SPR Spectroscopy as a Research Tool for Molecular Imprinting of NAD(P)+ and NAD(P)H Cofactors

Preparation of molecularly imprinted polymer

As a polymer base, a copolymer based on acrylamide and 3-acrylamidophenylboronic acid with a moderate content of a crosslinker, iV,ЛГ-methylene bisacrylamide, has been used, which makes the polymer base rather durable and at the same time allows its swelling and shrinking. Each of the oxidized cofactors NAD⁺ or NADP⁺, and the reduced cofactors NADH or NADPH (Fig. 5.2), were imprinted in a crosslinked acrylamide-acrylamidophenylboronic acid copolymer associated with an Au-coated glass support. The method for the generation of the imprinted polymer is schematically depicted in Fig. 5.3.

As the NAD(P)⁺ or NAD(P)H cofactors include ribose units, the cofactors yield a complex with the 3-acrylamidophenylboronic acid. This linkage, together with complementary H-bonds between the different functional units of the cofactors and the acrylamide and 3-acrylamidophenylboronic acid monomers, leads, upon polymerization accompanied by crosslinking in the presence of N,ЛГ-methylene bisacrylamide, to the cofactor-embedded polymer matrixes. To improve the adhesion of the imprinted polymer film on the Au-coated glass support, a primary cystamine monolayer is assembled on the Au surface, and acrylic acid is covalently linked to the monolayer interface. Thus, upon the radical-induced polymerization of the film, the solution-solubilized monomers are copolymerized with the surface-confined monomer units, leading to a surface-bound crosslinked film. The elimination of the template cofactors with 1% NH₃ generates the imprinted sites for the respective cofactors. The existence of functional groups at the periphery of the imprinted molecular contours (boronic acid residues, amide-functionalities, etc.) is expected to yield selective sites for the accommodation of the respective oxidized or reduced cofactors.

Figure 5.3 Schematic preparation of the NAD(P)VNAD(P)H cofactors- imprinted acrylamide-acrylamidophenylboronic acid copolymer sensing film on an Au-coated glass support. The support was primarily modified with cystamine monolayer followed by its coupling to acrylic acid. The scheme exemplifies the imprinting of NAD⁺. Reprinted from Ref. [58], Copyright 2004, with permission from Elsevier.

Detection of NAD+, NADP+, NADH, and NADPH cofactors

Mathematical modeling of the SPR spectra for bare and thin-film- modified gold surfaces (Fig. 5.4) allowed estimating the NAD⁺- imprinted polymer thin film thickness, which was found to be 22 ± 3 nm. The shift in the minimum reflectivity angle observed, before (curve b) and after the imprinted polymer rinsing (curve c), corresponds to the elimination of the imprinted NAD⁺ from the polymer. To estimate the analyte content in the imprinted polymer,

Eq. (5.2) [64] was applied:

where Г is the surface coverage, d is the thickness of the polymer layer, and n₂ correspond to the refractive indexes of media near the gold surface before and after polymer interaction with an aqueous solution of NAD⁺, respectively, and dn/dc reflects the influence of the analyte concentration (c) on the refractive index of the surrounding medium. Using the derived values of n_x and n₂ (1.45 and 1.40, respectively), and dn/dc = 0.188 cm³/g [65], the loading of the imprinted NAD⁺ sites in the polymer was estimated as

22.6 ng/mm².

Figure 5.4 The SPR spectra of (a) the bare gold electrode, (b) the gold electrode after the assembly of NAD⁺-imprinted polymer film, and (c) the gold electrode with NAD⁺-imprinted polymer film after rinsing with 1% NH₃ solution. Adapted from Ref. [58], Copyright 2004, with permission from Elsevier.

Figure 5.5 shows the SPR spectra of the NAD⁺-imprinted polymer film in the absence of NAD⁺ (curve a), and after treatment with NAD⁺, lxlO'³ M (curve b). The minimum reflectivity angle is shifted to lower values. In a control experiment, an acrylamide-acrylamidophenylboronic acid copolymer film was assembled without the imprint of the NAD⁺ cofactor. Treatment of the nonimprinted film with NAD⁺, lxlO'³ M, does not yield any noticeable change in the SPR spectrum of the film. This control experiment clearly indicates that no non-specific binding of NAD⁺

to the polymer takes place. It also reveals that minute changes in the refractive index of the solution resulting upon solubilization of NAD⁺ do not affect the SPR spectrum. Thus, the changes in the SPR spectrum upon interaction with NAD⁺ originate from the specific association of the oxidized cofactor to the imprinted sites. The mathematical modeling of SPR spectrum shown in curve b indicates a decrease in the refractive index of the polymer upon the association of NAD⁺ from 1.45 to 1.40 and an increase in the polymer thickness of 3.0 ± 0.2 nm, as a result of the swelling of the polymer film. The changes in the SPR spectrum of the NADMmprinted polymer film are controlled by the bulk concentration of NAD⁺ in solution.

