Optically Assisted Surface Functionalization for Protein Arraying in

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Optically-assisted surface functionalization for protein arraying in aqueous media Ricardo Enrique Alvarado, Hoang Thanh Nguyen, Brigitte PépinDonat, Christian Lombard, Yoann Roupioz, and Loïc Leroy Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02965 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017

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Optically-assisted surface functionalization for protein arraying in aqueous media Ricardo E. Alvarado, Hoang T. Nguyen, Brigitte Pepin-Donat, Christian Lombard, Yoann Roupioz,∗ and Loïc Leroy Univ. Grenoble Alpes, CEA, INAC, SyMMES F-38000 Grenoble, France E-mail: [email protected] Phone: +33 04 38 78 98 79

Abstract Protein surface patterning is employed in a broad spectrum of applications ranging from protein micro-array analysis to 2D cell organization. However, limitations arise due to the highly sensitive nature of proteins requiring careful handling to ensure their structural and functional integrity during the grafting process. Here we describe a patterning protocol that keeps proteins in an aqueous environment during their immobilization, avoiding loss of their biological activity. The procedure is based on the UV-mediated removal of Poly-Ethylene Glycol (PEG) Self-Assembled Monolayers (SAMs) in a transparent microfluidic chamber, giving access to micrometric motifs of predefined geometries. Afterwards, modified proteins can be grafted on the photopatterned domains. We also studied the influence of reactive oxygen species on for a better understanding of the chemical mechanism involved in this process. Finally, as a proof of concept, a protein micro-array was created with this process using cell capturing antibodies to immobilize human blood cells, confirming the functionality of the arrayed proteins.

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Introduction For more than twenty years, considerable effort has been invested in the attempt of controlling the spatial deposition of proteins on surfaces. 1,2 Such engineered surfaces are mainly employed in (i) micro-array based biomolecule screening and (ii) micro/nano-patterned matrices for cell assays. 3,4 In contrast to other biomolecules (nucleic acids, peptides or sugars) most proteins require a permanent hydrated environment to ensure their structural integrity and functionality, which is rarely taken into account when designing most arraying processes. Common approaches to address this issue, such as the addition of hydrated sugars or other alcohols in the spotting solutions, have inconsistent and sometimes unpredictable results. 5,6 To avoid loss of their activity, an ideal grafting procedure should be performed completely in an hydrated environment. Under such conditions, spatio-temporal control of the patterning results considerably challenging. In this regard, photochemical strategies have become an attractive solution for protein immobilization, thanks to advances in micro-photolithography for mask generation. Duroux et al. 7 validated this approach for the UV-induced covalent grafting of Immunoglobulin fragments and cutinase enzyme on glass slides. In their work, the authors created an array of spots (25 µm in diameter) in aqueous conditions. The photochemical immobilization was done by direct UV-irradiation of proteins, absorbing light via aromatic amino-acids and disulfide bridge reduction. 8 However, direct UV-irradiation of proteins may also cause irreversible modifications, such as their cleavage into smaller polypeptides. 9 On a different approach, Weber et al. 10 described the UV-mediated deterioration of polyethylene glycol terminated SAMs in an aqueous environment. This allowed them to tune the antifouling properties of the surface and thus increase the adhesion of BSA on the exposed areas. Nevertheless, such approach was not evaluated for immobilization of functional proteins. The present study aims to develop a protein grafting experimental procedure that not only can be performed in an aqueous environment but it also avoids direct exposure of 2 ACS Paragon Plus Environment

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the proteins to UV light. This procedure is based on the photo-chemical deprotection of gold surfaces prior to the spontaneous adsorption of thiolated proteins, all carried out in a microfluidic chamber (Scheme 1).

