Nanoarchitectonics Approach for Hydrogel Surface Functionalization via Self-Assembly of Terminally Modified Cello-oligosaccharides

Abstract

Advances in hydrogel research have enabled the design of bulk networks with diverse functionalities; however, precise control of interfacial properties remains a key challenge to expanding their applications, particularly in biointerfaces. Here, we report a simple and versatile surface functionalization strategy based on the self-assembly of terminally modified cello-oligosaccharides. When azido-functionalized cello-oligosaccharides dissolved in 85% phosphoric acid are applied to hydrogel surfaces, mixing with the intrinsic water within the hydrogels induces rapid self-assembly, forming nanostructured granular coatings at the interface. This method is applicable to both chemically and physically cross-linked hydrogels. The resulting azido-functionalized surfaces enable postfunctionalization via click chemistry, allowing biomolecule immobilization. As a proof of concept, antigen-conjugated hydrogels were used for the detection of specific immunoglobulin G, exhibiting selective responses even in serum-containing environments due to the antibiofouling properties of the assemblies. These findings establish terminally modified cello-oligosaccharides as a new class of interfacial modifiers for hydrogels, providing a facile route to biofunctional and antifouling interfaces for sensing and diagnostic applications.

Introduction

Hydrogels are soft materials composed of polymeric or supramolecular networks that retain large amounts of water and are widely used in healthcare, medicine, food, and other fields. Extensive studies have established design principles for bulk network architectures, enabling the construction of hydrogels with high toughness and strength, − controlled gelation and degradation, − stimuli responsiveness, , and anisotropy. ,

Beyond bulk network design, increasing attention has been directed toward surface structures for the development of advanced hydrogels. − Recent studies have reported a variety of strategies to engineer hydrogel surfaces through chemical modifications, − physical modifications, − and microstructuring approaches. − These surface engineering strategies have enabled a wide range of functionalities, including controlled molecular release, ,, stimuli-responsive behavior, actuation, controlled water transportation, improved water retention, , underwater superoleophobicity, underwater adhesion, and osteointegration. However, the development of a versatile, simple, and mild strategy for hydrogel surface functionalization remains challenging.

Nanoarchitectonics is a promising strategy for the fabrication of advanced functional materials through the integration of diverse scientific fields, including nanotechnology, supramolecular chemistry, and biorelated sciences. , Cello-oligosaccharides, also referred to as low-molecular-weight cellulose, have emerged as building blocks for constructing environmentally friendly, robust, and biocompatible nanostructures. , The precipitation-driven self-assembly of cello-oligosaccharides in solution yields crystalline particulate assemblies with unique nanomorphologies, such as nanosheets, − nanoribbons, − and helical nanorods.

Remarkably, recent studies have demonstrated that cello-oligosaccharides can self-assemble via heterogeneous nucleation on material surfaces. For example, cello-oligosaccharides form nanospike structures on microfibers of paper and cotton gauze. ,, Moreover, terminally azidated cello-oligosaccharides synthesized via enzymatic oligomerization can also be employed, enabling the functionalization of microfiber surfaces of paper and nonwoven fabrics with versatile, click-reactive azido groups. , Notably, both the building blocksnamely, cello-oligosaccharidesand the solvents after the self-assembly process, including diluted phosphoric acid and aqueous sodium chloride solutions, are biocompatible and environmentally friendly, and the resulting cello-oligosaccharide assemblies are highly robust. Such self-assembly strategies based on cello-oligosaccharides are therefore attractive for hydrogel surface functionalization. However, interfacial tension between hydrogels and water is negligible, , making heterogeneous nucleation-driven self-assembly difficult to achieve at hydrogel–water interfaces.

In this study, we focused on the fact that the solvent of hydrogelsnamely, watercan serve as a coagulant for cello-oligosaccharides. We hypothesized that when a solution of cello-oligosaccharides is applied to hydrogel surfaces, the oligosaccharide chains would initiate self-assembly as a result of mixing with the intrinsic water content of hydrogels. This process differs from previously reported self-assembly of cello-oligosaccharides via heterogeneous nucleation on material surfaces, in which cello-oligosaccharide solutions were first mixed with water as a coagulant to prepare supersaturated solutions prior to application onto the materials. ,

Herein, we report hydrogel surface functionalization via the self-assembly of terminally azidated cello-oligosaccharides, and the use of the resulting azido-functionalized hydrogels for biomolecular sensing applications (Figure ). When a solution of terminally azidated cello-oligosaccharides in concentrated phosphoric acid (85%) was applied to polyacrylamide or agarose hydrogels, the oligosaccharide chains started to self-assemble on the hydrogel surfaces. The azido groups presented on the hydrogel surfaces were subsequently used to conjugate biotin via click chemistry. Detection of a model target, antibiotin immunoglobulin G (IgG), was investigated using the biotin-conjugated hydrogels.

