Programmable Self-Assembly of DNAâProtein Hybrid Hydrogel for

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Programmable Self-Assembly of DNA-Protein Hybrid Hydrogel for Enzyme Encapsulation with Enhanced Biological Stability Lan Wan, Qiaoshu Chen, Jianbo Liu, Xiaohai Yang, Jin Huang, Li Li, Xi Guo, Jue Zhang, and Kemin Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00233 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 25, 2016

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Programmable Self-Assembly of DNA-Protein Hybrid Hydrogel for Enzyme Encapsulation with Enhanced Biological Stability Lan Wan, Qiaoshu Chen, Jianbo Liu,* Xiaohai Yang, Jin Huang, Li Li, Xi Guo, Jue Zhang, Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, P. R. China KEYWORDS Super sandwich hybridization, DNA-protein hydrogels, Enzyme encapsulation, Enzyme immobilization ABSTRACT A DNA–protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA–protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 µm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel

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was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA–protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogelencapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents.


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1. INTRODUCTION Enzyme encapsulation1,2 and immobilization,3,4 which are applied with the goals of enhancing enzyme stability and convenience of use,5,6 are key technologies for developing biocatalytic processes.7,8 Among the different methods of enzyme immobilization, encapsulation inside a host semi-permeable membrane or entrapment in a network matrix such as hydrogel and other polymers, is of particular interest.9,10 Because of the easy programmability of DNA sequences and convenient chemical synthesis,11–14 DNA hydrogels are one of the most important bulk-scale materials because of their several advantages, such as designable responsiveness, biodegradability and high permeability of biomaterials.15–19 A variety of DNA building blocks such as branched double-stranded DNA (dsDNA), Y-shaped DNA and X-shaped DNA are elaborately designed to form DNA hydrogel structures through enzyme ligation, enzyme polymerization,20 intermolecular i-motif structures13 and DNA hybridization.21 Liang et al. reported the formation of gel nanofibers that provided a convenient approach for enzyme immobilization and most enzymes retained high catalytic activities similar to free enzymes.22 These DNA hydrogels show potential for application in protein-loading, drug release, cell-free protein production and DNA immunotherapy,23,24 especially widely used to encapsulate and load nanoscale species such as enzymes,7 proteins,8 nanoparticles9,10 and microscale cells.19 Proteins and DNA represent the two major classes of biomolecules in nature, which have been recently esteemed by materials scientists to design materials with unique functions. Compared with the flexible linear structure of DNA, proteins are more rigid and functionally diverse. The polypeptide backbones of proteins offer many unique features such as precisely defined chain lengths and amino acid sequences, secondary structures and many different functional groups as well as excellent biocompatibility.25 Mediation of the synchronous assembly of DNA and


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proteins could be an efficient way to fabricate hybrid hydrogels with multifunctional properties, allowing the generation of a hybrid hydrogel with convenient designing properties and diverse immobilization capabilities. The high biocompatibility and soft nature of hydrogels render them similar to natural living matrices and imitate the physiological environment. Their porous structure, along with their water content are extremely suitable for accommodating high loads of guest molecules such as enzymes, therapeutic proteins and peptides.26,27 In this study, a DNA–protein hybrid hydrogel was developed using a programmable assembly approach that utilized streptavidin as the cross-linker. Two short DNA chains were designed as a precursor to facilitate the long dsDNA building block via supersandwich hybridization chain reaction.28 The biotinylated terminus of one of the short DNA chain provides the dsDNA building block with a tailored precise arrangement of biotin. Addition of the streptavidin crosslinker results in the growth of the hierarchical DNA–protein network hydrogel.

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components embedded in a DNA hydrogel supply the hybrid hydrogel with a bracket of polypeptide that serves as an effective enzyme carrier or container. Accordingly, alcohol oxidase (AOx) was chosen as a representative enzyme that was loaded onto the DNA–protein hydrogel using a synchronous assembly approach. AOx loaded onto the DNA–protein hydrogel (DNA– protein AOx hydrogels) was highly active as an antidote for alcohol removal in blood plasma. Moreover, it showed sustained activity along with enhanced stability for a relatively long period of time. Furthermore, the hydrogel can mediate non-destructive enzyme immobilization onto a streptavidin-modified or positively charged substrate with improved stability in the presence of various denaturants such as elevated temperatures, freeze–thaw cycles and organic solvents. These self-assembled DNA–protein hydrogels show great promise for enzyme delivery and versatile biomedical applications.


