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Fabrication of Reversible Poly(dimethylsiloxane) Surfaces via HostGuest Chemistry and Their Repeated Utilization in Cardiac Biomarker Analysis Yanrong Zhang,† Li Ren,† Qin Tu,† Xueqin Wang,† Rui Liu,† Li Li,† Jian-Chun Wang,† Wenming Liu,† Juan Xu,† and Jinyi Wang*,†,‡ † ‡
Colleges of Science and Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China
bS Supporting Information ABSTRACT: On the basis of the hostguest interactions between azobenzenes and cyclodextrins, a new strategy for the preparation of a dually functionalized poly(dimethylsiloxane) (PDMS) surface was investigated using surface-initiated atom-transfer radical polymerization (SI-ATRP) and click chemistry. The PDMS substrates were first oxidized in a H2SO4/H2O2 solution to transform the surface SiCH3 groups into SiOH groups. Then, the SI-ATRP initiator 3-(2bromoisobutyramido)propyl(trime-thoxy)silane was grafted onto the substrates through a silanization reaction. Sequentially, the poly(ethylene glycol) (PEG) units were introduced onto the PDMSBr surfaces via SI-ATRP reaction using oligo(ethylene glycol) methacrylate. Afterward, the bromide groups on the surface were converted to azido groups via nucleophilic substitution reaction with NaN3. Finally, the azido-grafted PDMS surfaces were subjected to a click reaction with alkynyl and PEG-modified β-cyclodextrins, resulting in the grafting of cyclodextrins onto the PDMS surfaces. The composition and chemical state of the modified surfaces were characterized via X-ray photoelectron spectroscopy, and the stability and dynamic characteristics of the cyclodextrin-modified PDMS substrates were investigated via attenuated total reflection-Fourier transform infrared spectroscopy and temporal contact angle experiments. The surface morphology of the modified PDMS surfaces was characterized through imaging using a multimode atomic force microscope. A protein adsorption assay using Alexa Fluor594-labeled bovine serum albumin, Alexa Fluor594-labeled chicken egg albumin, and FITC-labeled lysozyme shows that the prepared PDMS surfaces possess good protein-repelling properties. On-surface studies on the interactions between azobenzenes and the cyclodextrin-modified surfaces reveal that the reversible binding of azobenzene to the cyclodextrin-modified PDMS surfaces and its subsequent release can be reversibly controlled using UV irradiation. Sandwich fluoroimmunoassay of the cardiac markers myoglobin and fatty acid-binding protein demonstrates that the cyclodextrin-modified PDMS surfaces can be repeatedly utilized in disease biomarker analysis.
s research in the field of surface-functionalization modification continues to progress, many materials are exploited in biomedicine and bioanalysis, such as metal, glass, silicon, and synthetic polymers.1 Among them, poly(dimethylsiloxane) (PDMS) has been gaining popularity due to its distinct advantages, such as nontoxicity, easy fabrication, practical scalability, optical transparency, and gas permeability. In particular, PDMS-based microfluidic devices have found increasing applications in biosynthesis, disease diagnostics, DNA sequencing, protein crystallization, and cell-based bioanalysis.2,3 However, because of the hydrophobic nature of its surface, biological components from blood and body fluids interact strongly with the PDMS surface when it is presented in biological environments.4,5 A significant amount of protein adsorption on the PDMS surface caused by such hydrophobic interactions is the most important problem to be addressed because it triggers many undesirable bioreactions and greatly decreases the experimental efficiency in many cases, such as cell sorting,