Figure 5.5 The SPR spectra of the NAD⁺-imprinted polymer recorded (a) after the removal of the NAD* template molecules and (b) after its treatment with NAD⁺ (lxlCT³ M) solution. Adapted from Ref. [58], Copyright 2004, with permission from Elsevier.

The NADMmprinted polymer film reveals selectivity upon analysis of the imprinted cofactor. Figure 5.6 shows the sensogram corresponding to the changes in the minimum reflectivity angles of the SPR spectra in the course of analyzing a range of concentrations of the different oxidized and reduced cofactors solutions by the NAD⁺- imprinted film. In this experiment, the NADMmprinted film was initially treated with solutions containing different concentrations of NAD⁺. Clearly, as the bulk concentration of NAD⁺ increases, the minimum reflectivity angle decreases, and bulk concentrations of NAD⁺ as low as lxlO'⁶ M can be sensed by the system. Upon binding of a substrate to an imprinted polymer film, two processes will affect the SPR spectrum change: a mass increase due to the association of NAD⁺ with the sensitive sensor surface and a decrease in the refractive index of the polymer layer due to the saturation of the polymer with water (the refractive index of water, n = 1.33, is essentially lower than the refractive index of polymer, n₁ = 1.45).

The increase in the mass of the polymer-cofactor complex should be reflected by an increase in the minimum reflectivity angle of the SPR spectrum, whereas the decrease in the refractive index of the film should lower the minimum reflectivity angle of the SPR spectrum. Experimentally, it was observed that the swelling of the polymer upon the binding of NAD⁺ to the imprinted sites results in a decrease in the minimum reflectivity angle, implying that the changes in the refractive index of the polymer, as a result of the swelling, contribute mostly to the overall features of the experimental SPR spectra.

Figure 5.6 The SPR sensogram corresponding to the analyses of lxlO'⁶ M to lxlO^-3 M solutions of (a) NAD⁺, (b) NADH, (c) NADP⁺, and (d) NADPH by the NAD⁺-imprinted polymer film. Run (e) corresponds to repetitive analyses of NAD* by the imprinted film. Adapted from Ref. [58], Copyright 2004, with permission from Elsevier.

After the completion of the analysis cycle of NAD*, the film was washed with water. The bound NAD* is washed off as evident by the fact that the original minimum reflectivity angle of the imprinted film that lacks bound NAD* is achieved. The resulting film was then interacted with the NADH cofactor using different concentrations (lxlO'⁶ M to lxlO'³ M). Only minute changes in the minimum reflectivity angle were observed. Similarly, washing off the bound NADH and treatment of the NADMmprinted film with NADP⁺ or NADPH, respectively, do not yield any changes in the minimum reflectivity angles of the SPR spectra of the film. To reveal that the NADMmprinted film remained functionally intact toward the sensing of NAD⁺, the system that was employed for the analysis of NAD⁺, NADH, NADP⁺, and NADPH was applied again for the analysis of variable concentrations of NAD⁺. Clearly, the changes in the minimum reflectivity angle of the SPR spectra characteristic to the film in the presence of variable bulk concentrations of NAD⁺ were reproduced, similarly to the first analysis cycle of NAD⁺. These experiments clearly indicate that the NADMmprinted polymer film reveals selectivity toward the imprinted substrate, and that the oxidized cofactor NADP⁺ or the reduced cofactors NADH or NADPH exhibit low affinity, or no affinity, for the imprinted sites.

Figure 5.7 depicts the calibration curves corresponding to the changes in the minimum reflectivity angles of the NADMmprinted film upon analysis of the different cofactors. It is obvious that in the concentration range of the cofactor, lxl0~⁵to lxlO'³ M, the determination of NAD⁺ by the proposed method can be carried out selectively. The NADMmprinted film revealed stability for at least 10 days upon daily operation (with only 10% decay in the outcome signal but with no lack in the selectivity).

Similar results of SPR registration were also obtained for polymer films imprinted with NADP⁺, NADPH, and NADH cofactors before and after interaction with lxlO'³ M solution of respective cofactors. As in the case of NAD⁺, binding of other cofactors to the imprinted polymer results in a shift in the SPR minimum reflectivity angle. Theoretical fitting of the SPR spectra after the association of imprinted polymer films with cofactors indicates a decrease in the refractive index of the polymer and an increase in the film thickness due to its swelling. Control experiments revealed that treatment with solutions containing different concentrations of cofactors does not yield any change in the SPR spectrum of a nonimprinted acrylamide-acrylamidophenylboronic acid copolymer film, implying that no non-specific binding of cofactors to the polymer film occurs.