1.

Schematic illustration of hydrogel surface modification via the self-assembly of terminally azidated cello-oligosaccharides and subsequent antigen conjugation to the azidated assemblies via click chemistry for the detection of corresponding antibodies.

Materials and Methods

Materials

2-Azidoethyl β-d-glucopyranoside (>98%), trifluoroacetic acid (ca. 0.5 mol L^–1 in water), and N-(1R,8S,9S)-bicyclo­[6.1.0]­non-4-yn-9-ylmethyloxycarbonyl-1,8-diamino-3,6-dioxaoctane (BCN-amine, >90%) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Bovine serum albumin (BSA, 98%, protease free), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (>85%), N,N,N′,N′-tetramethylethylenediamine, 40% sodium deuteroxide (NaOD)–deuterium oxide (D₂O) solution, and D₂O (99.8%) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Super DHB (≥99%), horseradish peroxidase (HRP)-conjugated antimouse IgG IgG (produced in rabbits, polyclonal, A9044, lot: 0000143670), ammonium persulfate (≥98%), acrylamide (≥99%), N,N′-methylenebis­(acrylamide) (≥99.5%), and ProteoMass Peptide and Protein MALDI-MS Calibration Kit (containing Bradykinin fragment 1–7, P₁₄R, ACTH fragment 18–39, and acetonitrile) were purchased from Sigma-Aldrich (Missouri, USA). HRP-conjugated antibiotin IgG (produced in rabbits, polyclonal, A150-109P, Lot No. 19) was purchased from Bethyl Laboratories (Texas, USA). Fetal bovine serum (FBS) was purchased from BioWest (Nuaillé, France) and subjected to heat treatment at 56 °C for 30 min to inactivate complement proteins before use. All other chemical reagents were purchased from Nacalai Tesque (Kyoto, Japan). Ultrapure water (>18.2 MΩ·cm) was obtained using a Milli-Q Advantage A-10 system (Merck Millipore, Massachusetts, USA) and used throughout this study.

Enzyme-Catalyzed Synthesis of Terminally Azidated Cello-oligosaccharides

Terminally azidated cello-oligosaccharides were synthesized from α-d-glucose 1-phosphate monomers using 2-azidoethyl β-d-glucopyranoside as a primer and cellodextrin phosphorylase as a catalyst, in accordance with our previous reports. , Cellodextrin phosphorylase from Acetivibrio thermocellus DSM 1313 was prepared using Escherichia coli BL21-Gold­(DE3) strain containing a plasmid including the cdp gene. For azidated cello-oligosaccharides’ synthesis, 200 mM α-d-glucose 1-phosphate and 50 mM 2-azidoethyl β-d-glucopyranoside were reacted in a 500 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (pH 7.5) in the presence of cellodextrin phosphorylase (1 U mL^–1) at 60 °C for 3 days. The resulting precipitate was purified with water and collected through five cycles of centrifugation (20,400g, 10 min, 4 °C) and redispersion. The purified terminally azidated cello-oligosaccharides were then dispersed in water and stored at 4 °C, or alternatively stored in a freeze-dried state at room temperature.

The structure of the synthesized azidated cello-oligosaccharides was confirmed through proton nuclear magnetic resonance (¹H NMR) spectroscopy. Lyophilized azidated cello-oligosaccharides were dissolved in 4% NaOD–D₂O solution to obtain a concentration ≥ 2% (w/v) and analyzed using AVANCE III HD spectrometer (500 MHz, Bruker Biospin, Massachusetts, USA) at room temperature. Chemical shifts were calibrated using the residual water signal at δ = 4.79. The average degree of polymerization $(\stackrel{\circledR }{\mathrm{DP}})$ was calculated using the following equation:

$$(\stackrel{\circledR }{\mathrm{DP}})=\frac{H_{1^{\prime }}}{H_1}+1$$ 1

where H _1′ and H ₁ are the integrals of the corresponding protons (see the chemical structure in Figure S1a).

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was performed using an FT/IR-4100 instrument (Jasco, Tokyo, Japan) equipped with an ATR PRO 450-S accessory (Jasco, Tokyo, Japan), with 500 cumulative scans and a resolution of 2.0 cm^–1.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was conducted using a MALDI-8030 instrument (Shimadzu, Kyoto, Japan) equipped with a nitrogen laser (λ = 337 nm) and pulsed ion extraction, operated in the linear positive-ion mode under ambient conditions. Spectra were calibrated at 757.3997 Da (Bradykinin fragment 1–7), 1,533.8582 Da (P₁₄R), and 2,465.1989 Da (ACTH fragment 18–39). For sample preparation, azidated cello-oligosaccharides (0.01% (w/v)), super DHB (2 mg mL^–1), and trifluoroacetic acid (0.5 mM or 0.1%) were mixed in an acetonitrile–water solution (1:1, v/v). The resulting mixture was spotted onto a MALDI target plate and dried overnight under ambient conditions prior to analysis. The $\stackrel{\circledR }{\mathrm{DP}}$ was calculated using the following equation:

$$\stackrel{\circledR }{\mathrm{DP}}=\frac{\stackrel{\circledR }{M_{\mathrm{n}}}-87}{162}=\frac{\frac{\sum _i(N_i{M}_i)}{\sum _i{N}_i}-87}{162}$$ 2

where $\stackrel{\circledR }{M_{\mathrm{n}}}$ is the number-average molecular weight, N _i is the peak area of the i-mer species, and M _i is the molar mass of that species.

