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Mar 16, 2016 - Abstract: Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in ...
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Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry Chenggang Zhu 1 , Xiangdong Zhu 2 , James P. Landry 2 , Zhaomeng Cui 3 , Quanfu Li 3 , Yongjun Dang 3 , Lan Mi 1 , Fengyun Zheng 4 and Yiyan Fei 1, * 1

2 3

4

*

Department of Optical Science and Engineering, Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Fudan University, Shanghai 200433, China; [email protected] (C.Z.); [email protected] (L.M.) Department of Physics, University of California, Davis, CA 95616, USA; [email protected] (X.Z.); [email protected] (J.P.L.) Key Laboratory of Metabolism and Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; [email protected] (Z.C.); [email protected] (Q.L.); [email protected] (Y.D.) Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; [email protected] Correspondence: [email protected]; Tel.: +86-21-6564-2092; Fax: 86-21-6564-1344

Academic Editors: Alexandre François, Al Meldrum and Nicolas Riesen Received: 1 February 2016; Accepted: 11 March 2016; Published: 16 March 2016

Abstract: Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%. Keywords: high-throughput screening; small-molecule microarrays; surface functionalization; label-free optical biosensor

1. Introduction Using large arrays of small molecules immobilized on a solid support (small-molecule microarrays or SMMs), the reaction of a protein probe with thousands of distinct molecules can be characterized simultaneously in a single experiment. As a result, SMMs have emerged as one of the high-throughput screening platforms for probing of protein-ligand interactions [1] in applications such as drug discovery [2,3], protein profiling [4], ligand discovery [5], and biomarker search [3,6]. The success of microarray-based screening platforms hinges on efficient immobilization of a wide variety of small molecules on solid supports. A number of immobilization schemes exist for SMM fabrication, based on selective or non-selective attachment methods. Examples of selective attachment schemes include immobilization of biotin groups on strepavidin functionalized surface [7–13], thiol groups on a maleimide functionalized surface [14], alcohol groups on a silyl chloride functionalized surface [15], phenol or carboxylic acid groups on a diazobenzylidene functionalized surface [16], hydrazide groups on an epoxide functionalized surface [17], azide groups on an alkyne [18] or Sensors 2016, 16, 378; doi:10.3390/s16030378

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phosphane functionalized surface [19], polyfluorocarbon groups on a fluoroalkylsilane functionalized surface [20] and peptide-nucleic acids on an oligonucleotide functionalized surface [21]. These selective attachment methods ensure that the surface-immobilized biomolecules retain most of their original activities uniformly. However, they are not applicable to libraries of natural products or other compound libraries without a common residue for anchoring. In these cases, non-selective attachment methods such as photo-activated cross-linking and capture of nucleophilic residues by isocyanate-functionalized surface are alternatives. The photo-activated cross-linking method provides universal immobilization via carbon-based random insertion, but it comes with relatively high false positive results [22,23]. An isocyanate-functionalized surface is covalently reactive to compounds containing nucleophilic residues and thus can be used to immobilize molecules non-selectively [24,25]. SMMs of natural products, commercially available compound libraries, and oligonucleotides have been successfully fabricated on isocyanate functionalized glass slides and used in drug discovery and genomics study [26–32]. One issue concerning the isocyanate capture method is the relatively low immobilization efficiency for some molecules due to their low reactivity with isocyanate. Among nucleophilic residues, primary amine, aryl amine, and thiol have high reactivity to isocyanate; primary alcohol, phenol, and secondary amine have medium reactivity to isocyanate; carboxylic acid, secondary alcohol, and tertiary alcohol have low reactivity to isocyanate. The immobilization efficiency of molecules with carboxylic acid and secondary alcohol is less than 10% that of molecules with primary amine [23]. In screening 8000 compounds on SMMs for ligands of vascular endothelial growth factor (VEGF) [27] Fei and coworkers identified 64 hits with nucleophilic residues that bound to VEGF. Among them, 21 (33%) compounds have high isocyanate reactivity, 36 (56%) compounds have medium isocyanate reactivity, and 7 (11%) compounds have low isocyanate reactivity. The number of hits with low isocyanate reactivity is much smaller than the number of compounds with medium or high isocyanate reactivity. It is feasible that compounds with nucleophilic residues of low isocyanate reactivity are much less immobilized than compounds with nucleophilic residues of medium or high isocyanate reactivity under the same microarray fabrication conditions and thus fewer hits out of compounds with carboxylic acid or secondary alcohol may reflect more on the immobilization efficiency on the solid support than the reactivity of these compounds with VEGF. It is thus important to find optimal ways to improve immobilization efficiencies for compounds with low isocyanate reactivity. In this paper we report experimental studies on immobilization efficiency and morphological quality of printed small molecules on isocyanate functionalized surface as functions of (1) nucleophilic residues on small molecules for surface attachment; (2) penultimate chemical groups after terminal isocyanate residues on the isocyanate functionalized surface; (3) the length of polyethylene glycol (PEG) as the spacer between the penultimate chemical group and the glass surface; and (4) post-printing treatment. We identified a set of conditions that enable immobilization of small molecules with a wide range of nucleophilic residues, with an overall immobilization percentage of at least 73%. 2. Materials and Methods 2.1. Reagents and Proteins Amine functionalized glass slides were purchased from CapitalBio Corporation (Beijing, China). Polyethylene glycol (n = 1) or (PEG)1 was from PolyPeptide Group (San Diego, CA, USA) and (PEG)6 was from Shanghai Plus Bio-Science and Technology Company (Shanghai, China). (PEG)12 , (PEG)24 , and (PEG)36 were from Biomatrik Incorporated (Jiaxing, China). (PEG)2 , N,N-Diisopropylethylamine (DIPEA), piperidine, pyridine, baicalin, hexamethylene diisocyanate (HDI), 1,4-Phenylene diisocyanate (PPDI), (benzotriazol-l-yloxy) tripyrrolidinophosphonium hexafluoro phosphate (PyBOP,) biotin-(PEG)2 -NH2 , biotin-(PEG)6 -OH and D-biotin were from Aladdin Industrial Corporation (Shanghai, China). Biotin-NH-NH2 and biotin4-nitrophenyl ester were from Shanghai Yuanye Bio-Technology Company (Shanghai, China). N,N-Dimethylformamide (DMF), tetrahydrofuran (THF)

