ARTICLES PUBLISHED ONLINE: 4 MAY 2014 | DOI: 10.1038/NCHEM.1937

Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel–enzyme hybrids Masato Ikeda1,2, Tatsuya Tanida3, Tatsuyuki Yoshii3, Kazuya Kurotani3, Shoji Onogi3, Kenji Urayama4 and Itaru Hamachi3,5 * Soft materials that exhibit stimuli-responsive behaviour under aqueous conditions (such as supramolecular hydrogels composed of self-assembled nanofibres) have many potential biological applications. However, designing a macroscopic response to structurally complex biochemical stimuli in these materials still remains a challenge. Here we show that redoxresponsive peptide-based hydrogels have the ability to encapsulate enzymes and still retain their activities. Moreover, cooperative coupling of enzymatic reactions with the gel response enables us to construct unique stimuli-responsive soft materials capable of sensing a variety of disease-related biomarkers. The programmable gel–sol response (even to biological samples) is visible to the naked eye. Furthermore, we built Boolean logic gates (OR and AND) into the hydrogel– enzyme hybrid materials, which were able to sense simultaneously plural specific biochemicals and execute a controlled drug release in accordance with the logic operation. The intelligent soft materials that we have developed may prove valuable in future medical diagnostics or treatments. here has been a growing interest in soft matter1, the macroscopic structure and physical properties of which undergo detectable changes in response to external stimuli. Particularly, soft materials with the ability to function under aqueous conditions, such as hydrogels, are attractive because of their potential applications in bioassays2, controlled drug release3,4 and regenerative medicine5. The majority of stimuli-responsive hydrogels developed to date respond to simple physical or chemical stimuli2–6. In sharp contrast, living systems use diverse structurally complex biochemical stimuli to maintain homeostasis7,8. These biochemicals are regarded as indicators of health status (for example, D-glucose in diabetes9) and thus as biomarkers10–12. Although soft materials with the ability to sense biomarkers selectively and respond appropriately are highly desirable, the extraordinary diversity and structural complexity of biomarkers has impeded the rational design and synthesis of stimuli-responsive materials. Recently, supramolecular hydrogels composed of self-assembled small molecules (hydrogelators) and large amounts of water (typically over 99%) have emerged13–17. Various biological applications of supramolecular hydrogels, which include bioassays18–21 and regenerative medicine22–24, have been actively explored. In such hydrogels, small structural changes of hydrogelators induced by stimuli are amplified to bring about macroscopic gel–sol phase transitions. This property has been exploited to design programmable materials based on the elaborate chemical synthesis of the hydrogelators. However, the majority of chemical-stimuli responsive supramolecular hydrogels reported so far have the limitation of being able to respond only to structurally simple chemical stimuli such as pH22,25,26, cations (or ionic strength)23,24,27,28 or anions27,29. This is ascribed mainly to the fact that, so far, most biochemicals are too complex for synthetic chemists to prepare small molecular

T

scaffolds capable of selectively recognizing and sensing them. For biochemical stimuli, supramolecular hydrogels responsive to ligand–receptor binding30,31 or enzyme activities32–34 have been developed. However, these examples exploited the peptide-based hydrogelators themselves as a ligand for the receptor or the enzyme substrates, which thereby demanded elaborate design and synthesis of specialized hydrogelator molecules. Very recently, several groups, including us35, have focused on the redox-controlled self-assembly of rather simple hydrogelators36–38. Based on these gelators, we report here a new and simple strategy for constructing, without tedious synthetic effort, supramolecular hydrogels responsive to a variety of structurally complex biochemical stimuli. Using hydrogen peroxide (H2O2)-responsive hydrogels35 as a universal matrix, it was demonstrated that programmable hybridization of hydrogels with oxidases enables these materials to respond to input stimuli ranging from simple H2O2 molecules to a variety of disease-related biomarkers, which include glucose, lactose, uric acid, choline, acetylcholine and sarcosine. Also, the hybrid hydrogel was demonstrated to respond to the higher glucose content in samples of human blood plasma. This strategy was validated further by developing a second hydrogel type that is responsive to physiological reducing agents such as NADH and lactic acid. Additionally, into these supramolecular hydrogels we were able to build successfully OR and AND logic-gate responses towards several biochemicals. The soft materials we developed here allowed us not only to detect the presence of various biomarkers in solution with the naked eye, but also to regulate logically the release of therapeutic antibodies. These intelligent hybrid hydrogels can be expected to be suitable platforms for the development of advanced soft materials for the diagnosis and treatment of various diseases, including diabetes, gout and cancer.

