Versatile and Nondestructive Photochemical Process for Biomolecule

Article pubs.acs.org/Langmuir

Versatile and Nondestructive Photochemical Process for Biomolecule Immobilization Pascal Viel,† Justine Walter,† Sophie Bellon,‡ and Thomas Berthelot*,† †

CEA, IRAMIS, SPCSI Chemistry of Surfaces and Interfaces Group, F-91191 Gif-sur-Yvette, France Horiba Jobin Yvon-Genoptics, Avenue de la Vauve - Passage Jobin Yvon, F-91120 Palaiseau, France

‡

ABSTRACT: Covalent immobilization of unmodified biological materials as proteins has been performed through a one-step and soft method. This process is based on a polyazidophenylene layer derived from the electroreduction of the parent salt 4-azidobenzenediazonium tetrafluoborate on gold substrates. The wavelength used (365 nm) for the photochemical grafting of a large variety of molecules as biomolecules is a key point to this nondestructive immobilization method. This simple process is also versatile and could be used for covalently binding a wide range of molecules such as polyethylene glycol moieties, for example. To validate this approach for biochip or microarray fabrication, a surface plasmon resonance imaging (SPRi) platform for immobilization of various antibody families was created by grafting G-protein through this process. This SPRi antibodies platform was tested with several consecutive cycles of antigen injections/regeneration steps without loss of activity.

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INTRODUCTION Biology as electronics is following a path of miniaturization and large-scale integration processes that will revolutionize biotechnology.1,2 These ongoing processes are compiled in devices known as biosensors and/or biochips, including microarrays and lab-on-a-chip microfluidic devices.2−4 In biosensors, the recognition species are either biomolecules as enzymes, receptors, antibodies, peptides, or nucleic acids (DNA, RNA, etc.).3,4 The key parameters that govern the overall performances of biosensors are the mass transport parameters of the analyte to the interface, the affinity binding constant between the receptor and the target analyte, and the total amount of available binding sites on the sensing area.5−7 These two last parameters can be modulated by both the surface chemistry and the method used for biomolecule immobilization.8−10 For this purpose, methods are classified into three categories: (i) noncovalent attachment, which includes sol−gel encapsulation, polymer entrapment, and physical adsorption (electrostatic and hydrophobic adhesion), (ii) covalent attachment, and (iii) noncovalent specific binding (avidin/biotin, NTA/HisTag, or protein A or G/IgG technologies).11,12 Concerning the first category, it is noteworthy that three-dimensional supports are generally plagued by problems relating to mass transport effects and high background signals, resulting in false kinetic rate constants. Concerning the second category, many chemical routes have been explored: the first one consists of preparing the surface to react with biological materials; the second one consist of randomly modifying the biological materials in order to react with the surface. Concerning the third one, this multiple-step strategy is generally based on a biomimetic approach (avidin/biotin13 and protein A or G/IgG methods) and uses specific recognition between a biological ligand and its © 2013 American Chemical Society

receptor. Contrary to previous approaches, the engineered NTA/His-Tag system is based on a metal chelation between a His-Tag-modified protein and a Ni2+-NTA system.14 This third strategy allows one to (i) control the biomolecule immobilization orientation, (ii) immobilize biomolecules in highly diluted concentration, and (iii) preserve the biomolecule activity. However, these approaches can also use a grafting step in order to introduce on the surface, for example, biotin, NTA groups, or protein A.15 The formation of irreversible covalent bonds between biomolecules and reactive groups on the support is one of the most widely investigated methods.11,12 However, this strategy presents some drawbacks: (i) the surface activation methods may be incompatible with biological medium, (ii) the coupling procedures may require harsh conditions or release unsuitable byproducts, and (iii) the covalent attachment can prevent the biological activity. The formation of surface functional groups (carboxyl, amino, etc.) through various chemistries such as silane,16,17 alkanethiol monolayers,18,19 conducting polymers,20−22 and electrochemical reduction of diazonium salt23,24 have been employed to improve surface biofunctionalization. The latter solution using diazonium salts becomes widespread. In this approach, either diazonium salt is used to create functional groups, which can be then activated to react with biomolecules.25−27 Either diazonium salt can be introduced on biomolecules and deposited directly.11,28,29 However, this method requires harsh conditions only compatible with antibodies. Furthermore, the biomolecule activity can be altered by the process. On the basis of the upReceived: July 5, 2012 Revised: January 13, 2013 Published: January 14, 2013 2075