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3 of 15 Shanghai Yuanye Bio-Technology Company (Shanghai, China). N,N-Dimethylformamide (DMF), tetrahydrofuran (THF) were from Sinopharm Chemical Regent Company (Shanghai, China). Dimethyl sulfoxide (DMSO) was from Shanghai Lingfeng Chemical were from Sinopharm Chemical Regent Company (Shanghai, China). Dimethyl sulfoxide (DMSO) Reagent Company (Shanghai, China). Bovine serum albumin (BSA) was from Sigma-Aldrich (St. was from Shanghai Lingfeng Chemical Reagent Company (Shanghai, China). Bovine serum albumin Louis, MO, USA) and mouse anti-biotin antibody was from Jackson ImmunoResearch Laboratories (BSA) was from Sigma-Aldrich (St. Louis, MO, USA) and mouse anti-biotin antibody was from (West Grove, United states). Streptavidin (SAVD) was from Life Technologies (Shanghai, China). Jackson ImmunoResearch Laboratories (West Grove, United states). Streptavidin (SAVD) was from Biotin conjugated BSA was from Beijing Bo Sheng Bio-Technology Company Limited (Beijing, Life Technologies (Shanghai, China). Biotin conjugated BSA was from Beijing Bo Sheng Bio-Technology China). Company Limited (Beijing, China).