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biological and nonbiological material patterning on substrates, and electrophoretic separation of biomolecules.6,7 The immediate use of PDMS-based microfluidic devices without any surface processing is thus always prevented.8,9 To modify the properties of PDMS surfaces and confer hydrophilicity and biomolecule-repelling properties on PDMS surfaces, various surface modification methods have been explored.1013 Numerous materials have also been employed for these purposes, such as hydroxylpropyl methylcellulose,14 poly(vinyl pyrrolidone),15 dextran,16 hyaluronic acid,17 polyacrylamide,18 poly(2hydroxyethyl methacrylate),19 poly(vinyl alcohol),20 poly(acrylic acid),21 poly(2-methacryloyloxyethyl phosphorylcholine),22 and poly(ethylene glycol).23 All these efforts have greatly improved Received: September 22, 2011 Accepted: November 1, 2011 Published: November 01, 2011 9651
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Analytical Chemistry the protein-repelling properties of PDMS substrates and expanded the applications of PDMS-based microfluidic devices. However, existing technologies have mainly relied on irreversible control; that is, once the original structure on the PDMS surface is altered for biological application, it cannot be regenerated for further use. This limitation has greatly hindered the development of molecularly defined PDMS surfaces that can reversibly control on-chip biological applications.2428 Previous studies2932 have demonstrated that the two isomers of azobenzene, the trans and cis forms, can be reversibly interconverted upon photoirradiation. In addition, trans-azobenzene can be well-recognized by cyclodextrins via hydrophobic and van der Waals interactions.29 However, the ability of trans-azobenzene to bind into the hydrophobic cavity of cyclodextrin (CD) is greater than that of cis-azobenzene. When trans-azobenzene is transformed into cis-azobenzene, cyclodextrins can no longer include the bulky cis form because of the mismatch between the host and the guest.31,32 Therefore, if azobenzenes are incorporated into certain biomolecules as switchable recognition components, desired molecule-linked photoactive switches can be built onto cyclodextrin-conjugated substrates to construct photocontrolled reversible systems. To date, this photoswitchable reversible interaction between azobenzene and cyclodextrin has been utilized to build molecular shuttles, motors, machines, surfactants, ion channels, hydrogels, and so on.3136 The photocontrolled bioelectrocatalysis of glucose by glucose oxidase in an “onoff” state was realized using the reversible immobilization and detachment of a ferrocene-labeled redoxpolymer as a mediator through this hostguest chemistry;33 and the controlled release of cargo molecules from mechanized silica nanoparticles was realized by taking advantage of the difference in the binding affinity between β-CD and trans-azobenzene (high) and β-CD and cis-azobenzene (low).34 These studies have greatly exhibited the potential of the hostguest chemistry in the construction of a reversible bioanalytical system. However, current studies on the applications of this machine-like switching of molecular recognition have mainly focused on glass, silicon, and gold substrates. Few studies have explored the field of PDMS-based microfluidic device fabrication. Robust control of the hostguest complexation via light is advantageous for the application of the switchable building blocks to microfluidics-based bioanalysis.25,27 In addition, current investigations of this system mainly or only focus on the construction of reversible systems with little consideration for protein-repelling properties. With the foregoing analysis in mind, as well as our previous studies on PDMS surface modification,4,10 in this work, we describe a new strategy for the preparation of a dually functionalized PDMS surface by conjugating β-cyclodextrins onto PDMS surfaces through surface-initiated atom-transfer radical polymerization (SIATRP) and click chemistry. The prepared PDMS surface allows azobenzene to reversibly bind to cyclodextrin-conjugated PDMS surfaces and possesses good hydrophilicity and protein-repelling properties due to the introduction of poly(ethylene glycol) (PEG) in the preparation. Using this approach, numerous photocontrolled reversible PDMS substrates were prepared and their surface hydrophilicity and protein adsorption were investigated. The reversible and repeated use of the prepared PDMS surfaces in small-molecule interactions and disease biomarker analysis were also analyzed.