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Molecular assembly of a functional hydrogel usually requires polyvalent molecular binding interactions to generate a hydrogel network structure. In this study, the supersandwich hybridization assay was performed to generate a long-chain polymer, which was decorated with precisely placed

biotin groups. Streptavidin, with its strong specific recognition and its

multivalent binding capacity with biotin, acts as a cross-linker to initiate the assembly of the DNA–protein hybrid network hydrogel. Our strategy, which was based on the streptavidinmediated programmable assembly of a hybrid hydrogel, is illustrated in Figure 1. We designed two single-stranded DNA (ssDNA) molecules, namely, ssDNA1 and ssDNA2 with70 nucleotides (nt) each. One terminus of ssDNA1 was labelled with biotin. Both ssDNA1 and ssDNA2 are dislocation-complementary, which means that the head part of ssDNA1 is complementary to the rear part of ssDNA2, and the rear part of the former is complementary to the head part of the latter. Thus, an equimolar mixture of ssDNA1 and ssDNA2 leads to their partial hybridization and formation of long dsDNA concatemers containing multiple repeated ssDNA molecules. Subsequently, the addition of streptavidin to the dsDNA supersandwich chain triggers the growth of the DNA–protein network hydrogel mediated by the strong binding affinity of streptavidin to biotin.


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Figure 1. Programmable self-assembly of DNA–protein hybrid hydrogels. Step I: Long doublestranded DNA concatemers form through the supersandwich hybridization chain reaction of ssDNA1 and ssDNA2; Step II: Streptavidin triggers the formation of the DNA–protein hybrid hydrogel. 2. EXPERIMENTAL 2.1 Materials and instruments Streptavidin,

AOx,

2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and

horseradish peroxidase (HRP) were purchased from Sigma-Aldrich. We purchased (3aminopropyl) triethoxysilane (APTS) from A Johnson Matthey Co. DNA oligonucleotides were synthesized and purified by high-performance liquid chromatography (HPLC) by Sangon Biotechnology Co. Ltd (Shanghai, China). The ssDNA sequences were as follows: ssDNA1: 5′CGA GAC TAG AAC GAG ACT GCC AGG AGT GTA GGA CGC AGG TGG AGA ATG GAT CAT GAC GGA GGT GCA ACG -3′ and ssDNA2: 5′- CGT CCT ACA CTC CTG GC A


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GTC TCG TTC TAG TCT CGC GTT GCA CCT CCG TCA TGA TCC ATT CTC CAC CTC G-biotin-3′. UV-vis

spectroscopy

measurements

were

conducted

using

a

Shimadzu

UV-1601

Spectrophotometer in transmission mode and a quartz cuvette (10 mm). Scanning electron microscopy (S-4800, Hitachi, Japan) was used to exam the sizes and morphology of the hydrogel. The hydrogel products were deposited on silicone matrices, dried, and coated with Au, followed by observation on scanning electron microscope. Agarose gel electrophoresis was obtained with an Edvotek M6Plus Electrophoresis Apparatus. 2.2 Self-assembly of DNA–protein hybrid hydrogels DNA supersandwich hybridization: Stoichiometric quantities of ssDNA1 and ssDNA2 were separately added to the Eppendorf tubes containing phosphate-buffered saline (PBS), yielding a final concentration of 50 µM. Each tube containing the mixture was heated to 95 °C for 5 min and cooled to room temperature for 4 h to form the desired dsDNA building block units. Thermal stability of the resultant dsDNA was analysed using the F7000 instrument by mixing the supersandwiched dsDNA solution (60 µL) with SYBR Green I dissolved in dimethyl sulfoxide (DMSO) (6 µL, 20×). The fluorescence data for melting curves were acquired in increments of 5 °C during the transition from 25 °C to 90 °C (15 s at each temperature). The fluorescence data were then converted to melting peaks using the software and then plotted as the negative derivative of fluorescence intensity as a function of temperature. Fabrication of DNA–protein hybrid hydrogels: The dsDNA supersandwich chain and streptavidin were mixed to generate the DNA–protein hybrid hydrogel. The molar ratio of streptavidin depends on the molar ratio of biotin. The ideal molar ratio of streptavidin to biotin is