Preparation of Hydrogels

Polyacrylamide hydrogels were synthesized by first preparing an aqueous solution containing 9.75% (w/v) acrylamide and 0.26% (w/v) N,N′-methylenebis­(acrylamide). Subsequently, 1 mL of this solution was diluted with 1.982 mL of water and deaerated. The solution was then mixed with 15 μL of a 10% (w/v) ammonium persulfate aqueous solution and 3 μL of N,N,N′,N′-tetramethylethylenediamine. Aliquots (200 μL) of the resulting mixture were transferred to individual wells of a 96-well plate (clear, flat-bottom, IWAKI, Shizuoka, Japan) and immediately overlaid with 48 μL of water-saturated 1-butanol. After polymerization for 1 h under ambient conditions, the remaining 1-butanol was removed. Subsequently, 100 μL of ultrapure water was added to each well and removed; this washing cycle was repeated four times. The prepared hydrogels were then soaked in a water bath for 2 h, after which the water was replaced. This soaking cycle was repeated five times. Finally, the hydrogels were stored under hydrated conditions at 4 °C until use.

Agarose hydrogels were prepared by autoclaving a 1% (w/v) agarose aqueous solution at 121 °C for 20 min. The autoclaved solution was then incubated at 60 °C for 1 h and subsequently dispensed into individual wells of a 96-well plate maintained at 25 °C. After incubation at 25 °C for 1 h, agarose hydrogels formed. The resulting hydrogels were stored under hydrated conditions at 4 °C until use.

The water contents of the hydrogels were calculated from the weight changes of the hydrogels through freeze-drying.

Self-Assembly of Terminally Azidated Cello-oligosaccharides on Hydrogel Surfaces

Terminally azidated cello-oligosaccharides were dissolved in 85% phosphoric acid at room temperature to prepare solutions at concentrations of 1%, 3%, and 5% (w/v). Subsequently, 5 μL of each azidated cello-oligosaccharide solution was applied to the surface of a hydrogel prepared in a well of a 96-well plate. Prior to this self-assembly process, excess water was removed from the hydrogel surfaces. The hydrogels were then purified by immersion in a water bath for 3 h, after which the water was replaced. This purification cycle was repeated five times. The purified hydrogels were stored under hydrated conditions at 4 °C.

The loading rate of azidated cello-oligosaccharides on the hydrogels was estimated by comparing the turbidity of the hydrogel with azidated cello-oligosaccharide assemblies in the hydrated state before and after the purification process. Turbidity was evaluated by measuring the optical density (or apparent absorbance, A) at 490 nm using a microplate reader (Model 680, Bio-Rad Laboratories, CA, USA) at room temperature. Prior to purification, 100 μL of ultrapure water was added onto each hydrogel, and the optical density was recorded as A _initial. After the purification procedure, the absorbance was recorded as A _after. The loading rate was calculated using the following equation:

$$\mathrm{Loading}\mathrm{rate}(\%)=\left(\frac{A_{\mathrm{after}}-A_{\mathrm{hydrogel}}}{A_{\mathrm{initial}}-A_{\mathrm{hydrogel}}}\right)\times 100$$ 3

where A _hydrogel is the optical density value of a pristine hydrogel without azidated cello-oligosaccharides. This experiment was conducted for hydrogels prepared using 1%, 3%, and 5% (w/v) azidated cello-oligosaccharide solutions in 85% phosphoric acid, with at least three independent measurements performed for each condition. Notably, experimental measurements confirmed that treatment with 85% phosphoric acid caused negligible changes in the optical density values of the hydrogels. Specifically, the optical density values at 490 nm were 0.096 ± 0.005 and 0.090 ± 0.003 for the untreated and phosphoric acid-treated hydrogels, respectively.

To prepare preformed azidated cello-oligosaccharide assemblies, 5 μL of azidated cello-oligosaccharide solutions (1%, 3%, or 5% (w/v) in 85% phosphoric acid) was mixed with 5 μL of water and allowed to stand at room temperature for 1 h. The resulting suspension was then applied onto the surface of a polyacrylamide hydrogel and incubated at room temperature for 1 h. Subsequently, the hydrogel was immersed in water for 1 min for washing. Photographs were taken before and after the washing process to evaluate the adhesion of the preformed assemblies to the hydrogel surface.

