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Supramolecular Platform with Switchable Multivalent Affinity: Photo-Reversible Capture and Release of Bacteria Wenjun Zhan, Ting Wei, Limin Cao, Changming Hu, Yangcui Qu, Qian Yu,* and Hong Chen* State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren’ai Road, Suzhou, 215123, People’s Republic of China S Supporting Information *

ABSTRACT: Surfaces having dynamic control of interactions at the biological system−material interface are of great scientific and technological interest. In this work, a supramolecular platform with switchable multivalent affinity was developed to efficiently capture bacteria and on-demand release captured bacteria in response to irradiation with light of different wavelengths. The system consists of a photoresponsive self-assembled monolayer containing azobenzene (Azo) groups as guest and β-cyclodextrin (β-CD)-mannose (CD-M) conjugates as host with each CD-M containing seven mannose units to display localized multivalent carbohydrates. Taking the advantage of multivalent effect of CD-M, this system exhibited high capacity and specificity for the capture of mannose-specific type 1-fimbriated bacteria. Moreover, ultraviolet (UV) light irradiation caused isomerization of the Azo groups from transform to cis-form, resulting in the dissociation of the host−guest Azo/CD-M inclusion complexes and localized release of the captured bacteria. The capture and release process could be repeated for multiple cycles, suggesting good reproducibility. This platform provides the basis for development of reusable biosensors and diagnostic devices for the detection and measurement of bacteria and exhibits great potential for use as a standard protocol for the on-demand switching of surface functionalities. KEYWORDS: Photoresponsive, host−guest interaction, multivalent effect, bacterial capture, bacterial release

1. INTRODUCTION Interactions between biological systems and artificial materials normally occur at their interface and play a crucial role in living processes and in the realization of biofunctions.1 In nature, organisms are in a complex and changing environment, and they have already developed unique dynamic interfacial functions with excellent adjustability and reversibility during the long time span of evolution.2 It is thus of considerable interest to endow synthetic materials with the ability to dynamically control the interfacial behaviors of biological entities such as proteins,3 bacteria,4 and mammalian cells.5 Stimuli-responsive interfacial materials, which possess the capability to change their physical and/or chemical surface properties in response to external stimuli (e.g., temperature, pH, light, solvent, etc.), have thus attracted great attention in building such “smart” biointerfaces,6−8 which not only provide ideal platforms to study biological processes triggered by changes in the environment but also hold the potential for applications including biomolecular detection and separation,9 cell sheet tissue engineering,10 and kill-and-release dualfunctional antibacterial surfaces.11 Among the often used external stimuli, light is of unique advantage because of its precise spatiotemporal control with high resolution.12 Furthermore, light is a “clean” stimulus that is relatively noninvasive and does not lead to sample contamination, making it particularly suitable for use with biological systems. Incorporation of photoresponsive molecules onto © 2017 American Chemical Society

material surfaces can change or tune the surface properties and functions in an accurate and predictable manner by irradiation with light of different wavelengths, enabling the dynamic modulation of biointerfacial interactions and thus showing great potential for biomedical and biotechnology applications.13 For example, azobenzene (Azo) is one of the most investigated photoresponsive molecules and can isomerize reversibly between the extended trans-form and the compact cis-form upon exposure to UV and visible light.14 In particular, Azo is well-known as a guest moiety with host molecules such as cyclodextrin (CD) or cucurbituril (CB) via photoresponsive reversible host−guest interactions, as these host molecules show a greater thermodynamic preference for trans-Azo over cis-Azo.15,16 Several dynamic supramolecular platforms based on Azo have been developed to remotely control biointerfacial interactions (e.g., the reversible capture and release of DNA,17 proteins,18 and bacteria,19 the photoswitchable bioelectrocatalysis,20 and the in situ modulation of cellular adhesion and migration21). For example, using Azo derivatives conjugated with bioactive ligands, Gao et al.22 and Jonkheijm et al.23 constructed a series of supramolecular polymer brushes and self-assembled monolayers containing CD moieties to study specific intercellular interactions and to optically control Received: December 1, 2016 Accepted: January 10, 2017 Published: January 10, 2017 3505