’ EXPERIMENTAL SECTION Materials and Reagents. The PDMS substrates were fabricated using the RTV 615 PDMS prepolymer and a curing agent
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(GE Silicones, Minato-ku, Tokyo, Japan).8 Oligo(ethylene glycol) methacrylate (OEGMA6) was obtained from SigmaAldrich (MO). The inhibitor 4-methoxyphenol was removed by passing the monomer through a column of activated, basic aluminum oxide.37 2,20 -Bipyridine and copper dibromide (CuBr2) were purchased from Alfa Aesar (Lancaster, England) and used as received. Cuprous bromide (CuBr), sodium ascorbate, sodium azide (NaN3), and β-cyclodextrin (β-CD) were purchased from Aladdin (Shanghai, China). Before utilization, β-CD was recrystallized twice from deionized (DI) water and dried overnight over P2O5 under vacuum. Monoclonal primary antibodies [antimyoglobin 7C3 and antihuman fatty acid-binding protein (anti-HFABP) 10E1], secondary antibodies (antimyoglobin 4E2 and anti-H-FABP 9F3), human heart myoglobin, and H-FABP were purchased from Hytest, Ltd. (Turku, Finland). Human serum was obtained from BioMag Ltd. (Beijing, China) and was used to prepare different concentration myoglobin and H-FABP samples.38,39 Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO). Chicken egg albumin and lysozyme were purchased from Dingguo Biotechnology, Ltd. (Beijing, China). The EZ-Label FITC protein labeling kit was obtained from Pierce Biotechnology (Rockford, IL) and was used to label antimyoglobin 4E2 and lysozyme. The Alexa Fluor594 labeling kit was from Molecular Probes (Eugene, OR) and was used to label anti-H-FABP 9F3, BSA, and chicken egg albumin. The SI-ATRP initiator 3-(2-bromoisobutyramido) propyl(trimethoxy) silane (BrTMOS) and alkynyl and PEGmodified β-CD were synthesized in our laboratory. For the detailed information on their synthesis and characterization, see the Supporting Information. All other materials were obtained from local commercial suppliers and were of analytical reagent grade, unless otherwise stated. Conjugation of β-CDs onto the PDMS Surface. The conjugation of β-CDs onto the PDMS surface was performed using a five-step reaction as follows. PDMS Surface Activation and Bromide Group Introduction. The PDMS slides (0.5 cm 0.5 cm) were first made hydrophilic via surface oxidation with a piranha solution [H2SO4H2O2 = 3:1 (v/v)] for 30 s at 40 °C.10 After rinsing with DI water and drying with N2, the hydrophilic silanol-covered PDMS (PDMSOH) slides were immediately transferred to a freshly prepared ethanolic solution with 5% (w/v) BrTMOS for overnight immersion. Afterward, the PDMS slides with bromidegrafted surfaces (PDMS-Br) were thoroughly rinsed with ethanol and water, dried with N2, and heated to 60 °C for at least 2 h.40 SI-ATRP. The introduction of PEG units to the PDMSBr surfaces was performed via the SI-ATRP reaction.37 Briefly, a mixture of the ATRP catalyst [CuBr (180 mg, 1.26 mmol), CuBr2 (14 mg, 0.063 mmol), bipyridine (490 mg, 3.15 mmol)] and OEGMA6 (17.5 mL, 63 mmol) in a mixture solution [DI water (14 mL) and methanol (3.5 mL)] were degassed via three freezepumpthaw cycles. After gradually warming the mixture to room temperature, the solution was transferred by syringe to the reaction vessel containing the PDMSBr slides, and polymerization was allowed to proceed for 6 h at 60 °C under N2. After rinsing with methanol and water and drying by N2, the PDMS slides with PDMSPEGBr surfaces were used in the subsequent study. Nucleophilic Substitution of Terminal Bromide Groups. To perform the subsequent click chemistry, the terminal bromide groups of the OEGMA6 polymer brush on surface PDMS PEGBr were converted to azido groups via nucleophilic substitution in the presence of NaN3.41 Briefly, the PDMSPEGBr slides 9652
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Analytical Chemistry were first immersed in a N,N-dimethylformamide (DMF) solution (15 mL) containing NaN 3 (97.5 mg, 1.5 mmol) for 24 h at 40 °C. Afterward, the prepared PDMSPEGN3 slides were washed with DMF and DI water and dried in a stream of N2 . Click Chemistry on Surface PDMSPEGN3. A 15 mL aqueous solution containing alkynyl and PEG-modified β-CD (114.4 mg, 0.08 mmol), sodium L-ascorbate (79.2 mg, 0.4 mmol), and CuSO4 3 5H2O (20.0 mg, 0.08 mmol) was transferred to a reaction vessel containing the PDMSPEGN3 slides, and the reaction was allowed to proceed for 24 h. A 10 mL aqueous solution of monoalkynyl-modified tetraethylene glycol (Compound S1 in the Supporting Information; 18.6 mg, 0.08 mmol), sodium L-ascorbate (79.2 mg, 0.4 mmol), and CuSO4 3 5H2O (20.0 