1

Department of Biomolecular Science, Graduate School of Engineering, Gifu University, Gifu 501-1193, Japan, 2 United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan, 3 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan, 4 Department of Macromolecular Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Kyoto 606–8585, Japan, 5 Japan Science and Technology Agency (JST), CREST, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. * e-mail: [email protected] NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry

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Figure 1 | BAmoc–peptide hydrogels. a, BAmoc–peptides (FFX: F, phenylalanine; X: OH, isoleucine (I), leucine (L) or phenylalanine (F)). b, Schematic representation showing the self-assembly of BPmoc–peptides to form a nanofibre network (gel) and H2O2-triggered gel degradation. c, CGC (wt%) values of BAmoc–peptide gels. Conditions: 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.1 for BPmoc-F2 and 7.0 for all other experiments). d,e, TEM (d) and CLSM (e) images of the BPmoc-F3 gel (staining was done with a fluorescent dye). (See Supplementary Fig. 1 for atomic force microscopy and Supplementary Fig. 2 for the fluorescent dye used in CLSM observations.).

Results Discovery of peptide-based hydrogelators that bear H2O2triggered degradable units. To develop H2O2-responsive supramolecular hydrogels consisting of amino acids, we constructed a small chemical library of di- and tripeptides that contained an H2O2-reactive boronoarylmethoxycarbonyl (BAmoc) group at their N terminal (Fig. 1a). Removal of the BAmoc unit through an oxidation/elimination reaction triggered by H2O2 was expected to induce destabilization of the self-assembled nanofibres so that a collapse of the gel and hence a gel–sol transition occurred (Fig. 1b). Using a conventional tube-inversion method, we found that five peptide derivatives from our library formed hydrogels efficiently. The critical gel concentration (CGC) varied depending on the nature and degree of the structural change. Thus, CGC values for BPmoc-F2 , BPmoc-F2L, BNmoc-F2 and BPmoc-F3 (BPmoc, boronophenylmethoxycarbonyl; BNmoc, borononaphthylmethoxycarbonyl; F, phenylalanine; L, leucine) were 1.5 wt% (31 mM), 0.4 wt% 512

(6.6 mM), 0.5 wt% (0.93 mM) and 0.05 wt% (0.78 mM), respectively (Fig. 1c). These results suggested that the increased hydrophobicity of the BAmoc group from BPmoc to BNmoc and/or the elongated peptide sequence with amino acids that bear hydrophobic side chains substantially decreased the CGC39. The gel formation was also pH dependent owing to the equilibrium between carboxylic acid and carboxylate forms at the C terminal. Accordingly, BPmoc–tripeptides (BPmoc-F2I, BPmoc-F2L and BPmoc-F3) and the BNmoc– dipeptide (BPmoc-F2) formed hydrogels under neutral pH (pH 7.0), whereas BPmoc-F2 formed a stable hydrogel only under acidic conditions (pH 6.1); this hampered us from utilizing a number of enzymes that functioned optimally at or near physiological pH (7.4). Because the CGC of BPmoc-F3 was the lowest under neutral pH, we primarily employed BPmoc-F3 hydrogels in our studies. We characterized the well-developed nanofibres and their entangled networks in the BPmoc-F3 hydrogels by transmission electron microscopy (TEM) and confocal laser-scanning microscopy (CLSM) experiments (Fig. 1d,e). NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry

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a [H2O2]/[BPmoc-F3] 0

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Figure 2 | H2O2-response sensitivity of BPmoc–peptide hydrogels. a, Chemical structure of BPmoc-F3 and photographs of BPmoc-F3 hydrogels after the addition of varying amounts of H2O2. Conditions: [BPmoc-F3] ¼ 0.10 wt% (1.6 mM), 100 mM MES (pH 7.0), V(gel):V(H2O2) ¼ 10:1 (V(gel) and V(H2O2) denote, respectively, the volumes of hydrogel and H2O2 solutions added), ambient temperature, two hours. b, H2O2 response sensitivities of BPmoc-F2 (1.5 wt% (31 mM)), BPmoc-F2L (0.60 wt% (9.9 mM)) and BPmoc-F3 (0.10 wt% (1.6 mM)). The H2O2 sensitivity of BPmoc-F2I gel could not be determined accurately because of the mechanical weakness of the gel. Conditions: 100 mM MES (pH 6.1 for BPmoc-F2 and 7.0 for the others), V(gel):V(H2O2) ¼ 10:1, ambient temperature, two hours. c, Photographs of BPmoc-F3 gels (0.10 wt%) after the addition of ROS. Conditions: 100 mM MES (pH 7.0), ROS: [O22, OCl2]/[BPmoc-F3] ¼ 1.0, V(gel):V(ROS) ¼ 10:1, ambient temperature, two hours.

