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Letter pubs.acs.org/NanoLett

Switchable Catalytic Acrylamide Hydrogels Cross-Linked by Hemin/ G-Quadruplexes Chun-Hua Lu,† Xiu-Juan Qi,†,‡ Ron Orbach,† Huang-Hao Yang,‡ Iris Mironi-Harpaz,§ Dror Seliktar,§ and Itamar Willner*,† †

Institute of Chemistry, The Hebrew University of Jerusalem and The Center for Nanoscience and Nanotechnology, Jerusalem 91904, Israel ‡ The Key Lab of Analysis and Detection Technology for Food Safety of the MOE, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, China § Faculty of Biomedical Engineering, TechnionIsrael Institute of Technology, Haifa 32000, Israel S Supporting Information *

ABSTRACT: Copolymer chains consisting of acrylamide units and guanine (G)-containing oligonucleotide-tethered acrylamide units undergo, in the presence of K+ ions, cross-linking by G-quadruplexes to yield a hydrogel. The hydrogel is dissociated upon addition of 18crown-6 ether that traps the K+ ions. Reversible formation and dissociation of the hydrogel is demonstrated by the cyclic addition of K+ ions and 18-crown-6 ether, respectively. Formation of the hydrogel in the presence of hemin results in a hemin/G-quadruplex-crosslinked catalytic hydrogel mimicking the function of horseradish peroxidase, reflected by the catalyzed oxidation of 2,2′-azinobis-(3ethylbenzthiazoline-6-sulfonic acid), ABTS2−, by H2O2 to ABTS.− and by the catalyzed generation of chemiluminescence in the presence of luminol/H2O2. Cyclic “ON” and “OFF” activation of the catalytic functions of the hydrogel are demonstrated upon the formation of the hydrogel in the presence of K+ ions and its dissociation by 18-crown-6 ether, respectively. The hydrogel is characterized by rheology measurements, circular dichroism, and probing its chemical and photophysical properties. KEYWORDS: Catalysis, chemiluminescence, G-quadruplex, hydrogel, switch

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separation of hydrogels through the formation of aptamer− substrate complexes,21 and the enzymatic22 or DNAzymecatalyzed cleavage of the hydrogel bridging units.23 One interesting DNA nanostructure that was extensively studied in the recent years is the G-quadruplex structure.24 Encoded G-rich nucleic acid sequences self-assemble in the presence of ions (e.g., K+, Pb2+, or NH4+) into parallel or antiparallel G-quadruplex structures. Also, hemin binds to the G-quadruplex, resulting in a catalytic horseradish peroxidasemimicking DNAzyme (HRP-mimicking DNAzyme).25 This DNAzyme was found to catalyze the oxidation of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS2−, by the reduction of H2O2 to H2O,25 to catalyze the oxidation of luminol by H2O2, while generating chemiluminescence,26 and to catalyze the oxidation of NADH by O2 or H2O2.27 Also, it was found that the G-quadruplex sequence may be split into subunits, and in the presence of the appropriate ions, the subunits assemble into the G-quadruplex that binds hemin and yields an active HRP-mimicking DNAzyme structure.28 Numerous DNA

ubstantial research efforts are directed to the development of new hydrogel matrices and to their application in various disciplines.1 Different natural,2 synthetic,3 and biohybrid4 hydrophilic polymers undergo gelation, and such matrices provide effective media for trapping macromolecular substrates5 and biomaterials, for example, enzymes6 or nanoparticles.7 Specifically, the switching of hydrogels to solid phases or solution phases by environmental stimuli attracts research interest. Different stimuli such as pH,8 temperature,9 light,10 and supramolecular receptor-ion complexes11 were used to control the formation of hydrogels. Different applications of hydrogel and stimuli-switchable hydrogels were reported, and these included controlled drug delivery,12 scaffolds for tissue engineering,13 the use of stimuli-regulated hydrogels as pumps or valves in microdevices,14 or their application as actuators15 or sensor matrices.16 One specific class of hydrogels includes DNA hydrogels and, specifically, stimuli-controlled DNA hydrogels.17 The cross-linking of the nucleic-acid scaffolds was also demonstrated by aptamer−substrate complexes18 or by the enzymatic ligation of nucleic acid tethers19 linked to DNA. Different stimuli-switchable DNA hydrogels were reported, such as the formation/separation of DNA hydrogels by means of pH (formation or dissociation of i-motif),20 the © XXXX American Chemical Society

Received: January 8, 2013 Revised: February 19, 2013

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dx.doi.org/10.1021/nl400078g | Nano Lett. XXXX, XXX, XXX−XXX