mg, 0.08 mmol) was subsequently added to the vessel. The reaction was allowed to proceed for another 6 h to deactivate the residual azide groups.37 The resulting PDMSPEGCD slides were removed from the vessel to stop the reaction, extensively rinsed with a solution of ethylenediaminetetraacetic acid disodium salt (0.05 mol/L) and DI water, and then dried by N2. X-ray Photoelectron Spectroscopy (XPS) Analysis. The XPS analyses were performed on an Axis Ultra X-ray photoelectron spectrometer with a Mg/Al X-ray source operating at 150 W (15 kV, 13 mA). The vacuum in the main chamber was kept above 3 106 Pa during XPS data acquisition. Specimens were analyzed at an electron takeoff angle of 70° with respect to the surface plane. General survey scans (01100 eV binding energy range, 160 eV pass energy) and high-resolution spectra (80 eV pass energy) in the C1s, Br3d, and N1s regions were recorded for all modified PDMS substrates (1 cm 1 cm). Binding energies were referenced to the C1s binding energy at 284.8 eV. Contact Angle Measurements. Contact angle measurements on PDMS specimens were performed via the sessile drops technique using a Dropmeter 100 equipment (Maist Vision, Ningbo, China).10 DI water (18.4 MΩ) was used. Each data point was based on 10 contact angle measurements at 5 different positions on the PDMS specimen. Atomic Force Microscope (AFM). The surface morphology and the roughness of the modified PDMS surfaces were investigated through imaging using a multimode atomic force microscope (AFM) (Veeco Instruments) in tapping mode with a standard silicon tip. The cantilever spring constant for imaging was 40 N/m. Images were recorded using height and phase-shift channels with 256 256 measurement points (pixels). Measurements were made three times on different zones of each sample in a scanning area of 3.0 μm 3.0 μm. Image processing and roughness parameter [arithmetic average (Ra), and root-meansquare (Rms)] calculation were performed using NanoScope Analysis software (Veeco Instruments). Stability Tests. To investigate the stability of the β-CDgrafted PDMS surfaces, the modified PDMS substrates were exposed to ambient conditions for 30 days.4,10 The dynamic surface characteristics of the CD-grafted PDMS surfaces were characterized using temporal contact angle measurements following the procedures described above as well as via attenuated total reflection-Fourier transform-infrared spectroscopy (ATRFT-IR). General Procedure for the Protein-Repelling Study. The freshly prepared PDMSPEGCD slides were stored under ambient conditions for 18 h; native PDMS slides were used as controls. After rinsing with phosphate-buffered saline (PBS, pH 7.4),
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the PDMS slides (0.5 cm 0.5 cm) were placed in 5 mL glass bottles, to which 2 mL of fluorophore-labeled protein (Alexa Fluor594-labeled BSA, Alexa Fluor594-labeled chicken egg albumin, or FITC-labeled lysozyme) solution with a final concentration of 1.0 mg/mL was added. Protein absorption was allowed to proceed at 37 °C for 1 h with gentle shaking.10 After washing with PBS five times and drying with a N2 stream, the fluorescence images of the PDMS surfaces were recorded and the amount of nonspecific absorbed protein was analyzed.40 Photocontrolled Reversible Interactions between Azobenzenes and PDMSPEGCD Surfaces. The prepared PDMSPEGCD slides (0.5 cm 0.5 cm) were first immersed in an aqueous solution (1.0 mmol/L) of rhodamine B-labeled trans-azobenzene or FITC-labeled trans-azobenzene for 12 h in the dark (for the detailed information on the synthesis, characterization, and conversion of the azobenzene moieties to the trans isomer, see the Supporting Information). After washing five times with DI water with gentle shaking, the fluorescence images of the postinteraction PDMSPEGCD surfaces were captured and their fluorescence intensity was analyzed.40 Afterward, the postinteraction PDMSPEGCD surfaces were exposed to UV light (365 nm) for 2 min,36 which converted the trans-azo compounds to the cis-isomer and released them from the PDMSPEGCD surfaces. After rinsing five times with DI water, the surface fluorescence images of the UV-exposed PDMSPEGCD surfaces were also captured and analyzed following the procedures described above. Each test was repeated at least three times on different PDMSPEGCD slides. Repeated experiments on the same PDMSPEGCD surface were performed at least four times following the previously described procedures. PDMSPEGCD Surface-Based Sandwich Fluoroimmunoassay for the Determination of the Cardiac Markers H-FABP and