Rapid Covalent Immobilization of Proteins by Phenol-Based

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Rapid Covalent Immobilization of Proteins by Phenol-Based Photochemical Cross-Linking Jun Ren,† Kaikai Tian,† Lingyun Jia,*,† Xiuyou Han,‡ and Mingshan Zhao‡ †

School of Life Science and Biotechnology and ‡School of Physics and Optoelectronic Engineering, Dalian University of Technology, Dalian, 116023, P. R. China S Supporting Information *

ABSTRACT: A strategy for photoinduced covalent immobilization of proteins on phenol-functionalized surfaces is described. Under visible light irradiation, the reaction can be completed within seconds at ambient temperature, with high yields in aqueous solution of physiological conditions. Protein immobilization is based on a ruthenium-catalyzed radical cross-linking reaction between proteins and phenol-modified surfaces, and the process has proven mild enough for lipase, Staphylococcus aureus protein A, and streptavidin to preserve their bioactivity. This strategy was successfully applied to antibody immobilization on different material platforms, including agarose beads, cellulose membranes, and glass wafers, thus providing a generic procedure for rapid biomodification of surfaces.

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excited state.18 A similar photoredox system has recently been reported to cross-link tyrosine-rich proteins such as gelatin19 and fibrinogen20 into hydrogels for tissue engineering applications. The Ru(II)-catalyzed reaction to immobilize cells on surfaces was also reported by Leubke and co-workers.21 All this research has revealed that tyrosine residues might play a major role in protein cross-linking, and the photooxidation product of phenol could be highly reactive for coupling with other proteins.22 Despite these achievements, direct information about possible protein residues involved in the reaction is still lacking. However, it still provides a feasible protein immobilization strategy by taking advantage of the highly efficient photochemical cross-linking between phenol groups and target proteins. This study aimed to establish a generally applicable photochemical method that could enable highly efficient cross-linking between active proteins and any phenol-functionalized material platform. The potential advantages of this method include no organic solvents required, reaction at physiological conditions, and light-dependent control. More importantly, covalent immobilization of proteins can be completed within seconds, thus providing a potential online strategy for biomodification, which would greatly facilitate the

he development of highly efficient bioconjugation chemistry has been greatly spurred by the growing demanding for various biodevices,1,2 such as biochips, enzymatic nanoreactors, and different types of biosensors. For these applications, an effective biomodification strategy that could achieve rapid and facile immobilization of bioactive molecules like enzyme and antibody is essentially important.3−8 However, due to the inherent drawback of protein reagents, mainly associated with low concentration of reactive sites and their steric hindrance in heterogeneous reactions, the methods commonly used for protein immobilization generally suffer from long reaction times, nonphysiological pH ranges and solvents, and even the necessity to employ UV-irradiation to activate the coupling reactions. These reaction conditions would threaten the activity of biomolecules, especially for those that are not stable in aqueous solutions. Naturally adopted photochemical processes, represented by photosynthesis of plants, have been found very effective for achieving highly efficient catalytic conversion in physiological conditions.9,10 Photoinduced immobilization of proteins has also been exploited.11−15 Fancy and Kodadek reported that native proteins rich in tyrosine residues can be chemically crosslinked into multiprotein complexes very rapidly using a visible light-irradiated system.16,17 The method is based on a photoinduced radical cross-linking reaction using ruthenium (Ru(II)) as photoredox catalyst, which was featured by absorbance in the visible range and chemical stability in the © XXXX American Chemical Society

Received: July 22, 2016 Revised: September 5, 2016

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DOI: 10.1021/acs.bioconjchem.6b00413 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Photoinduced immobilization of BSA-FITC on phenol-functionalized agarose beads. (a) Schematic representation of preparation of phenol-functionalized agarose beads and the photoinduced immobilization of BSA-FITC on them. (b) Micrographs of agarose beads under different reaction conditions, from left to right: (1) control group without photoredox catalyst and irradiation, (2) control group without irradiation, (3−6) reactions with exposure times of 0.2, 0.5, 1, and 5 s, respectively. All white light (front row) and fluorescence (back row) micrographs were taken under identical conditions: magnification 40×, exposure time 1/5.5 s. Scale bar represents 200 μm.

