SEARCH

SEARCH BY CITATION

Keywords:

  • fluorescence;
  • molecular recognition;
  • self-assembly;
  • sensors

Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information
Thumbnail image of graphical abstract

A fluorescent sensor based on guanidinium-tethered tetraphenylethene (TPE) has been investigated toward the differentiation of pyridine nucleotide cofactors (NAD+, NADH, NADP+, and NADPH). TPE selectively recognizes NADPH possessing the higher tetra-anionic net-charge, resulting in the steep “turn-on” fluorescence increase. The comparative aggregation behaviors and fluorescence response studies of TPE on the four cofactors reveal that the critical aggregate concentration of TPE against NADPH correlates directly with the concentration threshold for the fluorescence response. These results establish that TPE can selectively differentiate NADPH over the other three cofactors by the steep aggregation-induced fluorescence response accompanied by the high signal-to-background contrast.


1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information

The development of fluorescent sensing systems that recognize biologically important ions1 and molecules2 has been spotlighted because the fluorescence technique is highly simple and sensitive, facilitating its potential applications as bioprobes.3 Most of the fluorescent probes are mainly composed of the two constituent units, a fluorophore and a recognition site that binds target ions or molecules via noncovalent intermolecular interactions. This design principle makes it possible to report the binding (molecular recognition) events by a change in the fluorescence emission.4 One of the most prominent methods to detect targets is turn-on fluorescence sensing, which occurs along with the binding, although the fluorophores are intrinsically emissive and quenched at the higher concentration due to their aggregation (self-quenching).5 It seems rather disadvantageous that fluorescent probes based on this basic principle lead to “turn-off” sensing, which inevitably causes the low response as well as the low signal-to-background contrast in the fluorescence detection. However, improved molecular designs have provided target-specific “turn-on” fluorescent probes, which are coded to be “turn-on” switched by recognition-induced disassembling of the probes6 and by changing the electronic state of fluorophore.7 Their fluorescence responses inherently show a linear-saturation relationship until all of the probes are bound with the targets owning to the 1:1 binding mode between the target and the receptor. This intrinsic nature limits precise discrimination of targets from structurally similar biomolecules. For example, adenosine triphosphate (ATP), one of the most important biomolecules involving a universal energy source as well as an extracellular signaling mediator,8 has been difficult to discriminate from its structural analogue, adenosine diphosphate by conventional fluorescence sensors because the fluorescence response is based on the 1:1 binding between the receptor and the phosphate, leading to their concomitant detection.9 This difficulty in the recognition-based sensing seems common among probes for phosphates and nucleobases.10 To overcome this limitation, the development of a novel fluorescence sensing system other than the conventional 1:1 binding mode is indispensable.

The principle of molecular self-assembly is expected to afford a new dimension to the field of fluorescence sensing because subtle difference in the molecular structure can be amplified into the resulting self-assembled structures and spectroscopic properties as an output according to the molecular information encoded.11 The superiority of this strategy has been demonstrated in our previous work on the highly selective chiral recognition via the allosteric control attained by the self-assembly systems.12 Since the allosteric control exhibits nonlinear, sigmoidal information transduction, a small change in the input signal can be amplified into a large change in the output signal. This nonlinear response leads to successful chiral differentiation even if the enantioselectivity is low under the conventional 1:1 stoichiometric system.13 If such a sensing system is introduced to the fluorescence sensing system, precise detection and the differentiation of a concerned target molecule from others would be accomplished by a steep nonlinear fluorescence response. In this context, this system is intrinsically different from the conventional linearly transmitting fluorescence response based on the conventional 1:1 binding via molecular recognition. We have thus focused on a fluorophore exhibiting aggregation-induced emission (AIE) because AIE molecules behave in an opposite manner to intrinsic self-quenching dye molecules.14 Namely, the AIE molecules are endowed to show the “turn-on” fluorescence switching in response to self-assembly. Despite their remarkable advantages, the examples have so far been limited to apply the AIE molecules as sensors for biologically relevant polymers such as DNA,15 polysaccharide,16 and protein.17