Myoglobin. After rinsing with PBS, the PDMSPEGCD slides (0.5 cm 0.5 cm) were immersed in a trans-azobenzenegrafted primary antibody solution (trans-azobenzene-grafted antimyoglobin 7C3 or trans-azobenzene-grafted anti-H-FABP 10E1, 40 μg/mL) for 12 h (for the detailed information on the synthesis of azobenzene-grafted antimyoglobin 7C3 and azobenzenegrafted anti-H-FABP 10E1 as well as the conversion of the azobenzene moieties to the trans isomer, see the Supporting Information). Upon addition of a serial dilution of the antigen (cardiac marker) myoglobin or H-FABP (15 μL in blood serum) and incubation at 37 °C for 40 min, the antigen was subsequently assayed using fluorophore-labeled secondary antibody FITClabeled myoglobin 4E2 (50 μg/mL, 15 μL, 30 min) or Alexa Fluor594-labeled anti-H-FABP 9F3 solution (40 μg/mL, 15 μL, 30 min).38,42 To ensure that the azobenzene moieties were of the trans form, the entire assay was conducted in the dark. Caution was used in handling all human biological material. To demonstrate the reversibility and reusability of the prepared PDMSPEGCD surfaces, the postimmunoassay PDMS PEGCD surfaces were exposed to 365 nm UV light for 6 min.43 After rinsing with PBS, the PDMSPEGCD slides were reused in another round of fluoroimmunoassay following the previously described procedures. Microscopy and Image Analysis. An inverted microscope (Olympus, CKX41) with a CCD camera (QIMAGING, Micropublisher 5.0 RTV) and a mercury lamp (Olympus, U-RFLT50) was used to acquire fluorescence images of the postinteraction PDMS surfaces. Software Image-Pro Plus 6.0 (Media Cyternetics, Silver Spring, MD) and SPSS 12.0 (SPSS Inc.) were employed to perform image analysis and data statistical analysis, respectively. 9653
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Scheme 1. Schematic Presentation of the PDMS Modification Process Using Alkynyl and PEG-Modified β-CDa
(i) H2SO4/H2O2, 40 °C, 30 s; (ii) BrTMOS; (iii) CuBr, CuBr2, bipyridine, and OEGMA6; (iv) NaN3, 40 °C, 24 h; (v) sodium L-ascorbate and CuSO4 3 5H2O. a
Data are presented as means ( SD for the measured fluorescence intensity. The t-test for unpaired values was used to evaluate the significance of differences between the fluorescence intensities measured in the different tests. P < 0.01 was considered statistically significant.
’ RESULTS AND DISCUSSION Surface Modifications and Characterization. Scheme 1 depicts the schematic of the step-by-step surface modifications composed of PDMS surface activation (i), bromide group introduction (ii), SI-ATRP (iii), nucleophilic substitution (iv), and click chemistry (v). Generally, the construction of the dually functionalized PDMS surface started with the solution-phase oxidation reaction of the PDMS surfaces, which was conducted by treating the PDMS surfaces with H2SO4/H2O2 solution.10 Then, the SI-ATRP initiator BrTMOS was grafted onto the PDMSOH surfaces through a silanization reaction. Sequentially, the PEG units were introduced onto the PDMSBr surfaces via SI-ATRP reaction using OEGMA6. The use of OEGMA6 is critical for the generation of the protein-repelling properties of the CD-conjugated PDMS surface. Previous studies have demonstrated that the introduction of PEG-terminated selfassembled monolayer allows specific interactions between biological samples and surfaces and prevents nonspecific adhesion.23
Afterward, the bromide groups on the PDMSPEGBr surface were transformed into azido groups via nucleophilic substitution reaction with NaN3.41 Finally, the azido-grafted PDMS surfaces were subjected to a click reaction with the alkynyl and PEGmodified β-CD (i.e., Compound S5 in Scheme 1) to produce β-CD-conjugated PDMS surfaces. The residual azide groups on the β-CD-conjugated PDMS surfaces were deactivated using monoalkynyl-modified tetraethylene glycol via a further 6 h click reaction.37 The success of each PDMS modification step was confirmed through XPS, ATR-FT-IR, and AFM, which demonstrates that the β-CD was successfully conjugated onto the PDMS surface through SI-ATRP and click reaction. For the detailed discussion on the XPS, ATR-FT-IR, and AFM analysis, see the Supporting Information. Stability Tests. To investigate the stability of the β-CD-modified PDMS surfaces, numerous modified PDMS substrates were exposed to ambient conditions for 30 d.4,10 The dynamic surface characteristics of the modified substrates were monitored through temporal contact angle experiments. The results (Figure 1A) reveal that the introduction of PEG-modified β-CDs onto the PDMS surfaces greatly improved the hydrophilicity of the native PDMS substrates (the stabilized static contact angles were 114.7 ( 2.1 and 30.3 ( 0.9° for the native PDMS and PDMSPEGCD surfaces, respectively). The stability of the β-CD-modified PDMS surfaces tended to remain constant after 18 h. These results suggest that robust cross-linked β-CDPEGsilane layers were created on the 9654