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sensor29 and aptasensor30 platforms were developed using the hemin/G-quadruplex or the hemin/G-quadruplex subunits as catalytic labels. Inspired by the unique self-assembly properties of the Gquadruplexes and by the catalytic properties of the hemin/Gquadruplex, and realizing the stimuli-controlled functions of hydrogels, we addressed the synthesis of a bioinspired acrylamide/hemin/G-quadruplex hydrogel. This hydrogel reveals switchable HRP-mimicking catalytic functions and demonstrates unique luminescence properties. Previously, nucleic acid-functionalized acrylamide copolymers were crosslinked by aptamer-thrombin complexes to form a hydrogel, which was separated by a strand displacement process that removed the aptamer chains and released thrombin.31 Also, the incorporation of transition-metal catalysts into hydrogels and the implementation of the hybrids for catalytic transformations was reported.32 In the present study, we demonstrate a unique example where synthetic acrylamide chains self-assemble into a hydrogel that reveals catalytic and switchable catalytic functions. The synthesis of the acrylamide copolymer chains is outlined in Figure 1. A mixture of the acrylamide monomers and the

Figure 2. (A) Rheological characterization of the G-quadruplex-crosslinked hydrogel: (a) Time-dependent changes of the storage modulus, G′. (b) Time-dependent changes of the loss modulus, G″. Inset: Temperature-controlled changes of the storage modulus upon the thermal dissociation of the hydrogel into solution upon heating to 90 °C, and the recovery of the hydrogel upon cooling to 20 °C. (B) Fluorescence spectra corresponding to protoporphyrin IX zinc(II), 0.1 mM, (a) in pure 10 mM HEPES buffer solution, pH = 7.2. (b) In the presence of the (1)-tethered acrylamide copolymer, 0.3 mM, in the absence of K+ ions. (c) In the hydrogel generated by the K+-induced cross-linking of the (1)-tethered acrylamide copolymer chains.

Figure 1. Scheme for the synthesis of the oligonucleotide tethered acrylamide copolymer chains and their assembly into the Gquadruplex-cross-linked hydrogel.

acrydite-modified oligonucleotides (acrydite-AA GGG, 1) was polymerized to yield the copolymer chains that form a homogeneous aqueous solution. The resulting copolymers consist of an acrylamide:acrydite-(1) with a ratio of 40:1 (for the synthesis details, see the Supporting Information experimental section and Figure S1). Upon the addition of K+ ions, gelation of the copolymer chains proceeds. The formation of the gel is attributed to the formation of interchain G-quadruplexes that cross-link the hydrogel. Rheology studies confirm the formation of the hydrogel. Figure 2A depicts the time-dependent changes of the storage modulus (G′) and loss modulus (G″) upon addition of K+-ions to the mixture of the copolymer chains. A time-dependent increase in G′ is observed (saturated value ca. 5 Pa), whereas G″ is unaffected. The crossover between G′ and G″ immediately upon the addition of the K+-ions implies the formation of a continuous hydrogel polymer network. The resulting hydrogel undergoes reversible thermal dissociation. The Figure 2A inset depicts the storage modulus of the hydrogel as a function of temperature. At temperatures above 80 °C the storage modulus (G′) is lower than the loss modulus (G″), implying that the hydrogel dissociates. Accordingly, the reversible thermo-stimulated hydrogel-to-solution transitions were examined at 90 °C. Subsequent cooling of the solution restores the hydrogel phase, as shown by the G′ in Figure 2A, inset. Interestingly, the temperature-dependent formation of the hydrogel and its

dissociation into solution show a hysteresis curve. Such behavior could be used in the future to store and erase information in the hydrogel structure (for other systems demonstrating hysteresis of a physical parameter to store structural information, see ref 33). The cross-linking of the acrylamide copolymer chains by G-quadruplexes is supported by fluorescence studies (Figure 2B). Previous studies have demonstrated that the association of protoporphyrin IX zinc(II) to G-quadruplexes is associated with a substantial enhancement of the fluorescence of the complex.34 Figure 2B shows that the fluorescence of the protoporphyrin IX zinc(II) in the aqueous solution of the copolymer is 10-fold enhanced upon the K+-induced formation of the hydrogel. Circular dichroism (CD) measurements further support the formation of the G-quadruplex cross-linked hydrogel, and the spectrum of the hydrogel, Figure S2, is consistent with the formation of parallel G-quadruplex nanostructures. It should be noted that the formation of the cross-linked hydrogel is sequence-specific, and other G-containing mutants (Acrydite-AA GAG, 2, and Acrydite-AA AGG, 3) or non-G-containing nucleic acids (Acrydite-AA AAA, 4) do not form the hydrogels (for the images of the G-quadruplex-cross-linked hydrogel and for the B