The design of an AIE-based fluorescence sensor, tetraphenylethene (TPE) as shown in Figure 1, can be realized by a modular-type structure possessing multiple recognition sites so that it can attain the allosteric function. The TPE has indeed demonstrated selective fluorescence detection of ATP through the cooperative self-assembly that is characterized by the nonlinear fluorescence response together with the high signal-to-background ratio.18 This means that the TPE senses triphosphate out of the other structural analogues of monophosphates and diphosphates through self-assembly. In the present study, we demonstrate that the TPE sensor differentiates biologically relevant four structural analogues, i.e., those of a pyridine nucleotide family (NAD+, NADH, NADP+, and NADPH, shown in Figure 1), which behave as cofactors mediating cellular biocatalytic reactions. Although the four cofactors play an individual role in intracellular metabolic cycles, these cofactors have been probed only by their redox states (oxidized or reduced form).19 As a biologically important process, we can raise glucose-6-phosphate dehydrogenase in pentose phosphate pathway, which generates NADPH from NADP+.20 Since both NADPH and NADP+ are weakly fluorescent, fluorescence monitoring of this process inevitably causes concomitant assay. To the best of our knowledge, no example has ever demonstrated precise differentiation of the cofactors based on their net charges derived from the difference in the number of phosphate groups. More generally, we emphasize here that our methodology utilizing fluorescence sensing via molecular self-assembly promises the direct detection and differentiation of concerned target out of its structural analogues. This technique has so far been unable to be accomplished by a conventional fluorescence sensing based on a 1:1 binding system via molecular recognition, but now has become possible for the first time by focusing the synergistic marriage of fluorescence sensing with molecular self-assembly.

thumbnail image

Figure 1. Chemical structures of TPE (a) and pyridine nucleotide cofactors (b).

Download figure to PowerPoint

2. Results and Discussion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information

2.1. Selective Detection of NADPH among the Four Cofactors

The TPE probe was synthesized according to the previously reported procedure,18 and used for the evaluation of its fluorescence response upon addition of TPE to NAD+, NADH, and NADP+, with the fixed TPE/cofactor ratio ([TPE] = 6.0 × 10−6 M and [cofactor] = 15 × 10−6 M). No significant fluorescence enhancement was observed. In contrast, a strong blue emission was newly observed at 463 nm only when TPE was mixed with NADPH (Figure 2a). In its excitation spectrum, the maximum appeared at 335 nm (Figure S1a, Supporting Information), which is in agreement with the absorption maximum of the TPE–NADPH mixture (Figure S1b, Supporting Information). It must be noted here that the 1,4-dihydronicotineamide moiety of NAD(P)H is weakly emissive and that its excitation maximum and emission maximum observed at 340 and 460 nm, respectively, are overlapped with those of the TPE–NADPH mixture. This means that the excitation wavelength at 335 nm excites both TPE and NAD(P)H itself. However, as the emission from the TPE–NADPH mixture was incomparably stronger than that from the TPE–NADH mixture and increases according to a sigmoidal curvature while that from the TPE–NADH mixture increases according to a linear fashion (vide post), these two systems are clearly distinguishable with high contrast as observable even by our naked eyes (Figure 2b).

thumbnail image

Figure 2. (a) Fluorescence spectra (λex = 335 nm) of TPE (6.0 × 10−6 M) in HEPES buffer (5.0 × 10−3 M, pH 7.4) at 25 °C in the presence of cofactors (15 × 10−6 M). (b) Photograph of TPE (6.0 × 10−6 M) in the presence of the corresponding cofactors (15 × 10−6 M) in HEPES buffer (5.0 × 10−3 M, pH 7.4). The image was obtained under UV irradiation (λex = 300–320 nm).