Also, the rheological data of the BPmoc-F3 hydrogel showed the typical viscoelastic property of the hydrogel that consists of fibre networks (Supplementary Fig. 3)19,24,25,31,36–38. H2O2-responsive collapse of BPmoc–peptide hydrogels. The superior CGC of BPmoc-F3 (Fig. 2a) had a direct impact on its H2O2 sensitivity. The H2O2-response sensitivity of the BPmoc-F3 hydrogel was evaluated by following the gel–sol transition after the addition of 0.25–1.0 molar equivalent of H2O2 relative to the amount of BPmoc-F3 present in the gel. The results showed that more than 0.5 molar equiv. H2O2 (8 mM, 10 ml) was required to induce a complete collapse of the gel (0.1 wt% (1.6 mM), 100 ml) after two hours (Fig. 2a). The time required for the gel–sol transition ranged from one to six hours, dependent on the amount of H2O2 (Supplementary Fig. 6). In contrast to BPmoc-F3 , relatively larger amounts of H2O2 were required for the BPmoc-F2 and BPmoc-F2L gels (38-fold and tenfold, respectively) to collapse (Fig. 2b). The order of H2O2 sensitivity for the hydrogels was found to be BPmoc-F3 (8 mM, 80 nmol) . BPmoc-F2L (80 mM, 800 nmol) . BPmoc-F2 (300 mM, 3 mmol), in good agreement with the order of CGC. Analysis of the gel after the addition of H2O2 by using high-performance liquid chromatography/electron spray ionization mass spectrometry showed the presence of three major compounds, one of which was the F3 peptide (Supplementary Fig. 10)35. This revealed that the collapse of hydrogel occurred by gelator degradation that involved H2O2-triggered cleavage of the carbamate bond present in BPmoc-F3 , and thereby produced the F3 peptide accompanied by the generation of p-quinone methide, boric acid and CO2 (Fig. 1b). The selectivity of BPmoc-F3 gel towards H2O2 over other reactive oxygen species (ROS), such as O22 and OCl2, was also examined. These experiments confirmed that only H2O2 among the ROS tested induced the gel–sol transition (Fig. 2c). Our results are consistent with the reactivity of a boron-appended H2O2 chemosensor40. Expansion of chemical stimuli by encapsulating enzymes in H2O2-responsive BPmoc-F3 gels. H2O2 is generated by the action

of natural oxidases on a variety of physiologically important biochemicals. If these oxidases are entrapped in the aforementioned hydrogel matrices and retain their activities, H2O2 generated in situ by the oxidation of their substrates could trigger a gel–sol transition. Thus, stable encapsulation of glucose oxidase (GOx) in BPmoc–peptide gels (GOx,BPmoc–peptide hybrid gels) converted the H2O2 response into a glucose response (Fig. 3a). The minimal concentration of glucose solution required for complete gel collapse, when 10 ml glucose solution was used against 100 ml gel, decreased dramatically from 300 mM for GOx,BPmoc-F2 , 75 mM for GOx,BPmoc-F2L to 18 mM for GOx,BPmoc-F3 , in good agreement with the order of H2O2 sensitivity and CGC. As expected, the more H2O2 responsive the hydrogel was, the higher the glucose sensitivity of the enzymeencapsulated hydrogel. The time required for the gel collapse was dependent on the amount of glucose; that is, one hour for 24 mM, two hours for 18 mM and no gel–sol change after 24 hours for ,12 mM (Supplementary Fig. 6). The response time against glucose was almost comparable to that against H2O2. For a rapid gel–sol change, the gel was downsized by tenfold to construct a gel array (see Methods for the details). The response time became faster (for example, from two hours to one hour for 18 mM glucose) and the H2O2 response was faster than the glucose response (Supplementary Fig. 7), which suggests that the reaction rate between BPmoc-F3 and H2O2 , the diffusion rate of substances and the enzymatic production rate of H2O2 should be taken into account to regulate the response time. As the glucose sensitivity of the GOx,BPmoc-F3 hybrid gel was comparable to the blood glucose levels found in patients with diabetes mellitus, we next sought to analyse human blood samples. By carefully tuning the concentration of BPmoc-F3 , we succeeded to construct the hybrid gels that showed the threshold type of gel–sol change only on the addition of blood plasma containing glucose at the hyperglycaemic levels (Fig. 3b, Supplementary Fig. 12). This result revealed that the GOx,BPmoc-F3 hybrid gel is able to detect the analyte even in the presence of serum proteins and other