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1:1. The hydrogels were then washed with double-distilled H2O, precipitated using centrifugation and stored at 4 °C. In this study, the molar concentration of hydrogel referred to the ssDNA content in the hybrid hydrogel. 2.3 Encapsulation of AOx in hybrid hydrogel AOx was encapsulated in the DNA–protein hydrogel using a synchronous assembly approach. Briefly, AOx (1 mM) was incubated with the dsDNA supersandwich chain (from 20 µL of dsDNA supersandwich-chain products). Streptavidin was dissolved in 100 µL PBS and was dispersed onto the hydrogel at room temperature for 24 h followed by centrifugation at 10,000 rpm for 15 min. The unbound AOx in the supernatant was isolated for further analysis. The products were washed with double-distilled H2O, precipitated using centrifugation and stored at 4 °C. A fluorescence co-localization method was employed to confirm encapsulation. Total internal reflection fluorescence imaging was applied to determine the co-localization of multicoloured fluorescence. 2.4 Enzymatic assay of AOx The enzymatic activity of AOx was determined using the H2O2-ABTS colorimetric assay. In a 1-mL reaction mixture, the final concentrations were 100 mM potassium phosphate, 2 mM ABTS, 0.1% (v/v) ethanol, 2.5 units HRP and 0.01 unit AOx. The amount of H2O2 produced is estimated by monitoring the absorption of ABTS-diradical at 418 nm (ε418 = 36,800 M-1cm-1). Using distilled water as a reference, the increase in absorption at 418 nm for 5 min was measured. To determine the amount of enzyme activity, the changed absorption values at 418 nm for both test and blank samples were calculated according to the formula presented in Supporting Information.


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2.5 DNA–protein AOx hydrogels as an alcohol antidote To assess the catalytic activities of native AOx and AOx in DNA–protein hydrogels, equal amounts of native AOx and DNA–protein AOx hydrogels were incubated with human serum for 0, 4, 8, 12, 16, 20 and 24 h, and the reactions were monitored using a UV–Vis spectrophotometer. The reaction mixture (1 mL) contained 100 mM potassium phosphate buffer (pH 7.5), 0.1% (v/v) ethanol, 2 mM ABTS and 2.5 units of HRP. H2O2 production was estimated by monitoring the generation of the ABTS-diradical every 2 min until the absorption coefficient did not change. To compare the enzymatic activity of native AOx and AOx in the DNA–protein hydrogels, we recorded the time to oxidize a defined amount of ethanol. 2.6 Hybrid hydrogel-mediated surface immobilization Hybrid hydrogel-mediated enzyme immobilization to a streptavidin-modified 96-well plate was based on biotin–streptavidin binding. Native AOx and DNA–protein AOx hydrogel solutions were dropped into the wells of streptavidin-modified plates. After 24 h of incubation at room temperature, the samples were washed three times with PBS (pH 7.5). The ABTS colorimetric assay was performed to validate the cross-linking of AOx with streptavidin. In addition, hybrid hydrogel-mediated enzyme immobilization on an amine surface was performed using above similar procedures, and the corresponding substrate was used instead of amino-modified coverslips. 2.7 AOx in hydrogels with enhanced enzyme stability The catalytic activities of native AOx and AOx in DNA–protein hydrogels were determined at 55 °C in 100 mM PBS (pH 7.5). Samples were collected at 20 min of time intervals during the


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incubation period. The enzymatic stability in the presence of organic solvents was determined by exposing native AOx and DNA–protein AOx hydrogels to an organic solvent (DMSO)–water mixture at different ratios of 0, 20%, 40%, 60%, 80%, and 100%. Enhanced enzymatic stability was also demonstrated by subjecting native AOx and DNA–protein AOx hydrogels to five freeze–thaw cycles. The relative enzymatic activities of native AOx and DNA–protein AOx hydrogels were determined by monitoring ABTS-diradical production in 100 mM PBS. 3. RESULTS AND DISCUSSION 3.1 Characterization of the DNA supersandwich hybrid The supersandwich hybrid of ssDNA1 and ssDNA2 produced long linear dsDNA. The dsDNA supersandwich was analysed using agarose gel electrophoresis (Figure 2A). The dsDNA supersandwich chain (lane 4) migrated more slowly than each individual strand of ssDNA1 and ssDNA2 (lanes 2 and 3). The bands corresponding to the dsDNA supersandwich had large tails, indicating that the products comprised ladders of different lengths of linear dsDNA. In contrast to the marker DNA, the dsDNA supersandwich chain comprised approximately 1000 bases with a linear length measureable in micrometers. It can be estimated that an average of 14 biotin residues precisely modified the dsDNA chain. These results demonstrate that a supersandwich chain of dsDNA was formed as our design and that assembly was efficient, which is indicated by the detection of discrete single strands. The supersandwich hybrid ssDNA structure was further evaluated using a fluorescence-based thermal denaturation assay (Figure 2B). The melting curves of the dsDNA supersandwich structure were determined by mixing dsDNA with SYBR Green I. The fluorescence data for melting curves were acquired at increments of 5 °C during the temperature transition from 25 °C to 90 °C. The melting curve and its negative first derivative