Characterization of Azidated Cello-oligosaccharide Assemblies on Hydrogels

The functional groups and crystalline structure of the assembled azidated cello-oligosaccharides were analyzed by ATR-FTIR spectroscopy. Prior to characterization, the hydrogels were collected from the wells of 96-well plates, frozen in liquid nitrogen, and lyophilized for at least 15 h before ATR-FTIR analysis.

The morphology of the azidated cello-oligosaccharide assemblies on the hydrogels was examined by scanning electron microscopy (SEM). Hydrogels with azidated cello-oligosaccharide assemblies were sequentially immersed in 1 mL each of 25% (v/v) ethanol–water, 50% (v/v) ethanol–water, 75% (v/v) ethanol–water, ethanol (twice), 50% (v/v) ethanol–tert-butyl alcohol, and tert-butyl alcohol (twice) for 10 min per step under ambient conditions. The resulting organogels were then frozen and lyophilized to obtain dried samples. The dried samples were mounted on SEM sample stages using carbon tape (Nissin EM, Tokyo, Japan) and coated with osmium using a Neoc-Pro osmium coater (Meiwafosis, Tokyo, Japan) operated at 5 mA for 20 s. SEM images were acquired using a JSM-7500F microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV. The surface coverage of the hydrogel surfaces by azidated cello-oligosaccharide assemblies were evaluated from SEM images using ImageJ software (https://imagej.net/ij/). Additionally, hydrogels with azidated cello-oligosaccharide assemblies on their surfaces were frozen, fractured, and their cross sections were observed by SEM.

In addition, polyacrylamide hydrogels with azidated cello-oligosaccharide assemblies were observed using an ECLIPSE LV100ND microscope (Nikon, Tokyo, Japan).

Conjugation of Biotin to Assembled Azidated Cello-oligosaccharides on Hydrogels

To enable biotin immobilization onto azido groups via click chemistry, a strained alkyne group was first introduced into biotin. This was achieved by incubating biotin (48 mM) with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (48 mM) and BCN-amine (24 mM) in a dimethyl sulfoxide–water mixture (1:1, v/v) at 25 °C for 2 h. Subsequently, the reaction solution was diluted appropriately, and 5 μL of the diluted solution was added to the hydrogel to obtain different molar ratios of azidated cello-oligosaccharides to BCN-amine (10:1, 1:2, and 1:4) in a dimethyl sulfoxide–water mixture (1:2, v/v). After incubation at 25 °C for 1 h, the hydrogel was purified by immersion in water for 2 h, after which the water was replaced. This purification cycle was repeated five times. The purified hydrogels were stored under hydrated conditions at 4 °C.

The biotin conjugation rate, defined as the percentage of azido groups conjugated with biotin via the click reaction, was estimated using MALDI-TOF mass spectrometry. After the click reaction and subsequent purification, 100 μL of water was added to each well and vigorously pipetted over the hydrogel surface repeatedly for 15–20 min to extract the cello-oligosaccharide assemblies. The collected extract was centrifuged (20,400g, 25 °C, 20 min) to pellet the cello-oligosaccharide assemblies. After removal of the supernatant, the pellet was dried at 25 °C and subjected to MALDI-TOF mass spectrometry as described above. The biotin conjugation rate was determined from the ratio of the peak areas corresponding to unreacted azidated cello-oligosaccharides and biotin-conjugated cello-oligosaccharides in the mass spectra.

Detection of HRP-Conjugated IgG

Prior to IgG detection, the solvent within the hydrogels was exchanged for phosphate-buffered saline (PBS, pH 7.4). Briefly, 100 μL of PBS was added to each well and incubated at room temperature for 5 min, after which the supernatant was removed. This exchange cycle was repeated five times. Subsequently, 10 μL of an HRP-conjugated IgG solution (antibiotin IgG or antimouse IgG IgG) prepared in PBS containing 3% (w/v) BSA, with or without 10% (v/v) FBS, was added to the hydrogel surface before incubation at 25 °C for 10 min. After incubation, 100 μL of PBS was added and incubated for 5 min at room temperature, followed by removal of the supernatant. This washing procedure was repeated five times. To initiate the enzymatic reaction, 50 μL of an o-phenylenediamine solution (2 mg mL^–1) in McIlvaine buffer (pH 4.0) containing 0.11% (w/v) H₂O₂ was added to the hydrogel surface and incubated at 25 °C for 10 min. The reaction was subsequently terminated by the addition of 50 μL of 3 N aqueous sulfuric acid. The hydrogel-containing wells were then subjected to absorbance measurements at 490 nm using a microplate reader (Model 680, Bio-Rad Laboratories, California, USA) at room temperature.