DOI: 10.1021/acsami.6b15446 ACS Appl. Mater. Interfaces 2017, 9, 3505−3513

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Diagram of Supramolecular Platform with Switchable Multivalent Affinity To Capture and Release Bacteriaa

a

To switch the bacteria-capture state (green part, trans- form of Azo) to the bacteria-release state (red part, cis- form of Azo), the Au−Mix/CD-M surface was irradiated with 365 nm UV light for 30 min. To switch the bacteria-release state back to bacteria-capture state, the Au−Mix surface was irradiated with 450 nm visible light for 30 min and then immersed it into a fresh CD-M solution to incorporate CD-M.

recognition at the mannose moiety. Moreover, irradiation of UV light on this surface causes the isomerization of Azo from the trans-form to the cis-form, resulting in the dissociation of the host−guest inclusion complexes and thus the removal of captured bacteria from the surface. The bacterial release efficiency can be further enhanced by the introduction of the competitive compound adamantane (Ada), which has a higher binding affinity to β-CD than Azo.

protein and bacterial immobilization, respectively. It should be noted that in their systems each Azo derivative only contained one ligand; this monovalent ligand may affect the recognition of corresponding cells or proteins.24 Alternatively, previous studies indicated that compared to monovalency, the multivalency concerning ligands present on the surfaces strongly increased the binding affinity.25−27 CD is an appropriate template with multivalent binding sites for postmodification to increase the local density of ligands.28,29 Decoration of biorecognizable ligands on CDs produce homogeneous compounds with welldefined structures and multiple ligands, which is an effective way to enhance the recognition capability via the “multivalent effect”.30−33 However, there are few reports on photoresponsive planar surfaces using a combination of Azo and CD-based multivalent ligands to control biointerfacial interactions. Taking advantage of the photoresponsive host−guest interaction between Azo and CD and the multivalent effect of CD derivatives, a highly efficient supramolecular platform was developed for the dynamic modulation of biointerfacial interactions to specifically capture and release bacteria on demand (as illustrated in Scheme 1). Here, a photoresponsive self-assembled monolayer (SAM) was fabricated on a gold substrate with mixed thiols terminated by either an Azo moiety or oligo(ethylene glycol) (OEG). Introduction of OEG not only provides enough free space to allow the reversible photoisomerization of Azo but also offers a hydrophilic environment to suppress nonspecific surface interactions.34 Moreover, a multivalent ligand, β-CD decorated with seven mannose molecules on its smaller ring (denoted CD-M), was incorporated on the SAM via host−guest inclusion. Mannose is a glycoside ligand with a specific affinity to lectin proteins (such as Concanavalin A (ConA)35 and the type 1 fimbrial protein FimH, which is expressed on the surface of many Gramnegative bacteria36) via well-established carbohydrate−protein interactions. Although the individual binding interaction between mannose and protein is typically weak, it can be improved upon by the incorporation of multiple mannose ligands using β-CD as a template for individual proteins.27,37 It is thus anticipated that this system will exhibit a high capability for the specific capture of type 1-fimbriated bacteria (such as Escherichia coli, E. coli) by the formation of ternary complexes of trans-Azo/CD-M/FimH through simultaneous host−guest inclusion in the CD cavity and carbohydrate−protein

2. EXPERIMENTAL SECTION 2.1. Materials. A β-cyclodextrin derivative with seven α-Dmannose ligands (CD-M),38 a fluorescent β-CD derivative with Rhodamine B isothiocyanate (CD-RBITC, red color),39 and 23mercapto-3,6,9,12-tetraoxatricosan-1-ol (OEG-SH)40 were synthesized as reported previously. A photoresponsive thiol, 23-mercapto-3,6,9,12tetraoxatricosyl-4-(phenyldiazenyl)benzoate (Azo-OEG-SH), was synthesized as shown in Supporting Information (SI). All other organic solvents were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China) and purified before use according to standard methods. Na125I was obtained from Chengdu Gaotong Isotope Co., Ltd. (China). ConA and human serum albumin (HSA) were purchased from Sigma-Aldrich Chem. Co.. Gold-coated silicon wafers (80 nm Au on a 10 nm chromium adhesion layer) were cut into 0.5 cm × 0.5 cm pieces. Deionized water was purified by a Millipore water purification system to give a resistivity of 18.2 MΩ·cm. 2.2. Preparation of SAMs/CD-M Surface. The gold-coated wafers were washed with acetone then treated with ozone plasma for 30 min. After washing with deionized water and ethanol, the goldcoated wafers were cleaned in a mixture of ammonia, hydrogen peroxide, and deionized water (NH3·H2O/H2O2/H2O = 1:1:5 v/v/v) for 10 min at 75 °C and then rinsed with deionized water and dried under nitrogen. SAMs were formed by immersing the cleaned goldcoated wafers into an ethanol solution containing the studied compounds in OEG-SH/Azo-OEG-SH at different ratios (1 mM total thiol concentration) for 24 h and then rinsed thoroughly with ethanol and dried in a stream of nitrogen. The SAMs/CD-M surface was prepared by the following procedure. The above SAM surfaces were immersed in 1 mM CD-M aqueous solution for 12 h, rinsed with deionized water to remove unconjugated CD-M, and then dried with N2. 2.3. Binding/Release of CD-RBITC. The Au−Mix surfaces were incubated with 0.25 mL of CD-RBITC solution at room temperature for 6 h in the dark and rinsed with deionized water. For CD-RBITC release, the above surfaces were immersed in deionized water and exposed to 365 nm UV light (approximately 20 mW/cm2) for 30 min and then rinsed with sterile water. To assess the reversible binding/ release of CD-RBITC on the Au−Mix surfaces, the samples previously exposed to 365 nm UV light were then exposed to 450 nm visible light 3506