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indicate that the major melting temperature (Tm) of 80.8 °C was approximated to that from theoretical calculation of 35 bp domains of the DNA sandwich hybrid structure (78 °C). Fluorescence-attenuation values at temperatures lower than the Tm value validated the formation of the dsDNA. Our results further demonstrate that the dsDNA supersandwich possessed high thermal stability. To obtain longer supersandwich chains, we optimized the PBS buffer with different parameters such as Mg2+ concentration, ionic strength, pH, reaction time and concentration of ssDNA. Sandwich hybridization depends on the concentration of ssDNA precursor; the longer supersandwich chain prefers a higher concentration of ssDNA (Figure S1). Such dsDNA supersandwich structures composed of ssDNA fragments should be more flexible than a long dsDNA consisting of only two long intact single strands because in the selfassembled dsDNA, there is a nick in the sugar–phosphate backbone after each ssDNA repeating unit of 70 bases (23.8 nm). In our system, after generating the supersandwich hybrid of ssDNA, although increased viscosity was observed, the linear dsDNA did not behave as a hydrogel. However, the long dsDNA chain with precisely placed biotin residues served as a flexible building block for the hydrogel nanoassembly.


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Figure 2. (A) Agarose gel electrophoresis of the dsDNA supersandwich hybrid chain. Lane 1: biomarker; lane 2: ssDNA1; lane 3: ssDNA2; lane 4: long concatemers of the dsDNA supersandwich structures with maximum lengths of approximately 1000 base pairs. (B) Negative first derivative of the melting curve of dsDNA supersandwich hybrid chains. Inset: the melting curve of dsDNA. The fluorescence intensity of the dsDNA increased when the temperature decreased. 3.2 Self-assembly of DNA–protein hybrid hydrogels Streptavidin is a stable, tetrameric protein that binds with great affinity to as many as four biotin molecules and shows high heat tolerance and resistance to proteolysis. We developed streptavidin–biotin as a cross-linker and adaptor to mediate the self-assembly of DNA–protein hybrid hydrogels. Agarose gel electrophoresis was used to examine the formation of hydrogel (Figure 3A). After addition of streptavidin, the hydrogel product was retained at the origin, indicating the formation of a protein-mediated hybrid hydrogel with a large structure. The DNA– protein hybrid hydrogel stained with SYBR Gold sedimented at the bottom of the Eppendorf tube. Furthermore, when this Eppendorf tube was left at 4 °C overnight and then inverted, the gelatinous DNA–protein hydrogels emitted yellow fluorescence when irradiated with UV light (Figure 3B). In addition, the swelling ratio of the DNA-protein hydrogel was investigated and it was calculated as 3.9 water/hydrogel (w/w). We observed DNA–protein hybrid hydrogels under a scanning electron microscope (SEM) which indicated that the DNA–protein hybrid hydrogels had diameters of 6.7 ± 2.1 µm (Figure 3C). DNA–protein hybrid hydrogels were accumulated stacked, and most of the hydrogels were inseparably intertwined. The high-magnification SEM images (Figure 3D) indicate that the hydrogel formed flower-like morphology and the micrometre hydrogel contained numerous wrinkle on their surface, which indicated their porous


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structures. These internal hierarchical amorphous structures provided a promising carrier for high-payload encapsulation in porous structures or integration within the DNA–protein cavity of nanoscale or molecular functional moieties such as enzymes, proteins, therapeutics, imaging agents and catalytic agents.

Figure 3. (A) Agarose gel electrophoresis of a dsDNA supersandwich hybrid chain and a DNA– protein hydrogel. Lane 1: dsDNA supersandwich structures; lane 2: DNA–protein hydrogel. (B) Fluorescence imaging of DNA–protein hydrogels stained with SYBR Gold revealed an intermediate yellow fluorescence when irradiated with UV light. (C) Scanning electron microscopic (SEM) image of DNA–protein hybrid hydrogel. (D) SEM images in a zoomed-out view.


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3.3 Enzyme encapsulation and immobilization Compared with the conventional hydrogel matrix, the DNA–protein hybrid hydrogel exhibits novel features. First, the rigidity of the protein and dsDNA gives the hydrogel a porous structure, which is efficiently penetrated by the guest molecule. Second, the hybrid hydrogel composed of DNA and protein mimics the natural atmosphere of a cell matrix in living systems, enzymes are present in a concentrated physiological matrix. The flexible nature of the DNA–protein hydrogel provides an excellent carrier with properties similar to natural living matrices and minimizes the physiological environment. Third, the hybrid hydrogel contains the cross-linker of streptavidin– biotin, which is widely used as an affinity-tag to bind and immobilize molecules. The highaffinity binding site of the hybrid hydrogel may provide a versatile approach for efficient immobilization of the hydrogel carrier. AOx receives high interest for its use in industrial and clinical applications and attracts wide interest in the field of biosensors, biocatalytic synthesis of fragrant compounds as well as the organic synthesis of optically pure compounds. We chose AOx as a representative enzyme for the encapsulation and immobilization application. AOx enzyme was encapsulated in the DNA–protein hybrid hydrogel using a synchronous assembly approach (Figure 4). Supersandwich hybridized dsDNA was mixed with AOx, and streptavidin was subsequently added as the cross-linker, which formed an AOx-entrapped DNA–protein hybrid hydrogel (DNA–protein AOx hydrogel). Furthermore, we investigated the biological activities and stabilities of the encapsulated AOx in the homogeneous and solid-phase systems.