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Sol

Choline

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SOx

Glucose

OH

+ N

GOx

UOx

O Ox BPmoc-F3 hybrid gel

Figure 3 | Oxidase,BPmoc-F3 hybrid gels. a, Photographs of GOx,BPmoc-F3 hybrid gels after the addition of varying amounts of glucose. Conditions: [BPmoc-F3] ¼ 0.10 wt% (1.6 mM), 100 mM MES (pH 7.0), [GOx] ¼ 2.8 mM, V(gel):V(glucose) ¼ 10:1, 37 8C, two hours. b, Photograph of GOx,BPmoc-F3 hybrid gel array on a flat glass slide after the response to blood plasma that contained different amounts of glucose (see the Supplementary Information for details). Gel spots (4 mm diameter) incubated with blood plasma that contained glucose at hyperglycaemic levels resulted in gel degradation and were washed away. Conditions: [BPmoc-F3] ¼ 0.10 wt% (1.6 mM), 100 mM MES (pH 7.0), [GOx] ¼ 2.8 mM, V(gel):V(blood plasma) ¼ 10:1, 37 8C, three hours. c, Schematic representation of Ox,BPmoc-F3 hybrid gels. Information in the form of molecular input is received by the enzyme and converted into H2O2 , which eventually gives rise to a gel–sol change as output through the degradation of a matrix that consists of H2O2-responsive nanofibres. d, Photograph of the hybrid gel array on flat glass slide after the response to analytes (see Methods for the details). Conditions: [BPmoc-F3] ¼ 0.15 wt% (2.4 mM), 100 mM MES (pH 7.0), [analyte]/[BPmoc-F3] ¼ 1.0, [GOx] ¼ 2.7 mM or [SOx] ¼ 6.7 mM or [COx] ¼ 4.6 mM or [UOx] ¼ 3.6 mM, V(gel):V(analyte) ¼ 10:1, 37 8C, four hours.

contaminants and thus strongly suggested a potential for practical sensing applications. The formation of stable hydrogels from BPmoc-F3 under neutral pH allowed us to encapsulate a variety of oxidases (Ox), such as choline oxidase (COx), urate oxidase (UOx) and sarcosine oxidase (SOx), in a BPmoc-F3 gel matrix (Fig. 3c) and still retain their activities. The specific stimuli that induced the degradation of hydrogel, and hence a gel–sol transition, was successfully diverse to include a variety of other biomolecules. Thus, the COx,BPmoc-F3 hybrid gel underwent a selective gel–sol transition in response to 1.0 molar equiv. choline (1 ml analyte solution (24 mM) against 10 ml gel (2.4 mM)), but was unresponsive to other stimuli (Fig. 3d, column 3). Similarly, the UOx,BPmoc-F3 hybrid gel (Fig. 3d, column 4) underwent a selective gel–sol change induced by uric acid, and sarcosine selectively induced the gel–sol transition of the SOx,BPmoc-F3 hybrid gel (Fig. 3d, column 2). These results clearly indicate that H2O2-responsive BPmoc-F3 gels can be used as universal matrices for various oxidases to develop unique soft materials that will then undergo visible phase transitions in response to a variety of biologically important substances as the analytes. We also demonstrated that increasing the volume of analyte solution from 20 ml to 200 ml against a fixed volume of hybrid gel (100 ml) 514

decreased (from 1.0 mM to 0.4 mM) the threshold concentration of uric acid required for sensing, a value within the range of blood uric acid concentration found in patients with hyperuricaemia (Supplementary Fig. 13). An abnormally high concentration of uric acid in the blood (hyperuricaemia) leads to gout pain10,11 and the presence of sarcosine in urine could be used as a measure of the malignancy of prostate cancer cells12. The detection of analytes using hybrid gels is not restricted to the substrates of oxidases. For instance, coupling of hydrolases that produced a substrate for Ox with the corresponding Ox allowed further expansion of the analytes. When BPmoc-F3 gel encapsulated both COx and acetylcholine esterase (AChE) to produce the AChE/COx,BPmoc-F3 hybrid gel (Fig. 4a), the gel–sol transition could be induced by the addition of either acetylcholine or choline (Fig. 4bi, columns 1 and 3; Supplementary Fig. 14). In contrast, the gel devoid of encapsulated AChE failed to undergo gel–sol transition in response to acetylcholine (Fig. 4bi, column 2). In the AChE/COx hybrid gel, acetylcholine was hydrolysed into choline by the action of AChE, which subsequently was oxidized by COx. It was conceivable that such tandem reactions generated H2O2 in situ, which caused the gel collapse (Fig. 4a). The serial coupling strategy was applied successfully to develop hybrid gels that contained other NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry