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ACS Applied Materials & Interfaces (approximately 10 mW/cm2) for 30 min and once again incubated in a CD-RBITC solution for 12 h to obtain the regenerated substrate. The amounts of adsorbed CD-RBITC and remained CD-RBITC after release were determined as described as follows. Fluorescence images (10 pictures each sample) were taken by fluorescence microscopy (IX71, Olympus) after samples were dried with N2. The fluorescence intensity was obtained by analyzing these images using ImageJ software. Three replicates were measured for each surface. 2.4. Protein Adsorption. Prior to adsorption experiments, the surfaces were immersed in PBS solution containing NaI (0.002 mg/ mL, PBS−NaI) for at least 30 min; the “cold” NaI was added to the buffer to inhibit the adsorption of free 125I to the gold surface. ConA and HSA were radiolabeled with Na 125I according to our previous work.41 To measure ConA and HSA adsorption from PBS−NaI, labeled and unlabeled proteins were mixed (1/19 labeled/unlabeled) to give a total concentration of 0.1 and 1 mg/mL, respectively. For the ConA adsorption experiment, 1 mM Ca2+ and 1 mM Mg2+ were added to the PBS−NaI solution. The samples were immersed in protein (ConA or HSA) solution for 3 h at room temperature. The samples were then removed from the wells, rinsed with PBS−NaI three times (10 min each time), wicked onto filter paper, and transferred to clean tubes. A Wallac 2480 Wizard 300 automatic gamma counter (PerkinElmer Life Sciences) was used for radioactivity determination. Adsorption of ConA or HSA was expressed as mass per unit surface area. 2.5. Bacterial Assays. Details of bacterial culture and pretreatments were described in SI. The attachment of bacteria on the SAMs/ CD-M surfaces was assessed using an E. coli. suspension (1 × 108 cells/mL in phosphate buffered saline (PBS), pH 7.4). The sample surfaces were immersed in 500 μL of the bacterial suspension for 30 min at 37 °C in 48-well microtiter plate wells and then rinsed with PBS and sterile water to remove loosely attached cells and salts. For bacterial detachment, the above surfaces were immersed in PBS and exposed to 365 nm UV light (approximately 20 mW/cm2) for 30 min and then rinsed with PBS and sterile water. The staining assay was then carried out using the following procedure. The sample surfaces were stained with SYTO 9 (6.68 mM) for 15 min in the dark. After gently rinsing with sterile water and being dried with nitrogen, the bacteria attached to the surfaces were examined using a fluorescence microscope (IX71, Olympus, Japan) with a 40× objective, and images of 15 randomly chosen fields of view were captured. Each experiment was repeated at least three times and the number of bacteria in each image was analyzed with the Image-Pro Plus software to obtain the average and standard deviation. To assess the reversible attachment and detachment of bacteria on the sample surfaces, the samples previously exposed to 365 nm UV light were then exposed to 450 nm visible light (approximately 10 mW/cm2) for 30 min and once again incubated in a 1 mM CD-M solution for 12 h to obtain the regenerated substrate.

Figure 1. 1H NMR spectrum of Azo-OEG-SH.