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Figure 1. Assay of the stability and protein-repelling characteristics of surface PDMSPEGCD. (A) Temporal evaluation of the contact angles of the PDMSPEGCD slides with DI water; (B) one set of fluorescent images of the three parallel native PDMS slides and the three parallel PDMSPEGCD slides obtained by treating the slides with fluorophore-labeled protein solutions; (C) amount of nonspecifically absorbed protein detected (as revealed by the fluorescence intensity) on the native PDMS and PDMSPEGCD surfaces. Error bars represent (SD (n = 3).
PDMS surfaces, resulting in long-lasting hydrophilicity.4 Additionally, ATR-FT-IR analysis (see the Supporting Information) of fresh PDMSPEGCD substrates and those stored for 30 days also provides supporting evidence that the modified surfaces possessed long-lasting stability, because the composition and chemical state of the CD-modified surface was not affected even after long storage. Protein Adsorption Analysis. The study of the proteinrepelling characteristics of the modified PDMS surfaces was conducted using three fluorophore-labeled proteins: Alexa Fluor594-labeled BSA, Alexa Fluor594-labeled chicken egg albumin, and FITC-labeled lysozyme. A number of freshly prepared PDMSPEGCD substrates were first stored under ambient conditions for 18 h to stabilize their surfaces.4 Afterward, they were treated with the fluorophore-labeled proteins in a physiological media. Figure 1B shows one set of the three parallel protein-repelling tests on PDMSPEGCD and native PDMS surfaces. A direct comparison of the fluorescent images was made via visual inspection of the change in the fluorescence color of the modified and native PDMS surfaces. The introduction of the PEG-modified CD greatly improved the protein-repelling properties of native PDMS. The quantitative analysis of these images (as revealed by the fluorescence intensity) indicates that the amounts of protein adsorbed onto the native PDMS were significantly higher than those on the PDMSPEGCD surfaces, regardless of the types of proteins (Figure 1C). This characteristic is attributed to the presence of the PEG chains in the structure of the PDMSPEGCD surfaces.23
Reversible Interactions of Surface PDMSPEGCD and Azobenzenes. To evaluate the photocontrolled reversible as-
sembly and disassembly of surface PDMSPECD and azo compounds, two fluorophore-labeled azobenzenes (rhodamine B-labeled azobenzene and FITC-labeled azobenzene) were synthesized. The reason for the introduction of fluorophores in the structure of azobenzenes is that fluorescence imaging is a powerful and sensitive microscopic technique that allows the study of molecules at monolayers and gives the possibility of imaging sites on a restricted area for the detection of on-chip reaction.44 Scheme 2 shows the schematic of the interaction between azo compounds and surface PDMSPEGCD. First, the trans-isomer of the fluorophore-labeled azobenzene was reacted with surface PDMSPEGCD for 12 h in the dark.43 After rinsing and analyzing their fluorescent images, the postreacted PDMSPEGCD surfaces were exposed to UV light (365 nm) for 2 min.36 During this treatment, the trans-isomer azo compounds were converted to the cis-isomer and released them from the PDMSPEGCD surfaces because of the mismatch between the host and the guest.31,32 The results (Supporting Information) indicate that after incubation with fluorescent-labeled azobenzenes, the fluorescence intensity of the surface PDMSPEGCD was significantly higher than those of surface PDMSPEGN3 and native PDMS. In addition, the fluorescence intensity of the surface PDMSPEGCD decreased sharply after UV irradiation. However, no significant difference was observed between surface PDMSPEGN3 and native PDMS. 9655
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Scheme 2. Illustration of the Photocontrolled Reversible Switching between Surface PDMSPEGCD and FITCAzobenzene (Left) and Rhodamine BAzobenzene (Right)a
In the dark, the fluorophore-labeled azobenzenes with the trans-azobenzene groups attached on the PDMSPEGCD surface; under UV irradiation (365 nm, 2 min), the trans-azobenzene groups of the fluorophore-labeled azobenzene converted to cis and detached from the surface.