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DOI: 10.1038/NCHEM.1937

a

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Figure 4 | Serial coupling of enzymatic reactions in the BPmoc-F3 hydrogel for expansion of a chemical-stimuli response. a, Schematic representation of the AChE/COx,BPmoc-F3 hybrid gel. The serial coupling of enzymatic reactions of AChE and COx enables the conversion of both choline and acetylcholine (input) into H2O2 , which eventually gives rise to a gel–sol change as the output through the degradation of a matrix that consists of H2O2-responsive nanofibres. b, Photographs of the hybrid gel arrays on flat glass slides after the response to analytes (see Supplementary Fig. 14 for the comprehensive experimental details). Conditions: [BPmoc-F3] ¼ 0.15 wt% (2.4 mM), 100 mM MES (pH 7.0); i, enzymes [AChE] ¼ 1.5 mM and [COx] ¼ 4.3 mM; ii, enzymes [b-Gal] ¼ 0.77 mM and [GOx] ¼ 2.6 mM; [analyte]/[BPmoc-F3] ¼ 1.0, V(gel):V(analyte) ¼ 10:1, 37 8C, one hour.

a

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Figure 5 | Reduction-responsive hydrogel and its combination with enzymes. a, Chemical structure of NPmoc-F2. b, Photographs of NR,NPmoc-F2 hybrid gels after the addition of NADH or NADþ. Conditions: [NPmoc-F2] ¼ 0.40 wt% (8.1 mM), 100 mM MES (pH 6.6), [NR] ¼ 5.2 mM, [NADH]/[NPmoc-F2] ¼ 1.0, [NADþ]/[NPmoc-F2] ¼ 10, V(gel):V(NADH or NADþ) ¼ 10:1, ambient temperature, 30 minutes for NADH and nine hours for NADþ. c, Schematic representation of the LDH/NR(NADþ),NPmoc-F2 hybrid gel. With the assistance of NR, NADH (input) can induce a gel–sol change as output through the degradation of a matrix that consists of reduction-responsive nanofibres. Also, NADþ in the hybrid gel is reduced to NADH during the oxidation of lactic acid (input) by LDH (NADþ-dependent dehydrogenase), which eventually gives rise to a gel–sol change as output through the degradation of the matrix. d, Photograph of the hybrid gel array on a flat glass slide after the response to analytes. Conditions: [NPmoc-F2] ¼ 0.40 wt% (8.1 mM), 100 mM MES (pH 7.2), [NADH]/[NPmoc-F2] ¼ 10, [lactic acid]/[NPmoc-F2] ¼ 10, [NADþ]/[NPmoc-F2] ¼ 10, [NR] ¼ 2.5 mM, [LDH] ¼ 7.1 mM, V(gel):V(analyte) ¼ 10:1, 37 8C, two hours.

enzyme pairs such as b-galactosidase (b-Gal) and GOx, in which lactose is sensed via b-al-catalysed glycolysis into glucose (and galactose) (Fig. 4bii).

Expansion of chemical stimuli by encapsulating enzymes in NPmoc-F2 gel. Based on the strategy that we used to design the H2O2-responsive BPmoc–peptide gels and hydrogel–enzyme

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Figure 6 | AND or OR logic-gate response of supramolecular hydrogels encapsulating multiple enzymes. a, Schematic of the OR logic-gate response of the GOx/COx,BPmoc-F3 hybrid gel (left) with a photograph of the GOx/COx,BPmoc-F3 hybrid gel array on a flat glass slide after the response to analytes (centre) and the truth table (right). Conditions: [BPmoc-F3] ¼ 0.15 wt% (2.4 mM), 100 mM MES (pH 7.0), [GOx] ¼ 3.1 mM, [COx] ¼ 5.7 mM, [glucose]/[BPmoc-F3] ¼ 1.0, [choline]/[BPmoc-F3] ¼ 1.0, V(gel):V(analyte) ¼ 10:1, 37 8C, two hours. b, Schematic representation of the NR/GOx,NPmoc-F2 , BPmoc-F3 hybrid gel. The AND input (NADH and glucose) is necessary to induce a gel–sol change as the output through the degradation of the matrix. c, AND logic-gate response of the hybrid gel (left) with a photograph of NR/GOx,NPmoc-F2 ,BPmoc-F3 hybrid gel array on a flat glass slide after the response to analytes (centre) and the truth table (right). Conditions: [NPmoc-F2] ¼ 0.30 wt% (6.1 mM), [BPmoc-F3] ¼ 0.10 wt% (1.6 mM), 100 mM MES (pH 7.0), [NR] ¼ 2.4 mM, [GOx] ¼ 3.1 mM, [NADH]/[NPmoc-F2] ¼ 2.0, [glucose]/[BPmoc-F3] ¼ 2.0, V(gel):V(analyte) ¼ 10:1, 37 8C, two hours. d, Release of encapsulated rhodamine-IgG from the NR/GOx,NPmoc-F2 ,BPmoc-F3 hybrid gel after the trigger of the AND input (photographs, excitation wavelength at 530–550 nm, fluorescence filter .580 nm). Conditions: [NPmoc-F2] ¼ 0.40 wt% (8.1 mM), [BPmoc-F3] ¼ 0.10 wt% (1.6 mM), 100 mM MES (pH 7.0), [NR] ¼ 5.9 mM, [GOx] ¼ 9.8 mM, [NADH] ¼ 40.5 mM (buffer), [glucose] ¼ 3.2 mM (buffer), V(gel):V(buffer) ¼ 3:13, 37 8C.