Figure 2. Effect of irradiation time on protein photoimmobilization on agarose. (a) Influence of irradiation time on IgG capacity (black, left axis) and immobilization yield (red, right axis). (b) Influence of irradiation time on BSA capacity (black, left axis) and immobilization yield (red, right axis).

functionalized surface could remain reactive after several months of storage in PBS (pH 7.4). To initially examine the kinetics of the visible-light-induced protein immobilization, a series of samples containing fluorescently labeled bovine serum albumin (BSA-FITC) as model protein were subjected to irradiation, with the exposure time ranging from 0.2 to 5 s (Figure 1b). The results confirmed the phototriggered capture of BSA-FITC onto agarose beads, as evidenced by the gradual increase of fluorescence intensity with irradiation time. For the control group containing only BSAFITC, no fluorescence was detected on the agarose beads after being thoroughly rinsed, suggesting negligible physical adsorption of protein on phenol-functionalized agarose. An irradiation process of mere 0.2 s could result in detectable fluorescence on the beads, and the fluorescence intensity became much stronger for the sample undergoing irradiation treatment for 1 s. This strategy can be applicable for various material platforms. With a similar process, human immunoglobulin G (IgG) has been successfully immobilized on the surface of cellulose filter paper (Figure S2). Quantitative experiments were further conducted with a broader range of reaction times (Figure 2). It turned out that

fabrication and application procedure for many biological devices and flow reactor systems (for example, microfluidic chips or nanochannels). For most material platforms, phenol-functionalization of surfaces can be easily achieved with standard grafting methods using tyramine.19 In the proof-of-principle study, tyramine was coupled on agarose beads via epichlorohydrin reaction (Figure 1a). The density of the epoxy group was controlled to about 10 μmol/mL in the activation step to avoid potential nonspecific adsorption of proteins induced by phenol groups. Coupling of tyramine on agarose beads was verified by FT-IR (Figure S1). For the resultant resin, the coupling density of phenol groups was detected to be 4 μmol/mL by elemental analysis. Protein immobilization on the prepared phenol-functionalized agarose was induced by the illumination of visible light (using a 200 W incandescent lamp) in a solution of 10 mM PBS (pH 7.4) containing 3 μM Ru(bpy)3Cl2 and 60 μM ammonium persulfate (APS). After the reaction, the resin was rinsed with 0.1 M EDTA solution to remove the remaining photocatalyst. Unlike other active groups for protein immobilization, such as surface-bound N-hydroxysuccinimide (NHS), which are usually unstable in aqueous solution, we found that the phenolB


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Figure 3. Evaluation of primary reactive groups involved in Ru (II)-catalyzed protein immobilization. (a) Schematic representation of photoinitiated reaction of phenol-functionalized surface with phenol, primary amino, and thiol groups. (b) Influence of phenol, ethanolamine, and mercaptoethanol, which were respectively added in the reaction solution as an additive, on BSA immobilization capacity; XPS characterization of coupled ethanolamine (c) and mercaptoethanol (d) on phenol-functionalized silicon substrates.

the coupling amount of proteins increased with irradiation period within the range of 2−15 s, while further prolonging reaction time could no longer increase the protein coupling amount significantly. For BSA, doubling the exposure time from 15 to 30 s could only result in an increase of yield by about 7%; thus, substantial yield could be achieved within 15 s of reaction, especially the first 2 s. For a solid−liquid heterogeneous reaction system, the efficiency of protein immobilization can also be affected by the concentration of reactants, so the influence of initial protein concentration was also investigated together with irradiation time (Figure S3). The results showed that the immobilization amount of BSA on agarose had a remarkable correlation with the initial protein amount (Figure S3a), and the reaction rate could remain at a rather stable level for the samples with the initial BSA concentration ranging from 4 to 10 mg/mL, which were around 35%, 45%, and 50% for 2, 10, and 30 s of reaction, respectively (Figure S3b). For the reaction with initial BSA concentration of 10 mg/mL, the irradiation period of 2 s could result in a coupling amount of 11 mg/mL resin, while prolonging reaction time to 30 s can only increase the amount by 27% (Figure 2b). However, in the case of IgG, longer reaction time could cause a substantial increase of coupling amount within the range of 2−15 s (Figure 2a). The larger size of the IgG molecule (MW 150 kDa) may have bigger mass transfer resistance when compared to BSA (MW 66 kDa) in porous media like agarose beads. In addition, the difference in the type and amount of reactive groups on the two proteins may also contribute to the different efficiency of the coupling reaction.