Results and Discussion

Self-Assembly of Terminally Azidated Cello-oligosaccharides on Hydrogel Surfaces

Terminally azidated cello-oligosaccharides were prepared as previously reported via a cellodextrin phosphorylase-catalyzed oligomerization reaction using α-d-glucose 1-phosphate as the monomer and 2-azidoethyl β-d-glucopyranoside as the primer (Scheme S1). , The synthesized azidated cello-oligosaccharides exhibited an $\stackrel{\circledR }{\mathrm{DP}}$ of approximately 7, as confirmed by ¹H NMR spectroscopy and MALDI-TOF mass spectrometry (Figure S1a,b). In addition, ATR-FTIR spectroscopy confirmed the presence of azido groups, as evidenced by a characteristic peak at 2112 cm^–1, and revealed cellulose II allomorph of cello-oligosaccharide chains, indicated by distinct peaks at 3443 and 3488 cm^–1 (Figure S1c). Cellulose II is the thermodynamically stable allomorph of cellulose and typical of in vitro assembled cellulose and cello-oligosaccharides. , The formation of cellulose II is consistent with previous reports on terminally azidated cello-oligosaccharides. , These results collectively confirm the successful synthesis of terminally azidated cello-oligosaccharides via enzyme-catalyzed oligomerization.

The synthesized azidated cello-oligosaccharides were applied to the surface functionalization of hydrogels. Polyacrylamide hydrogels were selected as substrate materials because of their simple preparation, structural stability under ambient and physiological conditions, and intrinsically low biofouling characteristics of polyacrylamide, , which enable a clear evaluation of biomolecule-binding performance in the sensing system (see section Detection of IgG). Deposition of azidated cello-oligosaccharides onto polyacrylamide hydrogel surfaces was achieved through self-assembly. An 85% phosphoric acid solution was used as the solvent, because mixing a cello-oligosaccharide solution in 85% phosphoric acid with water has been reported to induce self-assembly. ,

Azidated cello-oligosaccharides were dissolved at the molecular level in 85% phosphoric acid, and self-assembly was initiated by simply applying the azidated cello-oligosaccharide solutions at concentrations of 1%, 3%, and 5% (w/v) onto the hydrogel surfaces (Figure a). Notably, this self-assembly process requires no additional reagents, highlighting its simplicity and practical utility. Through the self-assembly process, phosphoric acid is diluted upon mixing with water from the hydrogels, making this hydrogel surface functionalization system potentially suitable for biological applications. Additionally, the application of concentrated phosphoric acid caused no apparent changes in hydrogel integrity. The surfaces of hydrogels became turbid, indicating the formation of azidated cello-oligosaccharide assemblies (Figure a). For the 1% and 3% (w/v) azidated cello-oligosaccharide solutions, the turbidity gradually increased over the course of 1 h. In contrast, for the 5% (w/v) solution, the turbidity appeared to reach saturation within a few minutes. The turbidity remained after purification by immersion in water, demonstrating stable surface functionalization of hydrogels with azidated cello-oligosaccharides. Moreover, the turbidity increased with increasing concentration of azidated cello-oligosaccharides, suggesting a higher amount of assemblies on the hydrogel surfaces.

2.

Self-assembly of terminally azidated cello-oligosaccharides on polyacrylamide hydrogel surfaces. (a) Photographs of polyacrylamide hydrogels before and after the self-assembly of terminally azidated cello-oligosaccharides and subsequent purification. Solutions of azidated cello-oligosaccharides at concentrations of 1%, 3%, and 5% (w/v) in 85% phosphoric acid were applied to the hydrogel surfaces to induce self-assembly. (b) Estimated loading rates and loading amounts of azidated cello-oligosaccharides on polyacrylamide hydrogels. The values were estimated based on the simplified assumption that the assemblies formed on the hydrogel surfaces and the dispersed assemblies exhibited similar light-scattering behavior. Data are presented as the mean ± standard deviation (n = 3). (c) ATR-FTIR spectra of polyacrylamide hydrogels before and after the self-assembly of terminally azidated cello-oligosaccharides at a concentration of 5% (w/v) and subsequent purification.

The loading amount of cello-oligosaccharide assemblies on the hydrogel surface was roughly estimated from turbidity. A high loading rate of approximately 90% was achieved when a 1% (w/v) azidated cello-oligosaccharide solution was used (Figure b). Lower loading rates were observed at higher concentrations, with values decreasing to approximately 36% and 30% for 3% and 5% (w/v) solutions, respectively. This suggests that higher concentrations promote assembly formation through homogeneous nucleation in the solution phase. Based on these estimated loading rates, the amount of cello-oligosaccharides immobilized on the hydrogel surface was calculated. Higher solution concentrations resulted in increased amount of azidated cello-oligosaccharide assemblies on hydrogels (Figure b).