Figure 1, indicating successful synthesis. Moreover, the reversible trans-cis photoisomerization of this compound was investigated in solution using UV−vis spectroscopy (Figure S2, SI). After the solution was illuminated with 365 nm UV light for 2 min, a remarkable decrease in the intensity of the transAzo characteristic peak at 324 nm was observed in the UV−vis spectrum, indicating the trans- to cis- conformational conversion of the Azo group.42 When the solution was then irradiated with 450 nm visible light for 5 min, the intensity of this peak almost reverted back to its original state. This photoresponsive switching could be repeated several times without deterioration of the molecule, providing the foundation to control surface properties in response to light when using this Azo derivative to prepare SAMs as described below. 3.2. Preparation and Characterization of SAMs/CD-M Surfaces. A photoswitchable thin layer was prepared by immersing gold surfaces in a solution of photoresponsive thiol (Azo-OEG-SH) and diluent thiol (OEG-SH) with different ratios at room temperature for 24 h using a mixed self-assembly strategy. Before forming the mixed SAM, the solution containing Azo-OEG-SH was irradiated with visible light for 24 h to ensure the Azo moieties were in the trans-form. Compared with the homogeneous SAM of Azo-OEG-SH, the mixed SAM composed of loosely packed Azo end groups provided enough free space for the reversible photoisomerization of the Azo moieties, which is necessary for the photocontrolled binding and release of CD-M.20 The successful formation of a series of mixed SAMs with different Azo-OEGSH/OEG-SH ratios was confirmed by the results of ellipsometry and water contact angle measurements (Figure S3, SI). On the basis of the preliminary results (Figure S5, SI), it is found that among all the surfaces prepared using mixture of Azo-OEG-SH/OEG-SH with different ratios (from 1/4 to 1/ 20), the surface prepared with a ratio of 1/10 mixture exhibited the best binding and release capability of E. coli and thus it was chosen as the model surface (referred to as Au−Mix) for further experiments. As controls, gold surfaces deposited with homogeneous SAMs of either OEG-SH or Azo-OEG-SH were prepared in a similar fashion, and the resultant surfaces are referred to Au−OEG or Au−Azo, respectively. The surface properties of these three samples were characterized by a series of techniques. As shown in Figures 2 and S4, the Au−Mix surface showed an intermediate layer thickness, water contact

3. RESULTS AND DISSCUSSION 3.1. Synthesis of Azo-OEG-SH. To construct a dynamic platform for the capture and release of bacteria in response to light irradiation, an Azo derivative was designed for the formation of a photoswitchable SAM. This compound is composed of four functional parts including a thiol group to anchor the compound to the gold surface, an undecane chain to promote the order and packing density of the SAM, an OEG chain to suppress undesired nonspecific interactions, and finally the photoswitchable Azo unit (denoted Azo-OEG-SH, where the chemical structure is shown in Figure 1). Synthesis of this compound involved several steps, as illustrated in Scheme 2, and the composition of the product was determined by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) (Figure 1 and Figure S1, SI). Characteristic peaks at 1.26, 1.30, 3.51−4.55, and 7.46−8.24 ppm were attributed to the protons of the undecane chain, thiol group, OEG chain, and azobenzene unit, respectively, and can be clearly observed in 3507


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ACS Applied Materials & Interfaces Scheme 2. Synthetic Route to Azo-OEG-SH

Figure 2. Water contact angle and thickness of different SAMs samples. Data are presented as the mean ± SD (n = 3).

angle, and nitrogen composition (based on X-ray photoelectron spectroscopy (XPS) analysis) between those of the Au−OEG and Au−Azo surfaces, suggesting a successful mixed selfassembly process. To confirm that Azo groups in the Au−Mix surface could be recognized by β-CD derivatives reversibly in response to irradiation with light of different wavelengths, we first used βCD decorated with rhodamine B isothiocyanate (CD-RBITC, red color) as a model molecule and evaluated the binding/ release of CD-RBITC on the surface using fluorescence microscopy equipped with an appropriate filter set (ex 460− 550 nm, em 590 nm). As shown in Figure 3, after immersion in CD-RBITC solution the fluorescence intensity of the Au−Mix surface was approximately 1015 ± 77 au, suggesting successful inclusion. After irradiation with 365 nm UV light for 30 min, the fluorescence intensity decreased dramatically to 389 ± 25 au. We also conducted the same experiments on the control surfaces (Au−OEG and Au−Azo) but no remarkable changes were observed (Figure S6, SI), suggesting that the change in fluorescence intensity resulted from the dissociation of CDRBITC from the surface due to the transition of trans-Azo to cis-Azo, which is sterically hindered from fitting into the cavity of β-CD.12 Furthermore, we treated the same Au−Mix surface with the irradiation of 450 nm visible light for 30 min and then immersed it into a fresh CD-RBITC solution. The surface exhibited strong red fluorescence with a similar intensity (1005 ± 53 au) as the original state, suggesting that cis-Azo fully transformed to trans-Azo to reform the inclusion complex with CD-RBITC. Three cycles were repeated, and the reproducibility was found to be good, providing the potential to use the Au−Mix surface for the photoswitchable inclusion/dissociation of other functional β-CD derivatives to modulate surface bioactivity.