The reactivity of possible protein residues that might be involved in photochemical cross-linking with immobilized phenol groups was examined. In the proposed mechanism,17 Ru(II) (bpy)32+ is photolyzed in the presence of a persulfate, generating Ru(III) and sulfate radical. Ru(III) is a potent oneelectron oxidant and would be expected to oxidize immobilized phenol groups (Figure 3a). If another phenol residue on protein is nearby, then diphenol coupling would be expected. This mechanism of diphenol cross-linking has been demonstrated in several reaction systems aiming to develop photocontrolled hydrogel biomaterials.18,22,23 Besides tyrosine residue, nucleophilic attack by lysine or cysteine groups was also inferred by Fancy and Kodadek in proposing mechanistic hypotheses of Ru (II)-catalyzed protein cross-linking. 16 However, direct experimental evidence is still lacking. Phenol, ethanolamine, and mercaptoethanol were added into the reaction solution, respectively, to evaluate the influence of phenol, primary amino, and thiol groups (Figure 3b). The results showed that the addition of phenol could seriously hinder the BSA immobilization onto tyramine-modified agarose. The reaction could be almost totally blocked when 0.1 M phenol was present. The addition of ethanolamine or mercaptoethanol could also affect the reaction, confirming the involvement of lysine and cysteine in the protein immobilization, which was also proven directly by XPS analysis using silicon platform (Figure 3c,d). Figure 3c shows a highresolution N 1s region of silicon surface after reaction with ethanolamine, and Figure 3d shows S 2p region of silicon surface after reaction with mercaptoethanol. In the figures, peaks of Ph-N (400.10 eV) and Ph-S (163.50 eV) were C


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Figure 4. Capture of tumor cells on anti-EpCAM-functionalized glass substrates. (a) Micrographs of cells captured by different surfaces. Cell capture assays were carried out on four different surfaces: glass substrate without any surface modification (Glass), glass substrate coupling tyramine after silanization (Glass-Tyramine), glass substrate further modified with anti-EpCAM antibody mediated by streptavidin that was immobilized through GMBS (Glass-GMBS-SA-EpCAM), and glass substrate modified with anti-EpCAM antibody mediated by streptavidin that was immobilized through tyramine-based photochemical conjugation (Glass-Tyramine-SA-EpCAM). MCF7 cell line was used. The incubation time was 1 and 3 h, respectively. (b) Quantitative analysis of cell attachment on each surface. The statistics of immobilized cells was performed on three replicate substrates after DAPI staining (** p < 0.01). (c) Schematic representation of Glass-Tyramine-SA-EpCAM.

detected.24,25 These data demonstrate the presence of a heteroatom−arene linkage as the result of amino and thiol coupling on surface-immobilized phenol groups. However, it is noteworthy that there is still about half the amount of BSA being immobilized when 0.1 M primary amino or thiol (about 600-fold molar excess compared to BSA in the solution) was present, as shown in Figure 3b. The results reveal that tyrosine residues might dominate the coupling reaction of proteins on immobilized phenol groups, with much higher reaction activity than amino and thiol groups. To evaluate the potential impact of photochemical reaction condition on the bioactivity of proteins, lipase and Staphylococcus aureus protein A (PA) were chosen as model proteins. After a typical photochemical process for 30 s, the catalytic activity of lipase was detected and compared with that of original enzyme without treatment. It turned out that 80.7% activity was preserved for the treated lipase (Figure S4). PA was immobilized on agarose beads through the described method, and the immobilized PA could also retain the antibody binding activity. The resultant PA resin was found to have a stable binding capacity toward human immunoglobulin G (Figure S5). Furthermore, the antibody immobilization strategy on glass surfaces was also established through tyramine-based photochemical conjugation, and compared with the traditional method (Figure 4). Immuno-capture of cells expressing the epithelial cell adhesion molecule (EpCAM) is frequently used

to enrich circulating tumor cells (CTC) from blood. In our strategy, biotinylated anti-EpCAM antibody was immobilized on glass surface through biotin−streptavidin (SA) conjugation, and SA-fuctionalized glass substrates were initially prepared by photochemical cross-linking between SA and tyraminemodified glass substrates (Figure 4c). As shown in Figure 4a, compared to traditional methods to prepare anti-EpCAMfunctionalized surface that normally use N-succinimidyl 3maleimidopropionate (GMBS) for SA coupling, photochemical reaction could yield a bigger coupling amount of SA. The resultant surface has proven more sufficient for antibody immobilization, and could capture significantly larger number of MCF7 cells, a kind of breast cancer cell line (Figure 4b). The results proved the feasibility to employ phenol-based photochemical reaction for producing a highly efficient avidinmodified surface, which is the basis for antibody immobilization in most immunoassay applications. In summary, we have presented a highly efficient and facile method for protein immobilization. The reaction is initiated by visible light irradiation, and proceeds for only seconds at room temperature. Proteins can be immobilized covalently onto various material platforms with considerable coupling amount and bioactivity preservation. To the best of our knowledge, it is the fastest strategy for protein immobilization, especially applicable to the proteins that are not stable in aqueous solutions at room temperature. D


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00413. Experimental procedures, additional data, and characterization data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: (86) 411 84706125. Fax: (86) 411 84706125. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Fundamental Research Funds for the Central Universities (DUT15LAB17) and International Science & Technology Cooperation Program of China (No. 2014DFG32590).

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Rapid Covalent Immobilization of Proteins by Phenol-Based

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