Because the self-assembly process of cello-oligosaccharides is triggered by the intrinsic water content of hydrogels, this surface functionalization strategy is expected to be applicable to a wide range of hydrogels, irrespective of their chemical composition. To validate this universality, physical hydrogels of agarose were employed as substrates for the self-assembly of azidated cello-oligosaccharides. Figure S2a shows images of agarose hydrogels taken 1 min and 1 h after the precipitation procedure, revealing that the assemblies were visually heterogeneous on a local scale. Notably, assemblies of azidated cello-oligosaccharides were already formed within 1 min, and only minimal changes in the locally heterogeneous morphology were observed after 1 h. These results indicate that self-assembly on agarose hydrogels occurs predominantly and almost instantaneously upon deposition of the solution onto the hydrogel surface, proceeding more rapidly than that observed on polyacrylamide hydrogels.

The loading rates of azidated cello-oligosaccharides on agarose hydrogel surfaces were estimated by turbidity measurements and are shown in Figure S2b. A consistently high loading rate was observed across different concentrations of azidated cello-oligosaccharides, which contrasts with the concentration-dependent loading behavior observed for polyacrylamide hydrogels. Taken together with the faster assembly kinetics, agarose hydrogels appear to promote oligosaccharide assembly more effectively than polyacrylamide hydrogels. This behavior may be partly attributed to the higher water content of agarose hydrogels (98.9 ± 0.1% (w/w)) compared with that of polyacrylamide hydrogels (90.6 ± 0.1% (w/w)). Nevertheless, these results demonstrate that hydrogel surface functionalization via the self-assembly of cello-oligosaccharides is broadly applicable across different hydrogel materials, with the assembly efficiency being influenced by the water content and other characteristics of the substrate gels.

Given that successful surface functionalization was achieved for both polyacrylamide and agarose hydrogels despite their substantially different structures and properties, physical entanglement between the assemblies and the hydrogel networks appears to play an important role in the surface modification process. Intermolecular interactions, including hydrogen bonding, are also expected to contribute to the adhesion. Notably, the application of assemblies preformed in solution to hydrogel surfaces resulted in only limited adhesion of the assemblies (Figure S3). This suggests that self-assembly occurring in the confined environment near the hydrogel surface may promote physical entanglement between the assemblies and the hydrogel networks, thereby resulting in stable surface modification.

Assembled Structure of Terminally Azidated Cello-oligosaccharides

Following the self-assembly process, the assembled azidated cello-oligosaccharides were characterized. ATR-FTIR spectra of polyacrylamide hydrogels with assembled azidated cello-oligosaccharides are shown in Figure c, where a characteristic peak corresponding to the azido group is observed at 2113 cm^–1. The crystal allomorph of the cello-oligosaccharide chains was identified as cellulose II, as indicated by characteristic peaks derived from hydrogen bonding at 3438 and 3483 cm^–1. These results suggest that the assembled azidated cello-oligosaccharides adopted the thermodynamically most stable crystalline structure at the hydrogel surface. Meanwhile, characteristic peaks of cellulose II and azido groups were absent in the ATR-FTIR spectrum of pristine polyacrylamide hydrogels, confirming that the observed signals originated exclusively from the azidated cello-oligosaccharide assemblies. Consequently, the preparation of cello-oligosaccharide-functionalized hydrogels can be achieved through a simple precipitation-based approach employing phosphoric acid as the solvent.

The nanomorphology of assembled azidated cello-oligosaccharides on polyacrylamide hydrogel surfaces was examined by SEM. In contrast to the smooth surface observed for pristine polyacrylamide hydrogels (Figure a), assemblies of azidated cello-oligosaccharides exhibited characteristic nanomorphologies (Figure b–d). The extent of surface coverage strongly depended on the concentration of azidated cello-oligosaccharides used during the assembly process. Lower concentrations resulted in sparse assemblies with partially exposed hydrogel surfaces, whereas higher concentrations led to the formation of dense assemblies covering the hydrogel surface. The surface coverage by the assemblies was estimated from the SEM images to be approximately 90%, 98%, and 99% for the samples prepared using 1%, 3%, and 5% (w/v) azidated cello-oligosaccharide solutions, respectively.

3.

SEM images of hydrogel surfaces. (a) Pristine polyacrylamide hydrogel. (b–d) Polyacrylamide hydrogels with terminally azidated cello-oligosaccharides assembled on the surface via self-assembly. Solutions of azidated cello-oligosaccharides at concentrations of (b) 1%, (c) 3%, and (d) 5% (w/v) were applied to the hydrogel surfaces to induce self-assembly.