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Figure 1. Cyclic voltammetry recording of the 4-azidobenzene diazonium tetrafluoroborate (10−2 M) solution (CH3CN, TEAP 5 × 10−2 M) electrochemical reduction. The peak centered at −0.3 V on the first scan confirms the diazonum salt reduction on the gold surface. The peak disappearance on the second and on the last scans highlights the surface modification by the polyazidophenylene layer. v = 20 mV·s−1. underlayer (2 nm) followed by gold evaporation (200 nm) on microscopic glass slide (0.5 × 20 mm) or high refractive index glass for SPRi experiments (12 × 25 mm) (purchased from HORIBA). 4-Azidobenzene Tetrafluoroborate Salt Synthesis. 4-azidobenzene diazonium tetrafluoroborate was obtained by reacting 4aminophenyl azide hydrochloride (100 mg, 5.86 × 10−4 mol) with sodium nitrite (45 mg, 6.52 × 10−4 mol) in 1 mL of fluoroboric acid at 0 °C. After steering for 1 h, the resulting solution was poured in diethyl ether. A white precipitate was filtered off, dried under reduced pressure, and stored in the dark. Electrochemical Grafting. Before electrochemical grafting, acetonitrile water titration was performed with a Karl Fischer procedure and was measured at less than 20 ppm. Tetraethyl ammonium perchlorate (TEAP) was dried before use in vacuum oven. Electrochemical grafting of 4-azidobenzene diazonium tetrafluoborate salt (10−2 M) was performed in a mixture of acetonitrile and TEAP (5 × 10−2 M) as supporting salt. Cyclic voltammetry was performed using an EG&G potentiostat, model 273A. Experiments were carried out in a three-electrode electrochemical cell under argon atmosphere. The auxiliary electrode was a graphite plate sample of large surface area. The reference electrode was based on the (Ag/AgClO4, 10−2 M) couple. Working electrodes were gold substrates (0.5 × 20 mm or 12 × 25 mm) used afterward for azide photochemical grafting experiments. Azide Photochemical Grafting Process. A CN-15.LC UV viewing cabinet (Vilber Lourmat, Marne-la-Vallée, France) (1050 μW/ cm2, equipped with a two band-pass filter: 254 and 365 nm) was used for azide photochemical grafting process. Pure polyethylene glycol (PEG; Mw: 4000, 10 μL) or G-protein solution (10 μg/mL in phosphate buffered saline (PBS)) was deposited on the whole surface. In order to limit side reaction with the solvent, the deposited solution volume was reduced, before UV insulation, by submitting the sample to mild vacuum for 5 min. Afterward, to obtain reproductive thickness and surface homogeneity film of pure PEG and G-protein solution, UV insulations were performed through a cover quartz slide. To discard all the absorbed material after UV insulation, a cleaning procedure based on Milli-Q water, ethanol, and acetone thorough rinsing is used before surface analysis for PEG grafting. For G-protein grafting, the cleaning procedure is a thorough rinsing with PBS buffer. IR Spectroscopy. Infrared spectra were recorded on a Bruker Vertex 70 spectrometer controlled by OPUS software. The analytical device was an ATR Pike-Miracle accessory. The detector was mercury cadmium telluride (MCT) working at liquid nitrogen temperature. ATR spectra acquisitions were obtained at 4 cm−1 resolution and 256 scans. SPRi Experiments. An SPRi platform composed of a SPRi-Slide, a SPRi-Arrayer, and a SPRi-Plex II instrument from Horiba Scientific was used. The contact SPRi-Arrayer prints proteins spot arrays with low cross contamination. Here, 36 spots of 500 μm diameter were laid

listed constraints, our team has previously proposed an uncommon method to graft biological compounds through self-adhesive surfaces.30,31 These surfaces were based on grafted diazonium salt able to spontaneously react with biological molecules. However, in usual biological pH conditions, the diazonium salt stability is not ensured and thus prevents this self-adhesive coupling. Moreover, the scope of immobilizable biomolecules is limited to those reactive with diazonium salts (excluding all the alkyl chains of amino acids and lipids for example). Recently another approach was based on the use of benzophenone-modified substrates to photochemically (365 nm) control covalent coupling of solution-phase biomolecules.32,33 This photochemical approach seems promising to covalently attach biomolecules without altering their properties. Besides being used for click chemistry,34−36 azide derivatives, and more precisely phenylazide groups, they can also react through a photochemical process and give raise to nitrene moieties37,38 that are highly reactive and can immobilize biomolecules. Indeed, these nitrogen intermediates can be inserted in the R−H (R could be C, N or O atoms39) closest bond to restore their valence.40 On the basis of complicated synthetic ways, biomolecules have already been grafted through nitrene insertion via azide photochemistry.41,42 Herein, we report an easy and convenient route to immobilize biomolecules via a one-step surface functionalization with 4-azidobenzene diazonium tetrafluoborate followed by biomolecule photochemical grafting. This simple process is versatile and could be used to covalently bind a large variety of molecules. To validate this approach for biochips or microarrays fabrication, we have created a surface plasmon resonance imaging (SPRi) platform43 for immobilization of a large range of antibody families by grafting G-protein through the process described above. This SPRi antibodies platform was tested with several consecutive cycles of antigen injections/regeneration steps.