Scheme 1: UV-induced protein patterning of gold surfaces. (a) Experimental set-up used for the photo-patterning of SAMs. (b) Gold protection by SAM formation, UV-controlled localized deprotection, protein patterning by spontaneous adsorption on gold. Such approach gives access to features made of functional proteins at millimetric and even micrometric scales. Hydrophobic and hydrophilic thiolated molecules were used for the formation of SAMs over gold and their UV-mediated desorption kinetics were monitored via SPR. Functionalization of the unprotected regions was evaluated by injection of biotin-labeled thiolated molecules followed by Streptavidin/R-phycoerythrin and fluorescent visualization. Once efficient grafting of new molecules was observed, protein immobilization was evaluated. For this, cell capturing antibodies were arrayed using the same procedure as described above followed by a flow of human blood cells. Immobilization of cells on the surface in the desired pattern was confirmed by optical microscopy. The obtained results provide an useful strategy for establishing efficient surface arraying methods for functional protein patterning. 3 ACS Paragon Plus Environment

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Experimental Reagents and materials: Phosphate Buffer Saline (PBS), PEG350 SH, PEG2000 SH, 2methyl-2-propanethiol, 1-dodecanethiol and 6-mercapto-1-hexanol, 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), Bis-(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium (BSPP), mannitol and sodium pyruvate were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). Fluorescence revelation was performed with Streptavidin-Phycoerythrin (SAPE) purchased from Fisher Scientific (Illkirch, France) and PEG-biotin from PolyPure (Oslo, Norway). Gold nanoparticles of 20 nm diameter were purchased from BBI Solutions (Cardiff, UK). For cell capturing experiments; antibodies anti-human CD3 IgG (BD Bioscience, Le Pont de Claix, France) and anti-rabbit IgG (Sigma Aldrich, Saint Quentin Fallavier, France) were used, while T cells were purified from human blood samples bought from Établissement Français du Sang (Grenoble, France). Horiba gold coated prisms (Longjumeau, France) were used during all SPR experiments. Formation of Self Assembled Monolayers (SAMs): All gold surfaces were cleaned via plasma treatment (plasma system Femto Diener Electronic, Ebhausen, Germany) for 3 min with an atmosphere of 75% O2 and 25% Ar at 0.6 mbar and 80% power of its maximum intensity. Clean surfaces were stored at room temperature for 18h before their use. For gold coated prisms functionalized with either PEG350 SH, PEG2000 SH or 6-mercapto-1-hexanol, the prisms were mounted on the SPRi set-up with a microfluidic device and thermal regulation was set to 25◦ C. Functionalization of the gold surface was done via injection of the respective solution at 20 µM during 1h at a flow rate of 0.1 µL.s−1 . Afterwards, a stronger flow of PBS (1.7 µL.s−1 ) for 10 min was used to remove unbounded molecules. For gold coated prisms functionalized with either 1-dodecanethiol or 2-methyl-2-propanethiol, SAM formation was performed before setting the prism in the fluidic device. This was done by direct exposure of the surfaces for 2 h to the respective 1 mM solution. After incubation at room temperature, the surface was rinsed three times with 10 mL of 96% ethanol and then three times with 10 mL of distilled water. In case of gold coated glass slides, functionaliza4 ACS Paragon Plus Environment

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tion was done with either PEG350 SH or PEG2000 SH via immersion on 1 mM solutions for 2 h. The surfaces were rinsed with 10 mL of distilled water thrice before experiments. SPRi monitoring of UV-mediated desorption: A homemade optical device was used during the Surface Plasmon Resonance Imaging (SPRi) assays (Supporting Information, Figure S1). This hybrid set-up combined a SPR device with a commercial optical microscope (Olympus BX-URA2) allowing exposure of the samples to UV light while monitoring the SPR signal over time. For the UV irradiation, a high pressure mercury lamp was used as a light source, an Olympus filter (λ 330-380 nm, peak at 365 nm) and either a 10x or 20x lens (Olympus UPlanF) were used on the device. SPRi was performed using a λ 625 nm diode as light source. During UV irradiation, samples had a constant flow of PBS (0.5 µL.s−1 ). Evaluation of radical species in desorption mechanism: For Electro Paramagnetic Resonance (EPR) assays, functionalization of gold nanoparticles was performed as described elsewhere 11 (See Supporting Information). A solution of 4.9x1016 particles.L−1 coated with PEG2000 SH was mixed with a DMPO solution(800 mM in PBS) . The mixture was poured into quartz tubes (Wilmad Labclass, New Jersey, USA) and illuminated directly using a 365 nm diode (Thorlabs, Maisons-Laffitte, France) for 105 s. EPR spectra were recorded on an MX Brucker spectrometer operating at 9 GHz at room temperature. Simulation of radical species was performed with free Winsim2002 software 12 (NIEHS, North Carolina, USA). SPR was used to monitor the effect of ROS scavengers/inducers as well as iron salts in the desorption kinetics of PEG2000 SH (see Supporting Information). Goniometry measurements: Gold coated slides were functionalized with PEG350 SH and PEG2000 SH as previously described. After 15 min of UV irradiation, contact angles were measured using a Digidrop MCAT (GBX). Water drop volume was set to 0.5 µL and measurements were made on the irradiated and control surfaces. Photopatterning and fluorescence assay: A gold coated prism was functionalized with PEG2000 SH as described above. UV irradiation was performed on the hybrid SPR device, however, the exposed area was defined by a mask with a grid pattern (See Supporting