Download figure to PowerPoint

The fluorescence response from the TPE–NADPH mixture is attributable to the AIE involving the self-assembly of TPE with NADPH as confirmed by dynamic light scattering (DLS) measurement and fluorescence microscopy. Mixing the weakly emissive aqueous solution of TPE (6.0 × 10−6 M) and NADPH (15 × 10−6 M) enhanced the emission intensity by 14-fold when compared with that of NADPH itself (Figure 3a), which directly attained the bright emission with high signal-to-background contrast (Figure 3b). In DLS measurements, the aqueous dispersion showed the presence of aggregates with a mean diameter of 580 nm (Figure 3c). To visualize the origin of the fluorescence emission, the aqueous dispersion was subjected to fluorescence microscopy. As shown in Figure 3d, dispersed fluorescent aggregates were directly observed. These results evidently support the view that the fluorescence enhancement is attributable to self-assembly of TPE and NADPH followed by growing as the larger aggregates. As the aggregation is induced by the specific guanidinium–phosphate interaction,18 the differentiation between NADPH and NADH is attained by the number of phosphate groups. We consider, in particular, that the charge coincidence of TPE (4+) and NADPH (4-) facilitates their aggregation. It is noteworthy that the self-assembly-driven fluorescence enhancement realizes the selective NADPH sensing over NADH with the high signal-to- background contrast.

thumbnail image

Figure 3. (a) Fluorescence spectra (λex = 335 nm) of TPE in the presence of NADPH and NADPH itself. (b) Photograph of TPE (left), TPE in the presence of NADPH (middle) and NADPH itself (right). The image was obtained under UV irradiation (λex = 300–320 nm). (c) Size distribution of the aggregates measured by DLS. (d) Fluorescence image of the suspension obtained by the addition of NADPH to TPE. Conditions: [TPE] = 6.0 × 10−6 M, [NADPH] = 15 × 10−6 M, [HEPES] = 5.0 × 10−3 M (pH 7.4), 25 °C.

Download figure to PowerPoint

The concentration dependence of cofactors on TPE fluorescence is displayed in Figure 4a. Titration of TPE (6.0 × 10−6 M) with NAD+ and NADP+ resulted in no significant fluorescence increase. On the contrary, distinct fluorescence response was observed upon titration with NADH and NADPH. The plot of the TPE–NADH mixture afforded a linear fluorescence increase, which is yet comparable to that of NADH itself (Figure S2a, Supporting Information). This result clearly indicates that the fluorescence emission of the TPE–NADH mixture is originated from NADH itself. This implies that TPE and NADH are molecularly dispersed in water without aggregate formation, leading to the simple linear fluorescence enhancement (inset in Figure S2a, Supporting Information). In sharp contrast, TPE exhibited unique fluorescence response with a nonlinear fashion against the NADPH concentration, whereas NADPH itself showed a linear increase in the fluorescence intensity as is the case with NADH (Figure S2b, Supporting Information). From these results, one can propose that the steep nonlinear fluorescence increase observed only for the TPE–NADPH mixture stems from the aggregation behavior between TPE and NADPH.

thumbnail image

Figure 4. (a) Fluorescence titration curves (λex = 335 nm) of TPE (6.0 × 10−6 M) upon the addition of NAD+, NADH, NADP+ and NADPH in HEPES buffer (5.0 × 10−3 M, pH 7.4) at 25 °C. (b) Plots of scattering intensity as a function of cofactor concentrations ([TPE] = 6.0 × 10−6 M, [HEPES] = 5.0 × 10−3 M pH 7.4, 25 °C).

Download figure to PowerPoint

In order to correlate the fluorescence response with the aggregation behavior, we investigated the dependence of light-scattering intensity on the concentration of cofactors by DLS measurements (Figure 4b). No significant change in the scattering intensity was observed for NAD+ and NADH. This result confirms the view again that the origin of the linear fluorescence response from the TPE–NADH mixture is attributable to the molecularly dispersed NADH. As already shown in Figure 3, the TPE–NADPH mixture indeed formed aggregates attainable to the fluorescence enhancement. Most remarkably, a change in the light-scattering intensity as a function of NADPH concentration revealed that the scattering intensity jumped up at around 7.5 × 10−6 M of NADPH. This noncontinuous increase implies that the minimum aggregate concentration exists in this system (Figure 4b). This result indicates that the minimum aggregate concentration obtained from DLS is in good agreement with that obtained from the fluorescence response. It is undoubted, therefore, that our TPE probe is endowed by fluorescence sensing that possesses the concentration threshold for the fluorescence response.