Minute changes in the minimum reflectivity angles of the film are observed in the presence of nonimprinted cofactors, indicating that these cofactors exhibit very little affinity to the imprinted sites. For the reduced cofactor NADH, some decrease in the minimum reflectivity angles was observed upon interaction with the NAD⁺- imprinted polymer. This may be explained by the fact that the reduced cofactors NADH or NADPH always include, as impurities, the oxidized cofactors NAD⁺ and NADP⁺, and thus the observed responses may originate from these components present during polymerization. It is interesting to note that the NADMmprinted polymer is unaffected in the presence of NADP⁺, and the NADPMmprinted film is not affected by the NAD⁺ cofactor. Thus, the imprinting procedure leads to impressive selectivity where the single phosphate substituent differentiating NADP⁺ from NAD⁺ is sufficient to generate specific molecular contours for the respective cofactors.

Figure 5.7 Calibration plots corresponding to the changes in the minimum reflectivity angles at variable concentrations of the NAD(P)⁺/NAD(P)H cofactors: (a) for NAD⁺, (b) for NADH, (c) for NADP⁺, and (d) for NADPH. Adapted from Ref. [58], Copyright 2004, with permission from Elsevier.

These results demonstrate that SPR sensors based on polymers imprinted with NADH or NADPH cofactors can be used to selectively detect the NADH or NADPH molecules, respectively. NADH- and NADPH-imprinted films exhibit cyclic sensing activities, under continuous operation, for at least several days, with no observable degradation of the SPR signals.

SPR Monitoring of Biocatalytic Oxidation of Lactate with NAD+ Cofactor Using NADH-Imprinted Polymer

The NADH-imprinted film was also utilized for SPR analysis of a biocatalytic process that involves the oxidation of lactate to pyruvate by NAD⁺ in the presence of lactate dehydrogenase (LDH) (Fig. 5.8).

Figure 5.8 Biocatalytic oxidation of lactate to pyruvate in the presence of NAD* and lactate dehydrogenase (LDH). Reprinted from Ref. [58], Copyright 2004, with permission from Elsevier.

The NADH being formed in the biocatalytic process binds to the NADH-imprinted film and results in its swelling by the uptake of water. As discussed earlier, the binding of NADH to the imprinted polymer should lead to the increase in the polymer mass and an expected increase in the minimum reflectivity angle. The uptake of water and subsequent swelling, however, decreased the refractive index of the polymer, and concomitantly a decrease in the minimum reflectivity angle was observed similar to the above-mentioned cases.

The effectiveness of the imprinted polymer film toward the sensing of NADH is demonstrated in Fig. 5.9, run (a). Upon interaction with lxlCT³ M NADH, the minimum reflectivity angle of the NADH- imprinted polymer changed by A0_min = 8.2 min. Polymer wash-off with water resulted in its complete regeneration (Fig. 5.9, point (x)). At point (y), the analysis of the biocatalyzed oxidation of lactate was initiated, run (b). At this point, the sensor was brought into contact with a solution containing 0.1 pg/ml LDH and 1хЮ'³ M NAD⁺. It is evident that only a minute change in the minimum reflectivity angle is observed in this case. At point (z) of run (b), after the signal stabilization, lactate, lxlO'² M, was added to the solution. The drastic change in the signal indicates the binding of biocatalytically formed NADH to the NADH-imprinted polymer. Control experiments revealed that addition of lactate alone to the system had no effect on the minimum reflectivity angle. This implies that the observed changes in the minimum reflectivity angle originate from the biocatalyzed generation of NADH. As noted above, the change in the minimum reflectivity angle is due to the uptake of water, and, accordingly, a decrease in the refractive index of the polymer. Here, the minimum reflectivity angle of the polymer film stabilized at a value of A0_min = 7.9 min, which is similar to the signal change registered upon the detection of pure NADH (Fig. 5.9, run (a)). This result is consistent with the fact that the concentration of NAD⁺ in the system was lxlCT³ M, whereas the concentration of lactate was 10-fold higher, lxlO'² M. Thus, the oxidized cofactor NAD⁺ was essentially fully transformed to the reduced cofactor NADH, which was being sensed by the imprinted polymer. By analyzing the kinetic curve (Fig. 5.9, inset), the pseudo first-order rate constant к = 3.2xl0'³s'¹ was calculated for the LDH-biocatalyzed oxidation reaction of lactate by NAD⁺ cofactor.

Figure 5.9 The SPR sensogram corresponding to (run a) the analysis of NADH, and (run b) analysis of the LDH-mediated oxidation of lactate in the presence of NAD*, using the NADH-imprinted polymer film as sensing interface. Inset shows enlarged time-dependent SPR response of the sensing interface in the presence of lactate (lxlO'² M), LDH (lxlO'⁷ g/ml), and NAD* (lxlO'³ M). Adapted from Ref. [58], Copyright 2004, with permission from Elsevier.