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Figure 4. AOx encapsulation in a DNA–protein hybrid hydrogel using a synchronous assembly approach that improves the enzyme’s biological stability for application in homogeneous catalysis and solid-phase reactions. 3.3.1 Encapsulation of AOx in the DNA–protein hybrid hydrogel AOx was encapsulated in the DNA–protein hybrid hydrogel using a synchronous assembly approach. SEM observation indicated that hydrogels were several micrometers in size (Figure 5A). Compared with DNA–protein hydrogels, DNA–protein AOx hydrogels were more monodisperse and well distributed. After encapsulation of the enzyme, the size of the hydrogel became slightly larger with a diameter of 9.4 ± 4.3 µm (Figure 5B). As shown by highmagnification SEM images, DNA–protein AOx hydrogels were three-dimensional and were more densely packed into a nanostructure (Figure 5C). The special structure of DNA–protein AOx hydrogels could potentially be attributed to the complex conformation of AOx encapsulated in the hydrogel. The polypeptide backbone and secondary structure of AOx generated a more three-dimensional and hierarchical structure. In this study, we encapsulated AOx in the microhydrogel using a physical entrapment method and a synchronous assembly approach. To confirm the successful encapsulation of the enzyme, a fluorescence co-localization method was


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employed. The dsDNA and AOx were labelled with 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC), respectively. The fluorescence microscopic images acquired (DAPI, blue channel and FITC, green channel) showed that the blue of dsDNA and the green of AOx were co-localized, which indicated that the AOx enzyme was encapsulated in the DNA hydrogel (Figures 5D–F). Furthermore, the sizes of fluorescence spots demonstrated that the hydrogel was several micrometers, which is consistent with the SEM observations. Therefore, the results confirmed that AOx was successfully encapsulated in the hydrogel. In addition, the enzyme encapsulation efficiency and loading capacity of DNA-protein hydrogel was investigated. Different concentrations of AOx were encapsulated into the DNA-protein hydrogel. The unloaded AOx was removed from DNA-protein hydrogel by filtration membrane. The enzyme content was quantified through enzymatic colorimetric assay, and it found that as the added free AOx increased, the content of encapsulated enzyme gradually increased, and it reached the maximal encapsulation capacity of 23.1 µg when 55 µg AOx was added (Fig. S2). In this case, the encapsulation efficiency and loading capacity were 42% and 1.8 g AOx/g hydrogel.


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Figure 5. (A–C) Scanning electron microscopic (SEM) images of the DNA–protein AOx hydrogel. (B, C) High-magnification SEM images. (D–F) Fluorescence co-localization of DNA– protein AOx hydrogels. DNA and AOx were labelled with DAPI and FITC, respectively. Fluorescence imaging of DNA–protein AOx hydrogels. 4′,6-diamidino-2-phenylindole (DAPI), blue channel (D). Fluorescein isothiocyanate (FITC), green channel (E). Merged images (F). Scale bar = 10 µm. In our system, the DNA–protein matrix provided a mild, biomimetic physiological environment for the guest molecules. To demonstrate that AOx retained its conformation in the hybrid hydrogel, circular dichroism (CD) spectroscopy was employed.30 AOx contains a high percentage of α-helical structure, which exhibits characteristic ellipticity in CD at 208 nm and 222 nm. As shown in Figure 6A, after enzyme-loading the hydrogel, there were no detectable changes in the CD peaks of AOx at 208 nm and 222 nm. There were positive and negative peaks


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at 278 nm and 246 nm, respectively, which were assigned to DNA bases and the B-type DNA structure, respectively. Thus, these results demonstrate that AOx was encapsulated into the hybrid hydrogels, without disruption of its secondary structure (Figure 6A). DNA and proteins in living systems can provide a concentrated physiological matrix for a guest enzyme and guarantee a low cytotoxicity of the hydrogel system. The cytotoxicity of hybrid hydrogels was examined using MTT assays of SMMC-7721 cells treated with various concentrations of the samples (Supporting Information). As shown in Figure 6B, cell metabolic activity retained more than 90% even when the concentrations of the DNA–protein AOx hydrogels were as high as 2.0 µM. Cells treated with the DNA–protein AOx hydrogels for 24 h retained a regular, elongated morphology, which indicated low cytotoxicity of DNA–protein AOx hydrogels (inset).