hybrids described above, a reduction-responsive supramolecular hydrogel was developed successfully (NPmoc-F2)35 (Fig. 5a). Reduction of the nitro group could trigger the cleavage of the carbamate bond in NPmoc-F2 , and thereby lead to a collapse of the gel and result in a gel–sol transition. Interestingly, we found that NADH, and not NADþ, induced the gel–sol transition when nitroreductase (NR) was encapsulated in NPmoc-F2 gel (NR,NPmoc-F2 hybrid gel) (Fig. 5b). As NR required NADH as a cofactor to reduce nitro groups present in its substrates, NADH acted as a signal that triggered the collapse of the NR,NPmoc-F2 hybrid gel. NADH/NADþ is a common coenzyme for numerous redox enzymes, so the NADH response can be extended widely to biologically important analytes by coupling other redox enzymes. Thus, when the NPmoc-F2 hybrid gel encapsulated lactic acid dehydrogenase (LDH) together with NR and NADþ (that is, the LDH/NR(NADþ),NPmoc-F2 hybrid gel, Fig. 5c), the hybrid gel 516

exhibited a gel–sol transition dependent on lactic acid (Fig. 5d, columns 4 and 5). Such lactic-acid sensing by the gel collapse was clearly ascribed to the following cascade reactions: NADþ is reduced to NADH during the oxidation of lactic acid by LDH in the hybrid gel, which facilitates the subsequent reduction of NPmoc-F2 by NR with the aid of the generated NADH. In contrast, no gel–sol change was observed without NADþ encapsulation (Fig. 5d, column 3). Excess amounts of NADþ and lactic acid (for example, [NADþ]/[NPmoc-F2] ¼ 10) were required to induce the gel–sol transition in an acceptable response time (several hours), most probably because of the rather slow rate of NADH production through the oxidation of lactic acid by LDH. Given that the lacticacid concentration increases around tumour tissues because of the elevated rates of aerobic glycolysis41,42, after further improvement in its sensitivity, this intelligent material responsive to lactic acid is expected to find applications in cancer diagnosis and treatment. NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry

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Logic-gate response by encapsulation of multiple enzymes or by coupling a BPmoc-F3 gel with a NPmoc-F2 gel. Finally, we sought to construct hybrid gels that exhibited a Boolean logic-gate response towards multiple biologically important stimuli as inputs. Two distinct strategies, namely simultaneous encapsulation of multiple enzymes or a combination of two different gelators, were tested. BPmoc-F3 gels that encapsulated two of the aforementioned four oxidases (for example, the GOx/COx,BPmoc-F3 hybrid gel) exhibited an OR logic-gate response towards two input stimuli (Fig. 6a). That is, either one of the inputs (glucose or choline) caused a gel–sol change as the output. Similarly, AChE/COx,BPmoc-F3 (Fig. 4bi) and LDH/NR(NADþ),NPmoc-F2 (Fig. 5d) exhibited the OR logic-gate response towards two distinct inputs, namely acetylcholine or choline, and lactic acid or NADH, respectively. In contrast, the AND logic-gate response was achieved by combining oxidation-responsive (BPmoc-F3) and reduction-responsive (NPmoc-F2) hydrogelators. Simple mixing of the GOx,BPmoc-F3 hybrid gel with the NR,NPmoc-F2 hybrid gel gave rise to a hybrid gel (that is, NR/GOx,NPmoc-F2 ,BPmoc-F3 hybrid gel (Fig. 6b)) that exhibited an AND logic-gate response towards both NADH (input 1) and glucose (input 2). Thus, the addition of NADH and glucose was necessary to induce a gel–sol transition (Fig. 6c). Preliminary structural analyses of this mixed gel revealed that NPmoc-F2 and BPmoc-F3 were mixed rather than perfectly segregated (or self-sorted) from each other43 (see Supplementary Fig. 5), which may suggest that the AND logic-gate response in this hybrid system did not require perfectly segregated gel fibres44,45. More significantly, the supramolecular hybrid gel (NR/GOx,NPmoc-F2 ,BPmoc-F3) executed a controlled drug release in response to the input patterns through AND logic-regulated macroscopic gel–sol changes. When the AND input (addition of both NADH and glucose) was applied to the hybrid gel, the gel collapsed within five hours, releasing about 90% of the rhodamine-labelled immunoglobulin G (rhodamine-IgG) initially encapsulated in the gel matrix (Fig. 6d). In contrast, neither the collapse of the gel nor substantial IgG release was observed by the addition of either one of the inputs or no input. The results demonstrate that our enzyme–hydrogel hybrids are promising intelligent biomaterials with potential applications in disease diagnosis and treatment.