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Information). After surface photopatterning, Biotin-PEG-SH (1 mM) was injected for 30 min followed by a solution of Streptavidin/R-Phycoerythrin Conjugate (SAPE) (50 µg ml−1 in PBS) for 15 min. After a gentle wash in PBS, visualization was performed at λ 520-550 nm on a Leica DMI 4000B microscope. Cell capturing assay: Lymphocytes were isolated from human blood samples via density gradient centrifugation with Ficoll-Paque as described elsewhere. 13 A gold coated prism was protected with PEG2000 -SH and photopatterned with a grid using two different magnification lens (10x and 20x). After performing UV-irradiation of PEG-SAM, the microfluidic chamber was filled with thiolated anti-human CD3 IgG antibodies (2.6 µM) and incubated for 2h at room temperature. Afterwards, the surface was washed with PBS and the chamber was filled with a suspension of T cells in PBS (2.2 x 106 cells.mL−1 ) for 20 min at room temperature. The array was washed twice with 1 mL of PBS and observed with an optical microscope.

Results and Discussion SPR monitoring of UV-mediated desorption. We carried out a series of experiments on thiolated compounds to follow their UV-induced desorption in real-time. Gold-covered SPR biochips were exposed to either PEG derivatives: a long chain thiolated-PEG (PEG2000 SH, MW= 2,000 Da), a short chain thiolated-PEG (PEG350 SH, MW= 350 Da); or one of three alkanethiols: 1-dodecanethiol (CH3 (CH2 )11 SH), 2-methyl-2- propanethiol (C(CH3 )3 SH) or 6-mercapto-1-hexanol (HO(CH2 )6 SH). Data analysis shows that only PEG-based SAMs are sensitive to UV-illumination (λ 365nm), while non PEG-based SAMs were not significantly affected (Figure 1). For PEG2000 SH and PEG350 SH SAMs desorption, a plateau was reached after 30 minutes of UV-exposure. The different level in reflectivity shifts observed for both thiolated-PEG species can be due to their different molecular weight inducing size-limited grafting densities. These results and

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the anti-fouling properties of PEG makes it attractive for the formation of protective SAMs. 0 −2 Reflectivity shift (%)

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Figure 1: SPR monitoring during UV irradiation of different SAMs: Polyethylene glycol (PEG2000 SH and PEG350 SH), 1-dodecanethiol (CH3 (CH2 )11 SH), 2-methyl-2-propanethiol (C(CH3 )3 SH) and 6-mercapto-1-hexanol (HO(CH2 )6 SH) Multiple cycles of SAM formation and desorption for both PEG derivatives were monitored by SPR. Each cycle consisted in the injection of the respective PEG-SH solution to form a new SAM followed by a stabilization time with PBS buffer and UV-irradiation. UVirradiation was performed exactly on the same area after each cycle allowing the study of SAM regeneration after each desorption phase. During the assays, PEG350 SH SAMs were only partially removed from the surface and, after each adsorption phase, it did not achieve the initial reflectivity value. This suggests that material is being left on the surface after each UV-desorption cycle and that this material hinders the attachment of new molecules on the surface (Figure 2). Contrary to what has been reported on alkanethiols, 14–17 our data suggest that this process is based on the degradation of the PEG chains (C-O cleavage) rather than the rupture of Au-S or C-S bonds. In case of PEG2000 SH SAM, such partial removal was not observed. Considering that PEG2000 SH forms SAMs with a relative lower molecular surface density, degradation of such SAMs will reveal large areas of gold previously blocked due to steric hindrance. Since the amount of material left on the surface is significantly lower than for PEG350 SH SAMs, the adsorption of new molecules would not be affected after only two 7 ACS Paragon Plus Environment