Figure 6. (A) Circular dichroism (CD spectra of AOx in DNA–protein hydrogels, native AOx and DNA–protein AOx hydrogel. (B) Cytotoxicity of DNA–protein AOx hydrogels. MTT assays were performed on SMMC-7721cells treated with different concentrations of DNA–protein AOx hydrogels for 24 h. Inset: Laser scanning confocal microscopy images of cells incubated with and without DNA–protein AOx hydrogels for 24 h. Scale bar = 20 µm. 3.3.2 DNA–protein AOx hydrogels as an alcohol antidote


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Typically, AOx is widely used in homogeneous systems and solid-phase reactions. For example, AOx has been used as an antidote for alcohol and as a biosensor. The DNA–protein matrix guaranteed good biocompatibility of the hydrogel that shows great promise for biomedical applications. Subsequently, we further investigated the feasibility of loading AOx into the DNA–protein hydrogel for these applications. Alcohol antidotes are a critical issue because excessive consumption and abuse of alcohol is associated with a range of organ injuries and social problems. The biocompatible DNA–protein AOx hydrogels were utilized to catalyse the oxidation of ethanol in a homogeneous solution. In this study, we demonstrated that the hydrogels served as an alcohol antidote. To explore whether the catalytic capacity and biological stability of the guest enzyme remained after loading the hydrogel, the catalytic rate and sustainable utilization of free or encapsulated AOx molecules were investigated. The DNA–protein AOx hydrogels and native AOx were incubated in human serum for different times and then tested for antidote activity. In this procedure, a defined ethanol with final concentration of 0.1% (v/v) was added to the enzyme-containing serum solution, and catalysis was monitored using an ABTS-colorimetric test at 2-min intervals (Fig. S3). The specific activity of the AOx embedded in hybrid hydrogel was determined using H2O2ABTS colorometric method.31 The activity of AOx was determined as a function of incubation time. After adding AOx to the serum solution for 24 h, the reaction time of native AOx increased from 15.2 min to 39.8 min, although that for AOx in the hydrogel was only slightly increased (Figure 7A). These results indicate that the catalytic activity and stability of native AOx were significantly decreased compared with the activity of AOx in the hydrogel. Furthermore, the AOx in DNA–protein hydrogels can be used for at least 24 h and retains as much as 78% of its initial activity, although native AOx lost as much as 38% of its activity (Figure 7B). These


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results clearly demonstrate that the DNA–protein AOx hydrogels exhibit higher efficiency and catalytic activity and that AOx encapsulated in a hydrogel is highly stable and reactive in a homogeneous system. Combined with enhanced stability, such highly active DNA–protein AOx hydrogels show great potential for alcohol prophylaxis as an alcohol antidote and as an agent for removing ethanol from plasma.

Figure 7. Ethanol oxidation catalyzed by native AOx and DNA–protein AOx hydrogels after incubation with serum at different times. (A) After incubation with human serum for 0, 4, 8, 12, 16, 20 and 24 h, native AOx and DNA–protein AOx hydrogels oxidized a certain amount of ethanol. (B) Relative activities of native AOx and DNA–protein AOx hydrogels. 3.3.3 Immobilization of DNA–protein AOx hydrogels Solid-phase surface immobilization is an another important process in applying AOx as a biosensor32, and one of the most critical problems for using an alcohol biosensor is maintaining enzymatic activity with high efficiency while immobilized on the solid matrix. In this study, AOx was incorporated into the interior of the hydrogel. Moreover, the hydrogel can provide high-affinity binding sites for efficient immobilization. For example, an excess number of biotin sites on DNA–protein hydrogels can be utilized to immobilize the hydrogel on streptavidin-