a
Figure 2. UV light-controlled cyclic fluorescence intensity of fluorophore-labeled azobenzene attached (top fluorescence images) and detached (bottom fluorescence images) on the same PDMSPEGCD surface: (A) FITC-labeled azobenzene; (B) rhodamine B-labeled azobenzene. Normalization of the fluorescence intensity was performed as the follows: the fluorescence intensity of the nonreacted PDMSPEGCD surface was first subtracted from those of the fluorophore-labeled azobenzene treated PDMSPEGCD surfaces. Then they were divided by the maximum fluorescence intensity obtained in the whole analysis. Error bars represent (SD (n = 3).
These results indicate that the photocontrolled attachment and detachment of azobenzene onto surface PDMSPEG CD were successfully realized through the hostguest chemistry between CD and azobenzene, which was also confirmed by
repeated tests on the same modified surface (Figure 2A,B). The increasing fluorescence background after each cycle of the attachment and detachment may be due to the diffusion of small molecules (i.e., fluorophore-labeled azobenzenes) 9656
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Scheme 3. Schematic Presentation of the HostGuest Interaction-Based Sandwich Fluoroimmunoassay of the Cardiac Markers Myoglobin and H-FABP and the Regeneration of the PDMSPEGCD Surface under UV Irradiationa
a
Left side for myoglobin [(i) f (ii) f (iii) f (iv)]; right side for H-FABP [(a) f (b) f (c) f (d)].
into the PDMS bulk.10,45 The AFM investigation and contact angle measurement (Supporting Information) of the modified PDMS surfaces after each cycle exhibited that there were no statistically significant differences (P > 0.01) in the surface morphology and contact angles between two repeated experiments. Sandwich Fluoroimmunoassay. To demonstrate the practical applications of the photocontrolled reversible PDMS surfaces, we applied the β-CD-conjugated PDMS surfaces to the study of an early biomarker immunoassay (Scheme 3) associated with acute myocardial infarction (AMI). Among cardiac markers, myoglobin and FABP have been demonstrated as highly suitable for early rapid screening for AMI. These markers have high sensitivity and high negative predictive value. A combined analysis of these two markers can discern injury to the myocardium after AMI onset.46 Therefore, the myoglobin-specific monoclonal primary antibody antimyoglobin 7C3 and H-FABP-specific monoclonal primary antibody anti-H-FABP 10E1 were, respectively, coupled with azo compounds (for the detailed information on their synthesis, see the Supporting Information). To prevent azo groups from wrapping in the biomacromolecules as well as to decrease nonspecific biofouling during the immunoassay,47 a long PEG spacer was introduced in the azo compound structure before the primary cardiac antibodies were grafted onto them. Afterward, the primary antibody-grafted azo derivatives with the trans-isomer were reacted with the PDMSPEGCD surfaces. Then, serials of blood serum samples with different concentrations of cardiac markers myoglobin or H-FABP were analyzed on the modified PDMS surfaces through the reaction between the
antibodies to the biomarkers and the antigen (biomarker) itself. The relationship between the amount of biomarker detected (as revealed by the fluorescence intensity) and the actual concentration of the biomarker is shown in Figure 3A,B, which is similar to those obtained using a conventional enzyme-linked immunosorbent assay (Supporting Information). The minimum detectable concentrations for myoglobin and H-FABP were 15 and 1 ng/mL, respectively. Statistical analysis showed that the differences in fluorescence intensity between different biomarker concentrations were statistically significant (P < 0.01). However, there were no statistically significant differences (P > 0.01) between two repeated experiments at the same myoglobin or H-FABP concentration. To compare with the expected concentrations of the two biomarkers in a healthy patient (17 ( 6 ng/mL for myoglobin; 3.0 ( 