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EXPERIMENTAL SECTION

Materials. Chemical reagents and solvent were purchased from Sigma-Aldrich and used as received. Proteins (G-protein from Streptococcus sp., antiovalbumine, antistreptavidine, antilectine antibodies, and mice-IgG) were purchased from Sigma-Aldrich and used without further purification. Antigens were purchased from SigmaAldrich for ovalbumine and lectine and from Pierce for streptavidine. Gold substrates were obtained after metallic evaporation of chromium 2076

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out on 1 cm2 of the SPRi-Slide. An indexing system coupled with software was used for multiplex analysis. SPRi data were acquired with a Horiba Scientific SPRi-Plex II system offering multiplex detection and real-time monitoring of kinetics curves. To be compatible with the SPRi detection chamber, SPRi-Slides were coupled to a glass prism thanks to a matching index liquid. A SPRi-Slide was functionalized by an electrografting process with 4-azidobenzene diazonium tetrafluoborate salt as previously described. This modified SPRi-Slide was UV functionalized in this whole surface with G-protein. Afterward, the Gprotein modified SPRi-Slide was rinsed with PBS buffer in order to discard ungrafted G-protein. The resulting G-Protein SPRi platform was used to bind antibodies. The running buffer was PBS, 10 mM at pH 7.4, with NaCl (138 mM) and KCl (2.7 mM). Antibody spots were deposited from 6 μM solutions of antiovalbumine, antistreptavidine, antilectine, and mice-IgG for negative control, prepared in running buffer, and incubated overnight at room temperature in a humidity chamber. No rinsing step was performed after the incubation, and the resulting antibody SPRi platform was directly used for SPR experiments. Injections of antigens (ovalbumine, streptavidine, and lectine) from solution at 10 μg/mL were performed with a sampling loop of 200 μL with a flow rate of 50 μL/min. The experiment was performed at 25 °C. After interaction measurements, the ligands were regenerated using Glycine-HCl pH 2 solution.

Figure 3. IR spectra of (a) pristine polyazidophenylene layer, (b) PEG grafted surface after 5 min of UV insulation and cleaning procedure, (c) thin layer of absorbed PEG on a gold surface as the reference, and (d) nonspecific binding of PEG molecules on a UV deactivated polyazidophenylene layer.

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RESULTS AND DISCUSSION Surface Modification of Gold Surface by Azidobenzene Diazonium Salt. Grafting of 4-azidobenzene diazonium

By taking account of coulometric analysis, current integration, and theoretical gyration diameter of an aryl group grafted on a surface, we estimate that the surface concentration of the azide group is about 7 × 10−10 mol/cm2. Azide Photochemical Grafting Process. It is well-known that phenyl azide groups are light sensitive with a typical absorption wavelength at 365 nm to convert the phenyl azide group in a nitrene function. This activation wavelength is a key point to graft biomolecules. In fact, the phenylazide wavelength activation is safe for biomolecules without altering their sequence or their structure and thus saving their biological activity compared to other photochemical processes at 254 nm. Degradation of the phenyl azide group starts at 430 nm.37,39 Figure 2 shows the IR spectra of a pristine polyazidophenylene layer and the resulting layers after 5 and 15 min of UV insulation in a laboratory atmosphere. The disappearance of the phenyl azide absorption band is almost completed after 5 min of UV insulation and is completed after 15 min. The out-ofplan deformation band of the C−H aromatic ring at 800 cm−1 and the low intensity band at 1100 cm−1 are maintained without loss of intensity. With this result, we assume that no important film degradation occurred in spite of the azide disappearance. The decrease of the vibrational band at 1500 cm−1 could be due to azido group removal. The infrared band at 1700 cm−1 could be assigned to an oxidative process generated by UV insulation under air atmosphere. To demonstrate the ability and versatility of a polyazidophenylene layer to graft different types of molecules, PEG and Gprotein have been chosen. PEG moieties are chemically inert compounds and present a great interest in biology for their antifouling and stealth properties.44,45 A pure PEG oil droplet (10 μL) was deposited on the polyazidophenylene layer and spread out under a cover quartz slide to achieve a uniform and thin PEG layer. The sample was then exposed to UV light for 5 min. Before infrared surface analysis, all samples were submitted to a cleaning procedure to discard absorbed material. Figure 3b shows the grafting of the PEG layer on the polyazidophenylene layer after UV insulation. The complete disappearance of the azide band at 2130 cm−1, the presence of vibrational bands at 1500 and 1600 cm−1