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modified 96-well plates. Furthermore, DNA gives the hybrid hydrogel a net negative charge, which allows us to directly immobilize the hydrogel to an amine substrate based on electrostatic binding. AOx encapsulated in hydrogel and native AOx as the control were deposited on the streptavidin-modified plate. After incubation for 24 h, the plate was washed, and the efficiency of immobilization was monitored using the ABTS colorimetric assay. When the AOx in hydrogel was successful fixed on the surface of the substrate, the DNA–protein AOx hydrogels triggered the ABTS colorimetric reaction and generated an obvious ABTS-diradical blue–green color. Otherwise, no obvious color could appeared. The sample of DNA–protein AOx hydrogels generated an obvious ABTS-diradical blue–green color (Figure 8A); however, no obvious colour was detected in the sample of native AOx. UV–Vis absorption at 418 nm also confirmed these findings. The results clearly indicate the immobilization of DNA–protein AOx hydrogels on streptavidin-modified plates (Figure 8A). As an alternative immobilization approach, DNA– protein AOx hydrogels can also be directly modified on the amino-modified slide because of the negative charge of the hydrogel. In order to verify the success of immobilization, an ABTS colorimetric assay was performed. As presented in Figure 8B, compared with native AOx, the samples of DNA–protein AOx hydrogels generated an ABTS-diradical that emitted strong blue– green color (Figure 8B), which was validated through UV–Vis absorption. Herein, compared with other enzyme-immobilized systems, an exceptionally convenient and stable immobilization is presumably attributable to the characteristics of the DNA–protein hydrogels. Conventional enzyme immobilization methods mainly depend on direct chemical modifications such as chemical cross-linking and surface adsorption. In this study, we used DNA–protein hydrogels to mediate the immobilization of AOx, which allowed target molecules to diffuse freely into the


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three-dimensional network while avoiding the possible influence on the conformation and spatial orientation of the guest enzyme.

Figure 8. (A) UV–Vis spectra and the colour change of the DNA–protein AOx hydrogels immobilized on streptavidin-modified plates based on the specific biotin–streptavidin binding. (B) UV–Vis spectra and the colour change of the DNA–protein AOx hydrogels immobilized on the amine groups of substrates. 3.4 An encapsulated enzyme with enhanced biological stability The catalytic activity and biological stability of the encapsulated enzyme are critical for practical use as a biosensor, particularly under the complex and extreme environments of industrial applications. The immobilized DNA–protein AOx hydrogels on a substrate provide improved biological stability against various denaturants, including elevated temperature, freeze–thaw cycles and organic solvents.33 The activity of the enzyme in the hydrogel was investigated in the presence of denaturants. As shown in Figure 9A, we investigated the catalytic activity of AOx before and after embedding it in the hydrogels at an elevated temperature of 55


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o

C. Unlike the fast and continuous inactivation of the native AOx, which lost 78% of its activity

after 30 min of incubation, AOx loaded in hydrogels underwent a slower decrease of catalytic activity in the first 30 min and maintained 60% of its original activity thereafter. The exceptional stability of the hydrogel could likely be attributed to several features of the hybrid hydrogels, including the high density of DNA packed in each hydrogel, which provides a rigid porous structure for the enzyme. Furthermore, extensive inter- and intra-strand weaving of long dsDNA building blocks effectively prevented a conformational change of AOx upon heating. Enhanced enzymatic stability in the presence of organic solvents was demonstrated by exposing the enzyme to an organic solvent–water mixture at different ratios. Compared with non-polar solvents such as hexane, polar organic solvents are more detrimental to enzymes because they compete with water to form hydrogen bonds with protein backbones, which disrupt the protein’s conformation. As presented in Figure 9B, AOx in hydrogels retained higher residual activity than native AOx in different volume fractions of the DMSO–water mixture. This improved the performance of DNA–protein AOx hydrogels, which can be attributed to the hydrophilic environment maintained by the hydrophilic hydrogel, which otherwise would be depleted by a polar organic solvent. Maintaining enzymatic activity in a non-aqueous environment is challenging but holds great promise for a broad range of industrial and environmental applications. The enhanced freeze–thaw effect is demonstrated in Figure 9C. After five freeze– thaw cycles, AOx in hydrogels retained its total activity, whereas that of native AOx was reduced. Enhanced freeze–thaw stability can provide a major advantage for practical applications because it can dramatically lower the cost of storing and replacing enzyme stocks Furthermore, in order to verify whether this AOx in hydrogel can retained for long time, the AOx enzyme activity was continues determined for 7 days. The enzyme still possessed high catalytic activity


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even after 7 days storage (Fig. S4). Prolonged shelf life can provide a major advantage in practical applications, since it can dramatically lower the cost to store and replace the enzyme stocks. The capability of fabricating hybrid hydrogels with significantly enhanced activity and stability provides a novel platform for various applications of the alcohol biosensor.