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Figure 2: Adsorption and UV-induced desorption kinetics of PEG-based SAMs followed by SPR. (a,b) SAM formation and UV-mediated desorption of (a) PEG350 SH and (b) PEG2000 SH ; (c,d) overlap of each UV-mediated desorption process for (c) PEG350 SH and (d) PEG2000 SH SAMs respectively. Data was normalized from 0% (reflectivity before first injection) to 100% coverage (reflectivity shift after first adsorption process) cycles of SAM formation/removal. Our data on partial desorption of SAMs upon UV irradiation are coherent with results published by Legett and Zharnikov who described experiments of similar compounds in air 18–21 and aqueous 10 conditions. In their work, they describe a dependence on the wavelength with the rate of degradation of the ethylene glycol chains while the alkanethiol part was primarily unaffected. Furthermore, they observed weak formation of sulfonates during UV irradiation, indicating rupture of Au-S bonds, but at a considerably slower rate. Evaluation of radical species in desorption mechanism: Knowing that in aqueous environment Reactive Oxygen Species (ROS) could be involved in the UV-induced SAM removal, Electron Paramagnetic Resonance (EPR) spectroscopy was used to analyze any radical species produced during our conditions. 22 In order to increase the amount of thio8 ACS Paragon Plus Environment

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lated molecules forming SAMs, 20 nm diameter gold nanoparticles were functionalized with PEG2000 SH and then exposed to UV light. After irradiation, the EPR spectra showed degradation products derived from the spin trapping agent (DMPO), while signals coming from expected hydroxyl radicals, thiyl radicals and carbon centered radicals were minor products (Figure 3). On the EPR simulation derived from data on the irradiated samples, signals from hydroxyl and thiyl radicals were very similar, while on control experiments without thiol containing molecules the observed peaks corresponded uniquely to hydroxyl radicals (see Supporting Information).

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Figure 3: EPR spectra of gold nanoparticles in a DMPO solution and after irradiation with UV light (365nm) for 1.75 min. The experimental spectrum (solid black line) together with a simulation for the expected radicals (DMPO degradation products*, hydroxyl/thiyl radicals † and carbon centered radicals ‡) (dotted red line). PEG degradation in presence of iron ions has been reported by hydroxyl radicals generated via a photo-Fenton reaction. 23 Considering the potential presence of ion traces in the solutions, control SPR experiments were conducted (see Supporting Information). Dissolved iron salts (either FeSO4 or FeCl3 ) were flowed on a microfluidic device during UV irradiation of a PEGylated gold surface. Nevertheless, instead of increasing the desorption rate, the desorption step was actually slower than in buffer (PBS). Iron ions can interact with dissolved oxygen forming complexes that absorb UV light and may reduce the amount of oxygen available. A slight reduction in irradiation efficiency as well as a decrease in the availability of oxygen could justify such results. However, the influence of dissolved oxygen was described 9 ACS Paragon Plus Environment

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by Weber et al. 10 who found that it is not required for UV-mediated PEG degradation in aqueous media. But its presence allows spontaneous desorption of SAMs due to oxidation of the thiol moieties, and thus, accelerates the desorption process. Finally, addition of EDTA solution did not affect the kinetics in any significant way compared to PBS suggesting that a photo-Fenton process does not take place under our experimental conditions. Further assays with either ROS scavengers or inducers were performed and monitored by SPR (see Supporting Information). For such experiments, either mannitol 24 (100 mM, reported to scavenge hydroxyl radicals), sodium pyruvate 25,26 (10 mM, which avoid formation of hydrogen peroxide) or hydrogen peroxide (0.25% v/v, producer of hydroxyl radicals when irradiated with UV light) were flowed during UV irradiation of a PEG2000 SH SAM. The production of hydroxyl radicals increased considerably the desorption rate, however, none of the scavengers had any significant effect on the desorption kinetics. These results suggests that ROS may be involved in the PEG degradation mechanism, however, prevention of their formation in the bulk solution do not affect the desorption process. Goniometry measurements: We measured the contact angles of water on the functionalized surfaces exposed to UV irradiation as well as those used as control (Figure 4a). Previous SPR assays based on UV irradiation of PEG derivatives showed either partial or complete removal of their corresponding SAM. Considering this, we expected to observe an increase in the contact angle on irradiated surfaces since degradation of the PEG chain would reduce its hydrophilicity. In case of the PEG2000 SH SAM after UV exposure, the contact angles should reach similar values to those of bare gold while PEG350 SH SAM could either be higher (formation of highly hydrophobic moieties on irradiated areas) or lower (relatively hydrophilic moieties). A drastic increase was observed in the contact angle for PEG2000 SH (from 30◦ to 61◦ ), reaching values similar to those of bare gold (63-65◦ ). For PEG350 SH, the increase was less radical (from 26◦ to 38◦ ). These results are coherent with our previous hypothesis. Degradation of PEG2000 SH unblocks large areas of unoccupied gold and therefore, measurements of the contact angle

