprinted with permission from Ref. [71]. Copyright (2011) American Chemical Society.

An attempt was also performed to develop an imprinted matrix for the sensing of EGDN (4) explosive. Based on the imprinting rationale described for the generation of imprinted matrixes for PETN or NG using the structurally related carboxylic acids, it was argued that fumaric acid (5), succinic acid (6), or maleic acid (7) may act as imprinting templates for EGDN. Accordingly, each of these dicarboxylic acids was used to generate the respective imprinted Au NP composites. Figure 5.13 shows the calibration curves observed upon the treatment of the imprinted Au NP composites (using (5), (6), or (7) as imprinting templates) with variable concentrations of EGDN. It was found that the three Au NP matrixes reveal comparable sensing capabilities for EGDN. Maleic acid as an imprinting template (7) yields the superior sensing matrix (highest reflectance changes along the EGDN concentration profile), while the second- best sensing matrix is formed upon the imprinting of (5) into the composite. The Au NP composite imprinted with (6) reveals the lowest sensing features. Thus, it was concluded that the imprinting of the dicarboxylic maleic acid in the Au NP composite is essential to yield an effective sensing matrix for EGDN.

Figure 5.13 Calibration curves relating the reflectance changes to the concentrations of EGDN on (a) the maleic-acid-imprinted, (b) the fumaric- acid-imprinted, and (c) the succinic-acid-imprinted bis(aniline)-crosslinked Au NP matrixes. Error bars correspond to a set of N = 5 measurements. All measurements were performed in ethanol. Reprinted with permission from Ref. [71]. Copyright (2011) American Chemical Society.

Finally, the selectivity of the carboxylic-acids-imprinted matrixes was examined toward the analysis of the three explosives: PETN, NG, and EGDN. Figure 5.14A shows the calibration curves corresponding to the analysis of PETN (curve I), NG (curve II), and EGDN (curve III) by the citric-acid-imprinted Au NP composite. While PETN is detected with a sensitivity that corresponds to 200 fM, the NG and EGDN explosives yield detectable reflectance responses starting at concentrations above 100 pM and 400 nM, respectively. These results clearly indicate that the citric acid template that includes three carboxylic acid substituents and one OH functionality yields an imprinted contour that allows an effective binding for the four nitrate ester substituents associated with PETN. The EGDN that is sterically smaller and includes only two nitro substituents does not fit to the resulting imprinted sites, leading to low binding affinities and to poor performance of the matrix toward sensing EGDN.

It was also found that, compared to the other carboxylic acid imprint molecules, fumaric acid exhibited the highest selectivity toward EGDN among the different explosives. Figure 5.14B shows the calibration curves corresponding to the analysis of EGDN (curve a), NG (curve b), and PETN (curve c) by the fumaric-acid- imprinted Au NP composite. Evidently, EGDN shows the best sensing performance on the fumaric-acid-imprinted composite, whereas both NG and PETN are inefficiently sensed by this matrix, as reflected by the lower reflectance changes measured for these explosives at a similar concentration range. These results clearly indicate that the imprinting procedure is highly efficient and facilitates the analysis ofpicomolar, or subpicomolar, concentration range of the explosives and that, by the appropriate selection of the imprinting template, highly selective matrixes for each of the three explosives can be easily prepared.

This specific study has demonstrated the fabrication possibility of molecularly imprinted Au NP composites for the detection of PETN, NG, and EGDN explosives. The molecular imprinting process of site-specific matrixes was successfully achieved by applying di- or tricarboxylic acids as structural analogues for the nitrate ester substituents associated with the target explosives. The study has demonstrated that the structure of the carboxylic acids and their steric rigidification are of utmost importance to yield high-affinity sites for

Figure 5.14 (A) Calibration curves relating the reflectance changes to the concentrations of (I) PETN, (II) NG, and (III) EGDN on the citric-

acid-imprinted bis(aniline)-crosslinked Au NP matrix. (B) Calibration curves relating the reflectance changes to the concentrations of (a) EGDN, (b) NG, and (c) PETN on the fumaric-acid-imprinted bis(aniline)-crosslinked Au NP matrix. Error bars correspond to a set of N = 5 measurements. All measurements were performed in ethanol. Reprinted with permission from Ref. [71]. Copyright (2011) American Chemical Society.

the respective explosives. The electropolymerization method used to synthesize the imprinted Au NP complexes provides an effective approach to prepare predesigned specific recognition matrixes for the respective explosives. Also, by employing the oligocarboxylic acid templates, sensing platforms for other explosives may be produced and, particularly, the multiplexed analysis of explosives may be performed.