Discussion By combining the redox sensitivity of peptide-based supramolecular hydrogels and their ability both to encapsulate enzymes and retain their activities, we have successfully developed a new generation of intelligent soft materials with the ability to undergo macroscopic gel–sol changes in response to a variety of physiologically significant biomolecules. The hydrogel–enzyme hybrids efficiently converted diverse physiological stimuli into H2O2 , either directly or through a series of enzymatic reactions, and thereby induced a gel–sol transition via H2O2-mediated decomposition of the peptide hydrogelators. Thus, in this strategy, one does not need to synthesize many hydrogels responsive to each chemical stimulus, but instead only one hydrogel that exhibits a H2O2 response is enough to construct advanced soft materials responsive to a variety of stimuli. A similar design strategy was applied to reduction-sensitive hydrogels by hybridizing with NADH/NADþ-dependent enzymes, so that the sensing chemicals were extended more widely. A combination of a pH-responsive hydrogel and enzymatic reactions that lead to pH change might be an alternative way to diversify sensing chemicals26; however, pH sensitivity of enzymatic activity or the presence of various substances capable of buffering pH change in biological fluids greatly restricted its utility. More interestingly, the oxidation- and/or reduction-sensitive hydrogel that encapsulates multiple enzymes showed a capacity for OR or AND logic-type sensing and intelligent drug-release responses

towards multiple biochemical substances as input stimuli. To date, the molecular logic-gate materials actively explored involved inputs that were rather simple, such as pH, cations or anions in most systems28,46, and outputs limited to physical signals, such as fluorescence intensity47,48 or electric signals48, in most cases, except for semibiological RNA- or DNA-based molecular devices49,50. The general strategy to couple supramolecular materials responsive to chemical stimuli and retain enzyme activities to install the flexible logic response to diverse biochemicals, described here, is a highly versatile approach for the design of intelligent soft materials.