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after UV irradiation should achieve values similar to those of bare gold. On the other hand, PEG350 SH SAM has a denser layer of remaining material with unknown moieties which, we can conclude by the results, are relatively hydrophilic (Figure 4b). Interestingly, previous work by Weber et al. 10 and Ducker et al. 27 described the generation of hydrophilic moieties (C=O) upon UV irradiation of PEGylated surfaces. In their studies, they used Infrared Reflection Absorption (IRRA) and X-ray Photoelectron Spectrometry (XPS) on PEG based SAMs before and after UV irradiation, observing an increase of C=O signal which can be ascribed to aldehyde moieties.

Figure 4: Measurements of contact angle of water on the functionalized surfaces. (a) Boxplot of measured contact angles for each SAM before and after UV irradiation. Lines inside the box represent that median value in the distribution; (b) scheme of surface packing for the different PEG-SH derivatives. Fluorescent labeling and cell capturing assays: Since SPR assays confirmed efficient removal of PEG2000 SH SAMs after UV exposure as well as its regeneration after incubation with the respective PEG-SH solution, experiments were conducted to extend 11 ACS Paragon Plus Environment

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this surface chemistry method to other biomolecules. A PEG2000 SH protected gold surface was UV-irradiated with a grid pattern using a mask. Afterwards, biotinylated-PEGSH was injected. After rinsing with PBS, the surface was incubated with Streptavidin/RPhycoerythrin (SAPE) in the microfluidic chamber. Efficient grafting of the biotin-derivative was confirmed by fluorescence microscopy which showed labeling closely matching the pattern used for the UV irradiation (Figure 5a). The ability to photo-pattern proteins on surfaces was assessed by the same protocol. Thiolated Immunoglobulin G (anti-CD3 IgG), specific to antigens anchored on the membrane of human T cells, was injected on the microfluidic cell after photo-deprotection and was incubated for 20 minutes. After washing with PBS, the patterned protein array could be used for human cell probing and detection. A suspension of human white blood cells (2x106 cells.mL−1 ) was then injected at room temperature for another 20 minutes. Afterwards unbounded cells were gently washed away with PBS. Observation of the biochip by optical microscopy confirmed the efficient capture of cells on each feature functionalized with anti-CD3 IgG. The feature size can be easily decreased to micrometric motifs by switching the microscope objective used on the illuminating setup (Figure 5b-d) giving access to motifs in the same size range of human cells (12-14 µm in diameter). A series of 10 µm-large features of anti-CD3 IgG (grafted with a 30 µm pitch) enabled the capture of individual cells on photo-patterned motifs. Control experiments performed with thiolated rabbit immunoglobulins used as negative control did not show any cell capture and thus confirmed the cell-type specific binding on photo-patterned proteins (see Supporting Information). This photo-chemical approach turns out to be fully compatible with surface patterning of proteins. By keeping a continuously hydrated medium while avoiding direct exposure of proteins to UV light, it is possible to guarantee the native functionality of delicate samples.

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Figure 5: UV-assisted protein patterning in aqueous conditions. (a) Fluorescence image of deprotected zones functionalized with biotin-PEG-SH and revealed with SAPE with a 20x objective; (b) patterns performed through 10x objective and functionalized with anti-CD3 IgG, multiple cells per feature can be observed; (c) pattern performed through 20x objective lens and functionalized with anti-CD3 IgG, individual cells are bound on the protein array.