The assemblies exhibited a granular morphology with characteristic sizes on the order of several micrometers (Figure b–d). Granular structures with sizes comparable to those observed by SEM were also observed by optical microscopy, confirming that the same assembled structures were detected across different imaging techniques (Figure S4). The apparent height of the cello-oligosaccharide assemblies formed on the hydrogel surfaces using a 1% (w/v) azidated cello-oligosaccharide solution was approximately 100 nm (Figure S5). These observations suggest that nucleation of azidated cello-oligosaccharide assemblies occurred sparsely on the hydrogel surface, followed by growth into granular structures composed of nanoscale features. Additionally, cross-sectional SEM images revealed no clear boundary between the cello-oligosaccharide assembly layer and the hydrogel layer (Figure S5), suggesting physical entanglement between the azidated cello-oligosaccharide assemblies and the hydrogel networks.

Conjugation of Biotin via the Click Reaction

Understanding and engineering materials–biological interfaces are central challenges in biomaterials science, as interfacial molecular interactions ultimately govern biological recognition and function. ,− Given the antibiofouling properties of cello-oligosaccharide assemblies, − hydrogels with azidated cello-oligosaccharides were explored as a platform for biomolecular sensing. In this study, biotin was employed to detect antibiotin IgG as a model target and was conjugated to the terminal azido groups of the cello-oligosaccharides via a Huisgen cycloaddition reaction (Figure a). Specifically, the biotin molecule was first modified by conjugation with BCN-amine through a condensation reaction, yielding biotin bearing a strained alkyne moiety (Scheme S2). This strained alkyne functionality enables efficient biotin conjugation to the azido groups in the cello-oligosaccharide assemblies via click chemistry.

4.

Biotin conjugation to azidated cello-oligosaccharides assembled on polyacrylamide hydrogel surfaces. (a) Schematic illustration. (b) MALDI-TOF mass spectra of azidated cello-oligosaccharides before and after biotin conjugation under conditions with azidated cello-oligosaccharide-to-BCN-amine molar ratios (denoted as azido-to-alkyne molar ratio in the graph) of 10:1, 1:2, and 1:4. Polyacrylamide hydrogels with azidated cello-oligosaccharides were prepared using a 1% (w/v) azidated cello-oligosaccharide solution prior to biotin conjugation.

The click reaction was performed using a dimethyl sulfoxide–containing solution (details are provided in section Conjugation of Biotin to Assembled Azidated Cello-oligosaccharides on Hydrogels). The biotin conjugation rate to azidated cello-oligosaccharides was controlled by adjusting the concentration of alkynylated biotin relative to the molar amount of azidated cello-oligosaccharides and was quantified using MALDI-TOF mass spectrometry (Figure b). Specifically, azidated cello-oligosaccharide-to-BCN-amine molar ratios of 10:1, 1:2, and 1:4 yielded average conjugation rates of 9 ± 10%, 51 ± 16%, and 95 ± 9%, respectively (n = 3). These results demonstrate that, although the assemblies are formed via intermolecular interactions, the cello-oligosaccharide-based assemblies on hydrogel surfaces are tolerant of postfunctionalization by chemical reactions conducted in organic solvent-containing solutions, owing to their inherent structural robustness.

Detection of IgG

To evaluate the biomolecule detection performance of the functionalized hydrogels, both target and nontarget antibodies conjugated with HRP were employed in an enzyme-linked immunosorbent assay (ELISA). In this system, antibiotin IgG served as the target antibody, whereas antimouse IgG IgG was used as a nontarget control. After binding of the target antibodies to the hydrogel surface, the conjugated HRP catalyzed the conversion of o-phenylenediamine with H₂O₂ into a dye, 2,3-diaminophenazine. The amount of antibody bound to hydrogel surfaces was quantified (semiquantitatively) by measuring the absorbance at 490 nm using a microplate reader, where higher absorbance values correspond to greater antibody binding. It should be noted that the measured absorbance is influenced by the turbidity of the hydrogel; therefore, hydrogels with lower amounts of cello-oligosaccharides were preferentially used to ensure accurate absorbance measurements (Figure S6).

Figure shows the absorbance signals obtained for antibiotin IgG and antimouse IgG IgG on biotinylated hydrogels at different biotin conjugation rates. Under all conditions examined, antibiotin IgG produced higher absorbance values than antimouse IgG IgG. The absorbance increased with increasing IgG concentration, and the corresponding slopes were higher for antibiotin IgG than for antimouse IgG IgG, irrespective of the biotin conjugation rates. The sensing performance was comparable to that of our previously reported biomolecular sensing system using paper functionalized with azidated cello-oligosaccharide assemblies. Notably, only low absorbance values were observed in the absence of biotin conjugation, indicating a low level of nonspecific protein adsorption in the present system (Figure S7). This result further supports the selectivity of the biomolecular sensing system and the antibiofouling properties of the cello-oligosaccharide assemblies. These results demonstrate the selective detection of antibiotin IgG and suggest that antibiotin IgG specifically recognizes biotin and binds to the surface-functionalized hydrogels.

5.