1.3 ng/mL for H-FABP)48 and the variability of upper reference limits across the population after the onset of AMI (49105 ng/mL for myoglobin; 5.312 ng/mL for H-FABP),49 the detectable ranges reported in the current study include them. In addition, specificity assay (Supporting Information) proves that the interactions between the cardiac markers and their corresponding monoclonal antibodies immobilized on surface PDMSPEGCD were specific. These results indicate that the PDMSPEGCD surfaces can be utilized to measure changes in the two biomarkers after the onset of AMI. Furthermore, repeated tests on the same PDMSPEGCD surface demonstrate that the PDMSPEGCD surface-based immunoassay can be repeatedly utilized in disease biomarker analysis because there were no statistically significant differences (P > 0.01) between two repeated experiments (Figure 3C,D). 9657
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Figure 3. PDMSPEGCD surface-based fluoroimmunoassay of the cardiac markers myoglobin and H-FABP. (A and B) Relationship between the average fluorescence intensity and cardiac markers concentration obtained from fluorescence images analyzed using software Image-Pro Plus 6.0 (A, myoglobin; B, H-FABP). (C and D) The UV light-controlled cyclic fluorescence intensity of postimmunoassay and post- UV irradiation PDMSPEGCD surface (C, myoglobin; D, H-FABP). Normalization of the fluorescence intensity was performed as follows: the fluorescence intensity of nonreacted PDMSPEGCD surfaces was first subtracted from those of the postimmunoassay PDMSPEGCD surfaces. Then they were divided by the maximum fluorescence intensity obtained in the whole analysis. Error bars represent (SD (n = 3).
’ CONCLUSIONS The current study presented a new strategy for the preparation of photocontrolled reversible PDMS surfaces via a hostguest interaction between azobenzene and CD. The reversible binding of azobenzene to the β-CD-modified PDMS surfaces and subsequent release from the surface were controlled by UV irradiation. Meanwhile, the β-CD-modified PDMS surfaces were proven to possess good hydrophilicity and protein-repelling properties. Sandwich fluoroimmunoassay of the cardiac markers myoglobin and H-FABP demonstrates that the β-CD-modified PDMS surfaces can also be repeatedly utilized in disease biomarker analysis. The smart photocontrolled reversible PDMS surfaces are expected to provide an excellent platform for potentially wide-ranging applications in stimuli-responsive biomedical technologies, controlled bioseparation, and biosensing devices. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: + 86-29-870 825 20. Fax: + 86-29-870 825 20. E-mail:
[email protected].
’ ACKNOWLEDGMENT Y.Z., L.R., and Q.T. contributed equally to this work. This study was funded by the National Natural Science Foundation of China (Grants 20975082, 20775059, and 21175107), the Natural Science Foundation of Shaanxi (Grant 2011JQ2006), the Ministry of Education of the People’s Republic of China (Grant NCET-08-0464), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, the State Education Ministry, and the Northwest A&F University. ’ REFERENCES (1) Bange, A.; Halsall, H. B.; Heineman, W. R. Biosens. Bioelectron. 2005, 20, 2488–2503. (2) Whitesides, G. M. Nature 2006, 442, 368–373. (3) Wong, I.; Ho, C. M. Microfluid. Nanofluid. 2009, 7, 291–306. (4) Sui, G.; Wang, J.; Lee, C. C.; Lu, W.; Lee, S. P.; Leyton, J. V.; Wu, A. M.; Tseng, H. R. Anal. Chem. 2006, 78, 5543–5551. (5) Zhou, J.; Yan, H.; Ren, K.; Dai, W.; Wu, H. Anal. Chem. 2009, 81, 6627–6632. (6) Ocvirk, G.; Munroe, M.; Tang, T.; Oleschuk, R.; Westra, K.; Harrison, D. J. Electrophoresis 2000, 21, 107–115. (7) Xiao, D.; Zhang, H.; Wirth, M. J. Langmuir 2002, 18, 9971–9976. (8) Liu, W. M.; Li, L.; Wang, X. M.; Ren, L.; Wang, X. Q.; Wang, J. C.; Tu, Q.; Huang, X. W.; Wang, J. Lab Chip 2010, 10, 1717–1724. (9) Li, L.; Liu, W. M.; Wang, J. C.; Tu, Q.; Liu, R.; Wang, J. Electrophoresis 2010, 31, 3159–3166. 9658
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