Conclusions To summarize, UV-photolithography in aqueous media of PEG SAMs can effectively be used to micro-pattern surfaces and create functional protein microarrays. To the best of our knowledge, this is the first reported fully-liquid patterning process for functional antibody arraying. This methodology can be employed with any thiolated protein and is compatible with cell culture conditions, making it suitable for a broad spectrum of applications. Furthermore, this technique overcomes limitations such as low throughput, difficult operation and dryness that can be encountered in technologies based on micro-contact printing or micro-cantilevers. 28

Acknowledgement This work has been partially supported by the French Labex ARCANE (ANR-11-LABX0003-01) ANR Program.

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Supporting Information Available The following files are available free of charge. • Description of hybrid SPR device, mask fabrication procedure, controls for antibody specificity as well as EPR experiments and SPR kinetics in presence of ROS scavengers or inducers. This material is available free of charge via the Internet at http://pubs.acs.org/.

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(16) Rieley, H.; Price, N. J.; Smith, T. L.; Yang, S. Photo-oxidation and photo-reduction in alkylthiol monolayers self-assembled on gold. Journal of the Chemical Society, Faraday Transactions 1996, 92, 3629. (17) Brewer, N. J.; Janusz, S.; Critchley, K.; Evans, S. D.; Leggett, G. J. Photooxidation of Self-Assembled Monolayers by Exposure to Light of Wavelength 254 nm: A Static SIMS Study. The Journal of Physical Chemistry B 2005, 109, 11247–11256. (18) Khan, M. N.; Zharnikov, M. Fabrication of ssDNA/Oligo(ethylene glycol) Monolayers and Patterns by Exchange Reaction Promoted by Ultraviolet Light Irradiation. The Journal of Physical Chemistry C 2013, 117, 24883–24893. (19) Jeyachandran, Y. L.; Weber, T.; Terfort, A.; Zharnikov, M. Application of Long Wavelength Ultraviolet Radiation for Modification and Patterning of Protein-Repelling Monolayers. The Journal of Physical Chemistry C 2013, 117, 5824–5830. (20) Jeyachandran, Y. L.; Meyerbröker, N.; Terfort, A.; Zharnikov, M. Maskless Ultraviolet Projection Lithography with a Biorepelling Monomolecular Resist. The Journal of Physical Chemistry C 2015, 119, 494–501. (21) Montague, M.; Ducker, R. E.; Chong, K. S. L.; Manning, R. J.; Rutten, F. J. M.; Davies, M. C.; Leggett, G. J. Fabrication of Biomolecular Nanostructures by Scanning Near-Field Photolithography of Oligo(ethylene glycol)-Terminated Self-Assembled Monolayers. Langmuir 2007, 23, 7328–7337. (22) Rowlands, C. C.; Murphy, D. M. Encyclopedia of Spectroscopy and Spectrometry; Elsevier, 2017; pp 173–179, DOI: 10.1016/B978-0-12-803224-4.00139-4. (23) Giroto, J. A.; Teixeira, A. C. S. C.; Nascimento, C. A. O.; Guardani, R. Degradation of Poly(ethylene glycol) in Aqueous Solution by Photo-Fenton and H 2 O 2 /UV Processes. Industrial & Engineering Chemistry Research 2010, 49, 3200–3206.

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(24) Shen, B.; Jensen, R. G.; Bohnert, H. J. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiology 1997, 115, 527–532. (25) Desagher, S.; Glowinski, J.; Prémont, J. Pyruvate protects neurons against hydrogen peroxide-induced toxicity. Journal of Neuroscience 1997, 17, 9060–9067. (26) Troxell, B.; Zhang, J.-J.; Bourret, T. J.; Zeng, M. Y.; Blum, J.; Gherardini, F.; Hassan, H. M.; Yang, X. F. Pyruvate Protects Pathogenic Spirochetes from H2O2 Killing. PLoS ONE 2014, 9, e84625. (27) Ducker, R. E.; Janusz, S.; Sun, S.; Leggett, G. J. One-Step Photochemical Introduction of Nanopatterned Protein-Binding Functionalities to Oligo(ethylene glycol)-Terminated Self-Assembled Monolayers. Journal of the American Chemical Society 2007, 129, 14842–14843. (28) Roupioz, Y.; Berthet-Duroure, N.; Lechlé, T.; Pourciel, J.-B.; Mailley, P.; Cortes, S.; Villiers, M.-B.; Marche, P. N.; Livache, T.; Nicu, L. Individual Blood-Cell Capture and 2D Organization on Microarrays. Small 2009, 5, 1493–1497.

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