ELISA-based detection of IgG using surface-biotinylated polyacrylamide hydrogels with biotin conjugation rates of approximately (a) 9%, (b) 51%, and (c) 95%. The polyacrylamide hydrogels with azidated cello-oligosaccharides were prepared using a 1% (w/v) azidated cello-oligosaccharide solution. The concentrations of antibiotin IgG (target) and antimouse IgG IgG (nontarget) were varied from 5 to 100 ng mL^–1. Data are presented as the mean ± standard deviation (n ≥ 6).

An increase in the biotin conjugation rate led to higher absorbance signals for antibiotin IgG, but also resulted in increased absorbance for antimouse IgG IgG (Figure ). At biotin conjugation rates of approximately 9% and 51%, the slopes of the IgG concentration-dependent absorbance were more than 7-fold higher for antibiotin IgG than for antimouse IgG IgG (Figure a,b). In contrast, at a biotin conjugation rate of approximately 95%, the difference between the two antibodies was substantially reduced to approximately 4-fold, owing to a pronounced increase in the background signal of antimouse IgG IgG (Figure c). This behavior suggests that a high biotin conjugation density promoted nonspecific adsorption of nontarget antibodies. These results indicate that maintaining a relatively low antigen conjugation rate is crucial for preserving the antibiofouling properties of the assemblies and, consequently, achieving high detection selectivity.

IgG detection was further evaluated in a solution containing 10% FBS, which serves as a complex biological medium with nonspecific proteins, in order to assess sensing specificity under physiologically relevant conditions. Even in the presence of a variety of protein species, selective detection of antibiotin IgG was successfully achieved (Figure ). Remarkably, at a biotin conjugation rate of approximately 9%, both signal intensity and detection selectivity were preserved, likely owing to the antibiofouling properties of the assemblies, even under protein-present conditions. Collectively, these results demonstrate that the hydrogel functionalization strategy enables specific biomolecular detection even under biologically relevant, protein-present conditions.

6.

ELISA-based detection of IgG in a solution containing 10% FBS. ELISA responses for 100 ng mL^–1 antibiotin IgG (target) and antimouse IgG IgG (nontarget) using surface-biotinylated polyacrylamide hydrogels with biotin conjugation rates of approximately 9%, 51%, and 95%. The polyacrylamide hydrogels with azidated cello-oligosaccharides were prepared using a 1% (w/v) azidated cello-oligosaccharide solution. Data are presented as the mean ± standard deviation (n ≥ 8). Statistical analyses were performed separately for each antibody using one-way ANOVA followed by Tukey’s multiple comparisons test. ns, **, ***, and **** indicate not significant, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Conclusions

We demonstrate a simple and versatile strategy for hydrogel surface functionalization based on the self-assembly of terminally azidated cello-oligosaccharides. The process relies on spontaneous mixing between the applied solution and the intrinsic water content of hydrogels, leading to the formation of nanostructured assemblies at the interface. The resulting surfaces are structurally stable and amenable to postfunctionalization via click chemistry, enabling biofunctional interfaces. The antibiofouling properties of the cello-oligosaccharide assemblies appeared to contribute to the high specificity observed in ELISA-based IgG detection, thereby opening potential applications in specific biomolecular sensing. Given that a wide variety of terminally modified cello-oligosaccharides are available, this self-assembly strategy provides substantial flexibility for designing hydrogel surfaces with tailored functionalities. ,,,, Moreover, this surface functionalization strategy is expected to be applicable not only to polymer hydrogels but also to biomass-derived hydrogels. − In conclusion, this approach provides a general route to engineering hydrogel interfaces and holds strong potential for applications in biomolecular sensing and other biointerface technologies.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acspolymersau.6c00076.

• Synthetic scheme of azidated cello-oligosaccharides; characterizations of terminally azidated cello-oligosaccharides; self-assembly of terminally azidated cello-oligosaccharides on agarose hydrogel surfaces; photographs of polyacrylamide hydrogels before and after washing, following the application of preformed azidated cello-oligosaccharide assemblies; optical microscopy images of polyacrylamide hydrogels with and without terminally azidated cello-oligosaccharides; cross-sectional SEM image of a polyacrylamide hydrogel surface functionalized with terminally azidated cello-oligosaccharide assemblies; synthetic scheme of alkynylated biotin; apparent absorbance at 490 nm of polyacrylamide hydrogels with azidated cello-oligosaccharides; ELISA-based detection of IgG using surface-biotinylated polyacrylamide hydrogels with biotin conjugation rates of 0%, 9%, 51%, and 95% (PDF)

D.M.S.: Methodology, validation, investigation, visualization, writingoriginal draft. H.M.: Methodology, investigation. Y.H.: Validation, writingreview and editing. K.S.: Validation, visualization, writingreview and editing. Y.-C.C.: Writingreview and editing, supervision, project administration, funding acquisition. T.S.: Conceptualization, resources, writingreview and editing, supervision, project administration, funding acquisition.

The authors declare no competing financial interest.