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results corresponding to the sequences 2, 3, and 4, see Supporting Information, Figures S3, S4 and Table S1). Further support that the acrylamide copolymer hydrogel is cross-linked by G-quadruplexes is obtained upon probing the catalytic activities of the hydrogel upon incorporation of hemin into the composite system. Similarly to DNA G-quadruplexes that bind hemin and form a HRP-mimicking DNAzyme, we find that the incorporation of hemin into the G-quadruplexcross-linked acrylamide hydrogel results in a catalytic gel that mimics the function of HRP. Figure 3A, curve a, shows the time-dependent oxidation of ABTS2− by H2O2 to ABTS•− (λmax = 414 nm) by the K+-induced hemin/G-quadruplexes catalysts that cross-link the acrylamide copolymer chains. Control experiments reveal the significance of the G-quadruplexes to generate the catalytic system. For example, an acrylamide copolymer that includes an oligonucleotide tether (2) that cannot form the G-quadruplex does not lead, in the presence of hemin, to the oxidation of ABTS2−, Figure 3A, curve b. Also, the acrylamide polymer chains that include the G-sequence (1) do not lead, in the presence of hemin, but in the absence of K+ ions, to any catalytic system, curve c. Furthermore, treatment of the hemin/G-quadruplex-cross-linked hydrogel with 18-crown6, a K+-ion chelator, results in the dissolution of the hydrogel and the formation of a solution that reveals a minute catalytic activity toward the oxidation of ABTS2−, Figure 3A, curve d. These results demonstrate that the formation of a catalytic hydrogel requires, indeed, the K+-induced formation of a Gquadruplex and that the association of hemin to the resulting G-quadruplex is essential to form the catalytic sites. It should be noted that the hemin/G-quadruplex-cross-linked hydrogel reveals improved catalytic properties, as compared to the hemin/G-quadruplex DNAzyme formed by the self-assembly of the monomer units consisting of the acrydite-modified oligonucleotide (1). Figure S5 shows the time-dependent absorbance changes of ABTS•−, generated by the catalyzed oxidation of ABTS2− by H2O2 by the hemin/G-quadruplex hydrogel, curve a, in comparison to a homogeneous system that contains the monomer units (1) at the same concentration existing in the hydrogel, curve b. The hemin/G-quadruplex exhibits a ca. 3-fold enhancement. This improved catalytic activity of the hydrogel is attributed to interchain hydrogen bonds between the acrylamide units that cooperatively stabilize the hemin/G-quadruplex bridging units. Furthermore, the observation that the elimination of the K+ from the Gquadruplex units by the crown ether receptor and the dissociation of the hydrogel to a catalytically inactive solution suggests that the catalytic hemin/G-quadruplex hydrogel can be switched to “ON” and “OFF” states by the addition or elimination of K+-ions, respectively. This is demonstrated in Figure 3B, where the cyclic “ON” and “OFF” activation of the hemin/G-quadruplex hydrogel is demonstrated. In this experiment, upon the addition of ABTS2−/H2O2 to the hemin/Gquadruplex-cross-linked hydrogel, the catalyzed oxidation of ABTS2− is activated. At the time-interval marked with an arrow (b), 18-crown-6 is added to the system. This dissociates the hydrogel and switches off the catalytic process. Readdition of K+ to the system, arrow (a), regenerates the catalytic hydrogel, resulting in the reoxidation of ABTS2−. By the cyclic generation of the hydrogel and its separation by the addition of the crown ether, the catalytic activities of the hydrogel are switched between “ON” and “OFF” states, respectively. It should be noted that, as the catalytic hydrogel is cycled between “ON” and “OFF” states, the rate of formation of ABTS.− slightly