2.2. Detection of NADP+ with the Aid of SO32− Ion

The concentration dependence of scattering intensity indeed indicates that the TPE–NADP+ mixture does provide the aggregates above 40 × 10−6 M of NADP+ with TPE (Figure S3, Supporting Information). However, no fluorescence enhancement was observed even at 15-fold higher concentration of TPE and NADP+ (Figure S3c, Supporting Information). This behavior is ascribed to fluorescence quenching induced by electron transfer owning to the presence of the electron-deficient pyridinium group as an acceptor. This makes it difficult to distinguish between NAD+ and NADP+ on the basis of the fluorescence technique with TPE. To sense NADP+ discriminating from NAD+, we considered a chemical modification of the pyridinium group in NAD(P)+. Some previous literatures gave us an idea that the addition of SO32− to the pyridinium ring could occur to afford its dihydro-form as a SO3 adduct.21 This reaction would cause the charge conversion from pyridinium cation to sulfonate anion, which is also a specific target of the guanidinium group. The formation of the dihydro-form with higher anionic charge is advantageous for the differentiation of NAD(P)+ by TPE because TPE severely senses the difference in the net charge leading to the formation of fluorescent aggregates. In this context, we examined the fluorescence response of TPE toward the SO3 adducts, i.e., NAD-SO3 and NADP-SO3 (Figure 5a). Addition of Na2SO3 into NAD(P)+ solution caused an increase in absorbance at 320 nm, indicating the generation of the SO3-adduct dihydro-form (Figure S4a, Supporting Information). Whereas addition of TPE to the NAD-SO3 adduct showed no fluorescence enhancement, a significant increase in the fluorescence intensity was observed for the NADP-SO3 adduct (Figure S4b, Supporting Information). Its dependence on Na2SO3 concentration revealed that 5.0 × 10−3 M of Na2SO3 afford the maximum fluorescence intensity, which indicates the importance of the balance between the favorable electrostatic interaction and the unfavorable inhibition by increasing ionic strength (Figure S4c, Supporting Information). These results indicate that the chemical modification of NAD+ and NADP+ to the SO3-adducts enables us to discriminate between these two cofactors by a single TPE probe (Figure 5b).

thumbnail image

Figure 5. (a) Chemical structures of sulfite adducts, NAD-SO3 and NADP-SO3. (b) Fluorescence intensity (λex = 335 nm) of TPE in the presence of NAD-SO3 and NADP-SO3. (c) Photograph of TPE in the presence of NAD-SO3 (left) and NADP-SO3 (right). The image was obtained under UV irradiation (λex = 300–320 nm). Conditions: [TPE] = 6.0 × 10−6 M, [cofactors] = 80 × 10−6 M, [Na2SO3] = 5.0 × 10−3 M, [HEPES] = 5.0 × 10−3 M (pH 7.4), 25°C.

Download figure to PowerPoint

3. Conclusion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information

We demonstrated that the differentiation of the four pyridine nucleotide cofactors by a single AIE-based fluorescent probe is possible. Particularly, TPE selectively recognized NADPH possessing the higher net charge leading to the fluorescence enhancement. The comparative aggregation behaviors and fluorescence response studies of TPE on a series of the four cofactors revealed that the critical aggregate concentration correlates directly with the concentration threshold for the fluorescence response, above which clear “turn-on” fluorescence is observable. We particularly emphasize that this steep fluorescence response accompanied by the high signal-to-background contrast undoubtedly realized clear fluorescence sensing of pyridine nucleotide cofactors. Such a differentiation based on molecular self-assembly has never been realized by conventional fluorescence sensing based on a 1:1 binding system via molecular recognition. Furthermore, we are going to envisage that the molecular strategy for the development of AIE-bioprobes can provide new opportunities to sense biologically important molecules on demand.

Supporting Information

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information

We are grateful to Dojindo Molecular Technologies, Inc. for helpful discussions. This work was financially supported, in part, by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research on Innovative Areas “Emergence in Chemistry” (20111011).

Supporting Information

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Results and Discussion
  5. 3. Conclusion
  6. Supporting Information
  7. Acknowledgements
  8. Supporting Information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
marc_201300015_sm_suppl.pdf4304Ksuppl

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.