Figure 9. The immobilized DNA–protein AOx hybrid hydrogels with enhanced enzymatic stability. (A) Relative activities of native AOx and DNA–protein AOx hydrogels incubated at 55 °C. (B) Relative activities of native AOx and DNA–protein AOx hydrogels exposed to solutions containing different concentrations of DMSO. (C) Relative activities of native AOx and DNA–protein AOx hydrogels subjected to five freeze–thaw cycles. 4. CONCLUSION In this study, we developed a DNA–protein hybrid hydrogel based on a programmable assembly approach, which served as a biomimetic physiological medium for the efficient encapsulation of AOx. The building blocks of dsDNA tailored with precisely placed biotin residues were prepared using supersandwich hybridization. AOx can be encapsulated into the DNA–protein network hydrogel using a physical entrapment method and a synchronous approach. The AOx-loaded hybrid hydrogel can be used as an antidote for alcohol. The encapsulation extended the enzymatic activity during incubation with plasma. The hydrogel can


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mediate the immobilization of enzyme on streptavidin-modified positively charged substrates in a non-destructive manner, which is very beneficial to solid-phase biosensor applications. In particular, this hydrogel allowed highly efficient immobilization of enzymes and imparted specificity and enhanced stability in the presence of diverse denaturants such as elevated temperature, freeze–thaw cycles and organic solvents. This study provides a reasonable programmable assembly approach to generate DNA–protein hybrid hydrogels, which might prove to be a powerful and indispensable tool for enzyme encapsulation and immobilization. In future, more exploration on their application to homogenous systems as well as to a solid-phase surface is needed.

ASSOCIATED CONTENT Supporting Information. Agarose gel electrophoresis, enzymatic assay of alcohol Oxidase. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * [email protected], [email protected] Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT This work was supported in part by the Natural Science Foundation of China (21190040, 21575037, and 21205033).


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ABBREVIATIONS AOx, alcohol oxidase; DMSO, dimethylsulfoxide; SEM, scanning electron microscopy ; TIRF, total internal reflection fluorescence; PBS, phosphate buffered saline; DAPI, 4',6-diamidino-2phenylindole; FITC, fluorescein-isothiocyanate; HPLC, High-performance liquid chromatography; CD, circular dichroism spectroscopy; dsDNA, double-stranded DNA ; ssDNA: single-stranded DNA. REFERENCES (1) Rusmini, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2007, 8, 1775–1789. (2) Feng, D.; Liu, T.; Su, J.; Bosch, M.; Wei, Z.; Wan, W.; Yuan, D.; Chen, Y.; Wang, X.; Wang, K.; Lian, X.; Gu, Z.; Park, J.; Zou, X.; Zhou, H. Nat. Commun. 2015, 6, 5979. (3) Douglas, S. M.; Bachelet, I.; Church, G. M. Science 2012, 335, 831–834. (4) Tran, D. N.; Balkus, K. J. ACS Catal. 2011, 1, 956–968. (5) Misson, M.; Zhang, H.; Jin, B. J. R. Soc., Interface 2015, 12, 20140891. (6) Sheldon, R. A.; van Pelt, S. Chem. Soc. Rev. 2013, 42, 6223–6235. (7) Zhu, Z.; Wu, C.; Liu, H.; Zou, Y.; Zhang, X.; Kang, H.; Tan, W. T. Angew. Chem., Int. Ed. 2010, 49, 1052–1056. (8) Bryan, W.; Immensee, C.; Luo, K. Q.; Yongli, M. Angew. Chem., Int. Ed. 2008, 47, 331– 333. (9) Tim, L.; Hendrik, D.; Bernard, Y.; Friedrich, S. Small 2007, 3, 1688–1693. (10) Yang, H.; Liu H.; Kang, H.; Tan, W. J. Am. Chem. Soc. 2008, 130, 706–714. (11) Jin, J.; Xing, Y.; Xi, Y.; Liu, X.; Zhou, T.; Ma, X.; Yang, Z.; Wang, S.; Liu, D. Adv. Mater. 2013, 25, 4714–4717. (12) Um, S. H.; Lee, J. B.; Park, N.; Kwon, S. Y.; Umbach, C. C.; Luo, D. Nat.Mater. 2006, 5, 797–801. (13) Cheng, E.; Xing, Y.; Chen, P.; Yang, Y.; Sun, Y.; Zhou, D.; Xu, L.; Fan, Q.; Liu, D. Angew. Chem., Int. Ed. 2009, 48, 7796–7799. (14) Xing, Y.; Cheng, E.; Yang, Y.; Chen, P.; Zhang, T.; Sun, Y.; Yang, Z.; Liu, D. Adv. Mater. 2011, 23, 1117–1121. (15) Ali, C.; Yanli, T.; Yue, L.; Huanxiang, Y.; Libing, L. Chem. Commun. 2013, 49, 5574–5576.


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We developed a DNA–protein hybrid hydrogel based on a programmable assembly approach, which served as a biomimetic physiological medium for the efficient encapsulation of alcohol oxidase. 248x77mm (150 x 150 DPI)


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