Figure 3. Light-induced reversible incorporation and dissociation of CD-RBITC on the Au−Mix surface for three cycles. To switch the incorporation state (green part, trans- form of Azo) to the dissociation state (red part, cis- form of Azo), the Au−Mix/CD-RBITC surface was irradiated with 365 nm UV light for 30 min. To switch the dissociation state back to incorporation state, the surface was irradiated with 450 nm visible light for 30 min and then immersed into a fresh CD-RBITC solution. The fluorescence intensity of surface on each state was determined using fluorescence microscopy equipped with an appropriate filter set (ex 460−550 nm, em 590 nm). Data are presented as the mean ± SD (n = 3).

3.3. Capture and Release of Protein. After confirming the photoswitchability of the Au−Mix surface, we further exploited the potential to use this surface to modulate biointerfacial interactions. As carbohydrate−protein binding is a typical supramolecular recognition process with specific affinity, which plays an important role in diverse biological events,35 we first investigated the capability of this surface to bind and release a typical mannose-affinity lectin, ConA (MW: 104−112 kDa). In addition, to test the binding specificity we chose human serum albumin (HSA, MW: 66.5 kDa) with a similar molecular weight to ConA as a model “nonspecific” protein. Au−Mix surfaces with and without the inclusion of CD-M were incubated in 0.1 mg/mL 125I-labeled ConA or 1 mg/mL 125 I-labeled HSA at room temperature for 3 h, and the amounts of adsorbed proteins were then quantitatively measured. As shown in Figure 4a, the original Au−Mix surface exhibited a relatively low level of protein adsorption regardless of above tested proteins; this protein resistance property mainly resulted 3508


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Figure 4. (a) Adsorption of HSA and ConA (3 h exposure) on Au−Mix surfaces with and without the inclusion of CD-M. (b) Amount of ConA on the Au−Mix surface before and after incubation in PBS for 30 min either in the dark or under the irradiation of 365 nm UV light. Data are presented as the mean ± SD (n = 3).

Figure 5. (a) Representative fluorescence images of bacteria attached to different sample surfaces; the quantified number of attached bacteria is summarized in (b). Data are presented as the mean ± SD (n = 3). (c) Representative SEM images of bacteria attached to Au and Au−Mix/CD-M surfaces.

from the hydrophilic OEG components.43 However, after inclusion of CD-M, the surfaces showed a remarkable increase in ConA adsorption (from 73.2 ± 6.0 to 251.6 ± 6.5 ng/cm2) but a slight decrease in HSA adsorption (from 79.0 ± 6.1 to 69.8 ± 7.1 ng/cm2). A similar trend was also found in the results of quartz crystal microbalance (QCM) measurements (Figure S7, SI), suggesting that ConA binding occurs specifically to the mannose ligand. We then tested whether the Au−Mix/CD-M surface exhibited switchable protein adsorption/desorption in response to the photoisomerization of Azo groups. After adsorption of ConA, the surfaces were incubated in fresh buffer solution either in the dark or under the irradiation of 365 nm UV light for 30 min, and the amount of remaining ConA was reevaluated. Less than 15% of the ConA released from the surface kept in dark, suggesting that

the physical desorption of ConA is limited. In contrast, more than 90% of the adsorbed ConA were released from the Au− Mix/CD-M surface exposed to UV light irradiation due to the photoisomerization of Azo groups from the trans- to cis- form, leading to the exclusion of CD-M (Figure 4b). Taken together, the results above demonstrated that the Au−Mix surface after inclusion of the CD-M ligand could specifically capture ConA and release the captured ConA on demand by UV irradiation. 3.4. Specific Capture of Bacteria. Previous studies have shown that bacteria carry carbohydrate-binding proteins, which are responsible for microbial adhesion to cells and organs.36 For example, type 1-fimbriated E. coli have organelles on their surface called fimbriae that are comprised of specialized lectintype proteins that recognize carbohydrate ligands, called FimH.53 Surfaces or particles that are capable of promoting 3509