Figure 3. (A) Time-dependent absorbance changes (at λmax = 414 nm) upon the catalyzed oxidation of ABTS2− to ABTS•− by H2O2 by: (a) The hemin/G-quadruplex-cross-linked hydrogel system. (b) The use of the mutated (2)-tethered acrylamide copolymer in the presence of hemin and K+ ions. (c) The solution that contains the (1)-tethered acrylamide copolymer, in the presence of hemin and the absence of K+ ions. (d) The (1)-tethered acrylamide copolymer in the presence of hemin, K+, and added 18-crown-6, 75 μM. In all experiments the following concentrations of the components were used: (1) or (2)tethered acrylamide copolymers, 0.3 mM, hemin, 1 μM, H2O2, 125 μM, ABTS2−, 500 μM, KOAc (where applicable) 0.1 mM. All experiments were performed in 10 mM HEPES buffer solution, pH = 7.2. (B) Switchable catalytic functions of the hemin/G-quadruplexcross-linked hydrogel monitored by the time-dependent absorbance changes originating from the catalyzed oxidation of ABTS2− by H2O2 to ABTS•− (λmax = 414 nm). At points (a) K+, 0.1 mM, is added to the mixture of (1) tethered acrylamide copolymer chains to yield the hemin/G-quadruplex-cross-linked hydrogel. At points (b) 18-crown-6, 75 μM, is added to the system to disassemble the hydrogel and yield the solution consisting of the different components. For details of experimental conditions see details in legend (A). The polymer:water ratio (0.2% w/w) implies a highly porous hydrogel matrix, and thus the diffusion of molecular units into the hydrogel and out of the hydrogel should be diffusion-controlled and relatively fast. To enhance the penetration of 18-crown-6 into the hydrogel, the system was vortexed for 5 s after addition of the ligands (points b). The porosity and assisted penetration of ligand lead to the fast switching off response.

decreases with the increase in the number of cycles. This is attributed to the fact that the concentration of ABTS2− decreases as the cycling process proceeds due to its consumption as well as to the slight dilution of the ABTS2− due to the repeated addition of the crown-ether ligand and the C


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K+ solution. In fact, the number of “ON”−“OFF” cycles will be limited by the concentration of ABTS2− and by the ability of the system to form a hydrogel, due to continuous dilution. The catalytic properties of the G-quadruplex-cross-linked acrylamide hydrogel are further reflected by the catalyzed and switchable generation of chemiluminescence. Figure 4, curve a,

platforms. For example, the comodification of the (1)functionalized copolymer chains with aptamer subunits might lead to the cooperative cross-linking of the hydrogel by Gquadruplexes and aptamer−substrate complexes, and the separation of the resulting hydrogel by the removal of K+ ions may lead to a solution that includes the separated substrate.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental section, determination of the acrylamide/ acrydite-(1) units in the copolymer chains, images of the Gquadruplex-cross-linked hydrogel, and CD spectra results corresponding to the sequences 1, 2, 3, and 4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +972-2-6585272. Fax: +972-2-6527715. Notes

The authors declare no competing financial interest.

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Figure 4. Chemiluminescence spectra generated in the presence of luminol, 0.5 mM and H2O2, 10 mM, by: (a) The (1)-tethered acrylamide copolymer 0.3 mM, in the presence of hemin, 1 μM, and absence of K+ ions. (b) The hemin/G-quadruplex cross-linked (1)tethered acrylamide copolymer hydrogel, 0.3 mM. (c) In the presence of the hemin/G-quadruplex cross-linked (1)-tethered acrylamide copolymer hydrogel, 0.3 mM, after the addition of 18-crown-6, 75 μM. Experiments were performed in 10 mM Tris buffer, pH = 9.0.

ACKNOWLEDGMENTS This research is supported by the Volkswagen Foundation, Germany. REFERENCES

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shows that the addition of luminol and H2O2 to a solution consisting of the acrylamide copolymer consisting of the oligonucleotide tethers, (1), and hemin leads to a very low chemiluminescence intensity (characteristic to hemin in the aqueous solution). The K+-induced formation of the Gquadruplex-cross-linked hydrogel with bound hemin, acts, however, as a catalytic matrix for the effective generation of chemiluminescence in the presence of luminol/H2O2, Figure 4, curve b. The addition of 18-crown-6 to the hydrogel separates the gel to a solution phase that lacks the catalytic properties to generate chemiluminescence, Figure 4, curve c. Control experiments confirmed that the K+-stimulated bridging of the acrylamide hydrogel by the hemin/G-quadruplex units yielded the catalytic hydrogel that generates the chemiluminescence. Oligonucleotides that are unable to form the G-quadruplex structures, and are tethered to the acrylamide copolymer chains, did not lead, in the presence of hemin, to any chemiluminescence in the absence or presence of K+-ions. To conclude, the present study has demonstrated the bioinspired assembly of a HRP-mimicking catalytic hydrogel. The rich catalytic properties of G-quadruplex DNAzymes suggest that such transformations could be similarly catalyzed by these hydrogels. The separation of the catalytic hydrogel by the crown ether elimination of K+-ions provides a general method to switch “ON”−“OFF” the catalytic function of Gquadruplex-cross-linked hydrogels. As stated, the thermal hysteresis curve of the dissociation of the hydrogel and its recovery might be used to implement the material for information storage. Nonetheless, other applications of the switchable G-quadruplex-cross-linked hydrogel may be envisaged, particularly in the stimuli-controlled release of drugs from the hydrogel or the development of new analytical separation D


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