Figure 2. IR spectra of (a) pristine polyazidophenylene layer, (b) polyazidophenylene layer after exposition to UV for 5 min, and (c) polyazidophenylene layer after exposition to UV for 15 min.

tetrafluoborate (10−2 M) was performed by cyclic voltammetry at a scan rate of 20 mV·s−1 between the open circuit potential and the final potential selected at −1 V. The well-defined reduction peak centered on −300 mV attests to the electrochemical reduction of the diazonium group as shown in Figure 1. The complete disappearance of this peak during the second scan attests to the success of the coating and, as widely admitted, the chemical grafting. The IR spectra displayed in Figure 2 highlight the electrochemical grafting of diazonium salt on the gold surface. The IR spectrum of the grafted polyazidophenylene layer shows two intense vibration bands for azide groups at 2130 and 1290 cm−1, aromatic ring breathing vibrations at 1600 and 1500 cm−1, and one out-ofplan deformation band of C−H aromatic ring at 800 cm−1. This spectrum and intensities are in accordance with a polyazidophenylene structure film23 of few nanometers thickness. 2077

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Figure 4. Proposed mechanism of PEG or protein grafting on a polyazidophenylene layer.

Figure 5. Synthetic scheme to obtain the SPRi chip platform.

control probe. In fact, if G-protein grafting by a polyazidophenylene layer destroys the G-protein structure, the system can lose its Fc binding domain. In this case, the resulting G-proteinmodified SPRi chip cannot bind the antibody, or the resulting complex with antibody is not stable during a cycle of regeneration step/antigen injection, for example. A droplet of 30 μL of G-protein solution (10 μg/mL) was deposited on a polyazidophenylene-modified SPRi chip (Figure 5). Afterward, the same procedure for UV insulation was used. The resulting modified SPRi chip was then rinsed with PBS buffer. As already mentioned above, the nitrene insertion mechanism is a nonspecific process and consequently could lead to immobilize any molecules in any adsorption geometry. However, this benefit is counterbalanced by the need to reduce the presence of a competitive mechanism with solvent.47,48 In order to overcome this drawback, the deposited solution volume was reduced, before UV insulation, by submitting the sample to mild vacuum for 5 min. As this work used sensitive materials (biomolecules), be careful to not submit the sample to the dryness state. However, the control of water or solvent quantity can allow to adjust the grafting density. Two IR

assigned to the resulting polyphenylene layer after UV insulation, and the appearance of vibrational bands centered at 2870 and 1120 cm−1 assigned to the PEG layer (Figure 3c) confirm this covalent grafting. Figure 3d shows the result of nonspecific binding of PEG pure oil on a UV deactivated polyazidophenylene layer. As highlighted by Figure 3d, PEG molecules have no nonspecific binding properties to the polyazido layer. We hypothesize that insertion mechanisms occur even if any IR bands are clearly assigned to the resulting primary amino group. Alkyl parts of PEG could be engaged in the attachment mechanism. Figure 4 depicts a proposed PEG grafting mechanism on the polyazidophenylene layer. In order to demonstrate the availability of our surface chemistry for biochip or microassay synthesis, a SPRi chip was modified with a polyazidophenylene layer. This activated chip was modified by grafting G-protein on its whole surface. In fact, G-protein is well-known to selectively and noncovalently immobilize the Fc region of the antibody and enables a more favorable orientation.46 By this way, we want to create a versatile SPRi chip to immobilize a large range of antibody families. Moreover, G-protein is also used here as a bioactivity 2078

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Figure 6. IR spectra of (a) unspecific binding of G-protein (10 μg/ mL) on a UV deactivated polyazidophenylene layer, (b) of G-protein (10 μg/mL) on a gold surface as the reference, and (c) the UV grafting of G-protein (10 μg/mL) on the polyazidophenylene layer. Cleaning procedure with PBS buffer and H2O DI.