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ACS Applied Materials & Interfaces the enrichment of bacteria have been developed based on the carbohydrate-FimH interaction.44−46 Therefore, we further tested whether Au−Mix/CD-M could selectively capture bacteria and release the captured bacteria on demand by UV irradiation. Au−Mix surfaces together with the other three control surfaces with and without the preinclusion of CD-M were incubated in a suspension of a type 1-fimbriated E. coli (1 × 108 cells/mL) at 37 °C for 3 h. After washing to remove loosely bound bacteria, the surfaces were stained using SYTO 9 to evaluate the amount of captured bacteria (Figure 5a). Without the inclusion of the CD-M ligand, few bacteria were found to be sparsely located on the relatively hydrophobic Au and Au− Azo surfaces, while the Au−OEG and Au−Mix surfaces exhibited bacterial resistance due to the nonfouling characteristics of the OEG component.47 The introduction of CD-M led to a dramatic increase (∼16 times higher) in bacterial attachment on the Au−Mix surface, while for the other control surfaces the increase was not obvious (Figure 5b). The attached bacteria exhibited a stretched morphology with spread fimbriae and formed clusters, as observed by scanning electron microscopy (Figure 5c). The bacterial capture showed a trend similar to that of the ConA adsorption, suggesting that bacterial adhesion was specific to the mannose moieties on CDM via the well-established mannose-FimH interaction.31,34 A similar experiment was performed using another bacterium without the FimH protein. Almost no attached bacterial cells were detected on the Au−Mix/CD-M surface (Figure S8, SI), further confirming the high specificity of bacterial capture. The sensitivity of this platform was also investigated by incubating the surfaces in serially diluted suspensions of E. coli (from 1 × 108 to 1 × 103 cells/mL). The captured bacteria were stained using SYTO 9 and observed by fluorescence microscopy. A concentration-dependent decrease in amount of captured bacteria was found (Figure S9, SI). The minimum concentration that could be detected was as low as 1 × 105 cells/mL, which is lower than or comparable with other previously reported methods.31,48 Taken together, these results suggest that Au−Mix/CD-M surfaces can be applied as platforms to capture E. coli cells with high specificity and sensitivity, showing great potential for bacterial detection or diagnosis. 3.5. Release of Bacteria and Regeneration. For practical applications, the ability to release captured bacteria is critical for the subsequent identification of pathogenic bacteria and sensor regeneration.4 Various strategies have been developed to modulate bacteria−surface interactions to obtain the ondemand release of attached bacteria from surfaces by changing environmental stimuli such as temperature,49,50 pH,51 solvent,33,52 light,19 and mechanical strain.53,54 Among them, light is of particular advantage as it is a “clean” stimulus with high speed and spatiotemporal precision, and thus light has been widely applied to switch the adsorption/desorption of biomolecules and attachment/detachment of cells. Because the above experiment demonstrated that the Au−Mix/CD-M surface exhibits the light-triggered desorption of ConA, we thus expected that this surface might also be suitable as a dynamic platform for the on-demand release of bacteria. After bacterial attachment, the sample surfaces were incubated in fresh buffer solution under irradiation of 365 nm UV light for 30 min, followed by rinsing with water. The amount of bacteria initially captured and bacteria remaining on the surface after treatment was recorded using fluorescence microscopy (Figure 6 and Figure S10, SI). The Au−OEG/CD-

Figure 6. Representative fluorescence images of bacteria on different sample surfaces before and after UV irradiation. The corresponding bacterial release ratio of Au−OEG/CD-M, Au−Mix/CD-M, Au−Azo/ CD-M surface was 23.7 ± 9.4%, 81.4 ± 3.7%, and 49.7 ± 7.5%, respectively. Data are presented as the mean ± SD (n = 3).