Methods Encapsulation of GOx into a BPmoc-F3 hydrogel and glucose assay. An aqueous solution of BPmoc-F3 (0.15 wt%, 100 mM MES buffer (pH 7.0)) was prepared by heating until a homogeneous solution was obtained and subsequently cooled to room temperature. Before gelation, to the aqueous solution of BPmoc-F3 (100 ml) was added an aqueous solution of GOx (50 mg ml21, 10 mM HEPES buffer (pH 7.2), 1.0 ml), and the solution was incubated at room temperature for 30 minutes to form a hydrogel. For the glucose assay, an aqueous solution of glucose (10 ml) was added to the GOx,BPmoc-F3 hydrogel hybrid and incubated at 37 8C to evaluate gel degradation. BPmoc-F3 hydrogels that encapsulated other enzymes were prepared according to a similar procedure and used for the respective assays. Preparation of an enzyme,BPmoc-F3 hybrid gel array and its assay. An aqueous solution of BPmoc-F3 (0.15 wt%, 100 mM MES (pH 7.0), 10 ml) was prepared as described above and an aqueous solution of GOx (50 mg ml21, 10 mM HEPES (pH 7.2), 0.5 ml) was spotted on a 24-well glass plate (f ¼ 4 mm) and mixed. The spot was allowed to stand at room temperature for at least 30 minutes to afford an enzyme,BPmoc-F3 hybrid gel array. For the analyte assay, 1.0 ml analyte solution was added to each gel spot and incubated for four hours at 37 8C. After this, the glass plate was washed carefully with water by immersion in a beaker of water (typically, 400 ml water in a 500 ml beaker) for approximately one minute at room temperature. Gel spots incubated with the target analytes (substrates of encapsulated enzymes) showed gel–sol transitions, and the corresponding spots were washed away with water. The gel array was photographed using a digital camera (Olympus, PEN E-PL2) before and after the addition of analytes. Encapsulation of NR into the NPmoc-F2 hydrogel for a NADH assay. An aqueous solution of NPmoc-F2 (0.45 wt%, 100 mM MES (pH 6.6)) was prepared by heating until a homogeneous solution was obtained, which was subsequently cooled to room temperature. To the aqueous solution of NPmoc-F2 (195 ml), an aqueous solution of NR (2.5 mg ml21, 10 mM HEPES (pH 7.2), 5.0 ml) was added, and the solution was incubated at room temperature for seven hours to form the hydrogel. For the NADH assay, an aqueous solution of NADH (20 ml) was added to the NR,NPmoc-F2 hybrid hydrogel, and this was incubated at 37 8C to evaluate the gel–sol transition. Preparation of the NR/GOx,NPmoc-F2 ,BPmoc-F3 hybrid gel array and its assay. An aqueous solution of NPmoc-F2 (0.30 wt%) and BPmoc-F3 (0.10 wt%) in 100 mM MES (pH 7.0) was prepared by heating until a homogeneous solution was obtained, which was subsequently cooled to room temperature. The aqueous solution of NPmoc-F2 and BPmoc-F3 (20 ml) and aqueous solutions of GOx (50 mg ml21, 10 mM HEPES (pH 7.2), 0.5 ml) and NR (2.5 mg ml21, 100 mM HEPES (pH 7.2), 0.5 ml) were spotted and mixed on a 24-well glass plate (f ¼ 4 mm). The spots were allowed to stand at room temperature for 30 minutes to afford a NR/GOx,NPmoc-F2 ,BPmoc-F3 hybrid gel array. For assays, a 2.0 ml analyte solution was added to each gel spot and incubated for two hours at 37 8C. Gel spots treated with the target analytes showed gel–sol transitions and were washed away by water. Arrays were photographed with a digital camera (Olympus, PEN E-PL2) before and after the addition of analytes. Release of rhodamine-IgG from the NR/GOx,NPmoc-F2 ,BPmoc-F3 hybrid gel. To an aqueous solution that contained GOx (50 mg ml21, 10 mM HEPES (pH 7.2), 1 ml), NR (2.5 mg ml21, 100 mM HEPES (pH 7.2), 5 ml) and rhodamine-IgG (5 mg ml21, 2 ml) in a quartz cuvette was added an aqueous solution (100 mM MES (pH 7.0), 92 ml) of BPmoc-F3 (0.10 wt%) and NPmoc-F2 (0.40 wt%), prepared as described above. The cuvette was incubated at room temperature and the contents formed a hydrogel. To the gel was carefully added an aqueous solution that contained GOx (50 mg ml21, 10 mM HEPES (pH 7.2), 1 ml) and NR (2.5 mg ml21, 100 mM HEPES (pH 7.2), 2 ml), followed by the addition of aqueous solutions (100 mM MES (pH 7.0), 47 ml) of BPmoc-F3 (0.10 wt%) and NPmoc-F2 (0.40 wt%). The mixture was incubated once again at room temperature to form a hydrogel. An aqueous solution of 100 mM MES (pH 7.0) that contained 50 mM NaCl was then carefully added to the top of the gel. The cuvette was incubated at 37 8C, and an aqueous solution of NADH or glucose, or an aqueous solution of NADH and glucose, was added at the desired time. The rhodamine-IgG released from the gel to the upper layer of the MES buffer was monitored by fluorescence spectroscopy (lex ¼ 530 nm, lem ¼ 574 nm). The concentration of rhodamine-IgG

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released was determined using a standard solution of rhodamine-IgG under the same conditions.

Received 16 October 2013; accepted 27 March 2014; published online 4 May 2014

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Acknowledgements This work was supported in part by the JST (Japan Science and Technology Agency), the CREST (Core Research for Evolutionary Science and Technology) program, a Grant-in-Aid for Young Scientists (A) (No. 23681022), the Scientific Research on the Innovative Areas ‘Molecular Robotics’ (No. 25104512), the global Centre of Excellence program, ‘Integrated Materials Science’ of the Ministry of Education, Culture, Science, Sports and Technology (Japan). M.I. thanks the Tokuyama Science Foundation for financial support. We acknowledge Y. Chujo and N. Kitamura (Kyoto University) for allowing us to use the transmission electron microscope and for their support, K. Kuwata (Kyoto University) for high-resolution mass spectroscopy measurements, E. Kusaka (Kyoto University) for NMR measurements and M. Ichihashi and M. Wagatsuma (Initium, ULVAC) for quartz-crystal microbalance measurements. We thank E. Ashihara (Kyoto Pharmaceutical University) and Y. Takaoka (Kyoto University) for their help in taking blood samples. T.Y. acknowledges the JSPS (Japan Society for the Promotion of Science) Research Fellowship for Young Scientists.

Author contributions M.I., T.T., T.Y., K.K., S.O. and K.U. performed the experiments and M.I. and I.H. conceived the project. The paper was written by M.I. and I.H. and edited by all the co-authors.

Additional information Supplementary information and chemical compound information are available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to I.H.

Competing financial interests The authors declare no competing financial interests. NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry

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