Figure 8. Streptavidin matrix SPRi response after streptavidin injection.

automatically deposited on the entire G-protein-modified SPRi chip surface. The picture in Figure 7 shows an image of the resulting spot pattern. Colored rings on the image refer to Table 1 in order to identify the nature and the localization of polyclonal antibodies on the chip. To improve the antibody immobilization, the SPRi chip is incubated overnight in a humidity chamber. The resulting SPRi chip was introduced directly without a rinsing step in the flow cell system of the SPRi-Plex II instrument. Afterward, typical SPR experiments were performed: (i) sequential injections of three antigens such as ovalbumin, lectin, and streptavidin to evaluate the interaction specificity and (ii) three consecutive cycles of regeneration step/ovalbumine injection in order to evaluate the effectiveness of our surface chemistry. The originality of SPRi technology is that it allows the interaction visualization in real time. Before the analyte injection, a reference image is taken. After antigen (streptavidin) injection, the corresponding antibodies react, and the spots appear as white spots, as shown in Figure 8. The image difference shows good homogeneity and reproducibility for all antistreptavidin spots. As show in the Figure 8, the other antibody spots and the negative control spots unreact with streptavidin, which highlights the specificity of this interaction. Same experiments

Table 1. Indexing Multiplex Matrix Table

absorption bands at 1663 and 1547 cm−1 (amide bands) were observed after the grafting process (Figure 6b). This result is supported by a comparative study with IR spectra of G-protein absorbed in a gold surface (Figure 6a,c) with or without the cleaning procedure. The conservation of the main amide bands and the general appearance of the spectrum corresponding to the protein structure attest to the protein grafting on the surface. A 36-spot matrix of three specific polyclonal antibodies and a negative control is prepared by using the contact SPRi-Arrayer. The microplate wells of the contact SPRi-Arrayer are filled by the four antibodies quoted in Table 1, and the matrix spot is

Figure 7. Image of the spot matrix pattern and specific antibody attribution for antigens analysis. 2079

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Figure 9. SPR response attesting to the interaction specificity through successive injections from left to right: ovalbumin, lectin, and streptavidin.

Table 2. Reproducibility Response with Three Consecutive Cycles of Regeneration Steps Followed by Ovalbumine Injections (10 μg/mL)a ovalbumin injection (10 μg/mL)

anti-ova antistrept anti-lect

ovalbumin injection (10 μg/mL)

ovalbumin injection (10 μg/mL)

average

standard deviation

average

standard deviation

average

standard deviation

0.473 0.012

0.103 0.045

0.460 0.013

0.109 0.031

0.461 0.009

0.112 0.037

0.005

0.024

0.010

0.008

0.008

0.006

a

The average and the standard deviation were performed on nine spots for each specific antibody.

antibodies on the G-protein layer. Afterward, no drift can be monitored during consecutive ovalbumin injection/regeneration steps, as shown in Figure 10A. This result highlights a rinsing step by the running buffer directly in the fluidic cell to obtain a stable immuno platform. Furthermore, the effectiveness and reproducibility of our surface chemistry was assessed by performing consecutive cycles of regeneration steps followed by ovalbumin injection. Results are presented in Table 2. The reflectivity variation obtained for these three cycles is depicted in Figure 10A,B. These results underline that the surface chemistry reported here allows covalent grafting of biomolecules. SPRi experiments highlight that, after phenylazide photochemical grafting, the resulting G-protein chip can still promote usual interaction with the Fc part of the antibody.

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CONCLUSION Polymer and unmodified biomolecule grafting were realized through a one-step photochemical method on a gold surface. This one-step process is based on photochemical grafting by a polyazidophenylene layer simply obtained by electroreduction of the corresponding diazonium salt. Photochemical activation of a phenylazide group in its nitrene moiety leads to the closest bond insertion mechanism with a wide range of molecules. Grafting results obtained with PEG demonstrate the potency and the versatility of this method. To validate this approach for

Figure 10. (A) Consecutive SPR responses of ovalbumin injection/ regeneration steps. (B) Reproducibility response with three consecutive cycles of regeneration step followed by ovalbumine injections.

were performed with other antigens such as ovalbumine and lectine, and the same results were obtained for each antibody/ antigen pair. As shown in Figure 9, a drift appears during the first 30 min of the SPRi experiment on each antibody spot. As no rinsing step was performed before the SPRi experiment, this negative drift is due to the dissociation of some unbinding 2080

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biochip or microarray fabrication, a SPRi platform for immobilization of a large range of antibody families was created by grafting G-protein through this process. This SPRi antibodies platform was tested with several consecutive cycles of antigen injections/regeneration steps. The SPRi data underline that the surface chemistry reported here allows covalent grafting of biomolecules. SPRi experiments highlight that, after phenylazide photochemical grafting, the resulting Gprotein chip can still promote usual interaction with the Fc part of the antibody.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Telephone number: +33 (0) 1 69 08 65 88; Fax number: +33 (0)1 69 08 40 44. Notes

The authors declare no competing financial interest.

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