M and Au−Azo/CD-M surfaces showed limited capability to release bacteria (the release ratio is lower than 50%), while the Au−Mix/CD-M surface released ∼81% attached bacteria after UV light treatment. The viability of released bacteria was investigated using live/dead staining assay. The results indicated that more than 93% bacterial cells were still live (Figure S11, SI), suggesting that the UV irradiation process did not affect the viability of bacteria.55 The release ratio of E. coli is slightly less than that of ConA (90%), which may be due to the obvious size difference between the bacteria and the protein. Taken into account that one E. coli bacterial cell has approximately 200 FimH receptors on its membrane surface and the fact that the photoisomerization of Azo groups has only 80% efficiency,23 it is thus supposed that ∼20% Azo groups remaining in the trans-conformation after UV irradiation maintained the inclusion complex with CD-M and thus preserved the binding of bacteria on the surface. To improve the release efficiency, a complementary guest molecule, adamantine (Ada), that has a higher affinity to β-CD compared with Azo was further introduced to synergistically dissociate the host−guest interaction between CD-M and Azo.22,56 It was found that after incubation in a solution of 20 μM 1adamantanamine hydrochloride and UV irradiation, more than 95% of the captured bacteria were removed from the Au−Mix/ CD-M surface (Figure 7). We also examined the regeneration ability of the surfaces to determine the long-term performance. After bacterial release, the Au−Mix surface was exposed to 450 nm visible light for 60 min followed by inclusion of fresh CDM. This regenerated Au−Mix/CD-M surface retained good bacteria capture ability, and the captured bacteria could also be released from the surface upon irradiation with 365 nm UV 3510


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bacteria clearly appeared on the half of the surface that was not exposed to UV irradiation, and almost no bacteria remained on the other half that was exposed to UV irradiation. This preliminary result suggested that the localized release of bacteria could be achieved. This light-modulated dynamic platform holds promise for the development of highthroughput bacteria sensing assays and downstream cellular analysis with the help of modern micropatterning technology. Importantly, the ability to mildly recover bacteria for analysis after these applications would eliminate potential interferences resulting from traditional bacterial release processes.

Figure 7. Amount of bacteria before and after release by various stimulations; the corresponding release ratio is shown in the inset. Data are presented as the mean ± SD (n = 3).

4. CONCLUSION In summary, we developed a dynamic supramolecular platform with the capability to specifically capture and release bacteria. This platform exploits the combination of the orthogonal photoresponsive host−guest interaction between Azo and βCD and the carbohydrate−protein interaction between mannose and the FimH protein on the surface of bacteria. This platform could selectively capture target bacteria and release the captured bacteria on demand by UV irradiation. The capture and release process could be repeated for multiple cycles, suggesting good reproducibility. Moreover, this supramolecular system is not limited to CD-M but can be applied to other β-CD derivatives with specific biofunctions. Reversible supramolecular interactions triggered by external stimuli provide a general strategy for controlling surface activity and biorecognition, paving the way for a variety of practical applications in the biomedical and biotechnology fields such as biosensors and diagnostic devices.

light. There were no significant changes in performance over four capture-and-release cycles, indicating good reproducibility (Figure S12, SI). Taken together, these results suggested that Au−Mix/CD-M surfaces can be regarded as effective platforms for the photoresponsive capture and release of bacteria, which can potentially be used to develop reusable biosensor chips for the detection and analysis of specific bacteria. One of the remarkable advantages of light as a stimulus is its high spatial resolution, providing the possibility to modulate the release of cells from predetermined regions via localized UV irradiation, which would enable the photopatterning or photoguiding of cells on the surface, and the study of growth, migration, and intercellular interactions.57,58 To test whether our platform possessed the capability for the localized release of bacteria, after the capture of bacteria, the Au−Mix/CD-M surface was partially exposed to UV light using a mask. As shown in Figure 8, a distinct boundary was observed where the

Figure 8. Localized release of bacteria on the Au−Mix/CD-M surface. Region (I), Region (II), and Region (III) refer to the area without UV irradiation, the boundary area, and the area with UV irradiation, respectively. Local UV irradiation was achieved by using a mask to shield half part of the sample surface during irradiation process. 3511


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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/acsami.6b15446. Details of synthesis of Azo-OEG-SH, surface characterization, statistical analysis, and figures showing characterization of compounds and surfaces, protein adsorption assays, bacterial assays, and other related characterization (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Qian Yu: 0000-0003-3612-6951 Hong Chen: 0000-0001-7799-4961 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. W.Z. and T.W. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21404076, 21334004, 21474071, 21674074, and 21504060), the Natural Science Foundation of Jiangsu Province (BK20140316), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Jiangsu Clinical Research Center for Cardiovascular Surgery.

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