2.2. Preparation Preparation of of Isocyanate Isocyanate Functionalized Functionalized Glass Glass Slides Slides 2.2. Figure 11shows showssteps steps preparing isocyanate functionalized from commercial Figure forfor preparing isocyanate functionalized glassglass slidesslides from commercial amine amine functionalized glass slides. First, the spacer PEG was introduced by immersing amine functionalized glass slides. First, the spacer PEG was introduced by immersing amine functionalized functionalized glass slides into a (PEG) n solution for 10 h with stirring. The (PEG)n solution glass slides into a (PEG)n solution for 10 h with stirring. The (PEG)n solution contained 1 mM contained 1 mM Fmoc-NH-(PEG) n-(CH2)2-COOH, 2 mM PyBOP and 20 mM DIPEA dissolved in Fmoc-NH-(PEG) n -(CH2 )2 -COOH, 2 mM PyBOP and 20 mM DIPEA dissolved in DMF. (PEG)n of DMF. (PEG) n of varying lengths (n = 1, 2, 6, 12, 24, 36) were used on respective slides. Second, the varying lengths (n = 1, 2, 6, 12, 24, 36) were used on respective slides. Second, the protecting Fmoc group protecting Fmoc group wasthe removed by incubating the slides(v/v in a1%) solution of was removed by incubating PEG-treated glass slides in PEG-treated a solution of glass piperidine in DMF piperidine (v/v 1%) stirring. in DMF Third, for 12 h with gentleisocyanate stirring. Third, terminal isocyanate group was for 12 h with gentle the terminal groupthe was added through incubation of added through incubation of de-protected glass slides in a solution of isocyanate for 1 hwith stirring. de-protected glass slides in a solution of isocyanate for 1 hwith stirring. Two isocyanate solutions at the Two isocyanate solutions at the same of 60residues mM were used to addpenultimate isocyanate chemical residues same concentration of 60 mM were usedconcentration to add isocyanate with different with different penultimate chemical groups to the (PEG) n spacer: the HDI solution in DMF was used groups to the (PEG)n spacer: the HDI solution in DMF was used to add hexyl-isocyanate residues to add and theto PPDI in THF wasresidues used to(Figure add phenyl-isocyanate and thehexyl-isocyanate PPDI solution inresidues THF was used addsolution phenyl-isocyanate 1). Afterwards, residues (Figure 1). Afterwards, isocyanate functionalized slides were rinsed with DMFofand THF isocyanate functionalized slides were rinsed with DMF and THF and dried with a stream purified and dried with a stream of purified nitrogen. If not used immediately, the processed slides were ˝ nitrogen. If not used immediately, the processed slides were stored in a ´20 C freezer until printing stored in a molecules. −20 °C freezer until printing with small molecules. with small

Figure 1.1.Steps Stepsof of chemical modification an amine functionalized glass slide toanproduce an Figure chemical modification of anof amine functionalized glass slide to produce isocyanate isocyanate functionalized slide for SMM fabrication. functionalized slide for SMM fabrication.

2.3. Fabrication Fabrication of of SMMs SMMs on on Isocyanate Isocyanate Functionalized Functionalized Glass Glass Slides Slides 2.3. Biotinylated compound compound microarrays microarrays were were fabricated fabricated as as follows. follows. Five Five biotinylated biotinylated compounds, compounds, Biotinylated biotin-(PEG)22-NH ester, biotin-NH-NH biotin-NH-NH22,, biotin-(PEG) biotin-(PEG)66-OH, D-biotin (with (with biotin-(PEG) -NH22,, biotin4-nitrophenyl biotin4-nitrophenyl ester, -OH, and and D-biotin intrinsic COOH group), were printed on as-prepared isocyanate functionalized glass slides with intrinsic COOH group), were printed on as-prepared isocyanate functionalized glass slides with aa contact microarray microarray printer printer (SmartArrayer (SmartArrayer 136 136 Microarray Microarray Spotter, Spotter, CapitalBio CapitalBio Corporation, Corporation, Beijing, Beijing, contact China). Each compound was diluted in DMSO and separately in a mixture of DMSO with ddH22O O China). Each compound was diluted in DMSO and separately in a mixture of DMSO with ddH (v/v 50%) to 4 concentrations of 1 mM, 4 mM, 8 mM, and 10 mM. Three control compounds (v/v 50%) to 4 concentrations of 1 mM, 4 mM, 8 mM, and 10 mM. Three control compounds (baicalein, (baicalein, and rapamycin, and diluted FK506)inwere in DMSO ofat5 mM. a concentration of 5compounds mM. The rapamycin, FK506) were DMSOdiluted at a concentration The biotinylated 2 biotinylated compounds were each printed in triplicate. There were a total of 135 printed spots over were each printed in triplicate. There were a total of 135 printed spots over an area of 3.75 ˆ 2.5 mm

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that formed one microarray. We printed 6 identical microarrays on one slide. The averaged diameter of printed spots was 100~150 µm and the center-to-center spot separation was 250 µm. After printing, 4 distinct post-printing treatments were used on respective glass slides to facilitate covalent attachment of nucleophilic residues to isocyanate functionalized slides: (1) drying at room temperature only; (2) drying at 45 ˝ C only; (3) catalyzation in pyridine vapor at room temperature; and (4) catalyzation in pyridine vapor at 45 ˝ C. After post-printing treatment, the microarrays were stored in a ´20 ˝ C freezer until use. A small molecule microarray of 3375 bioactive compounds (FMSCL: Fudan MolMed-Selleck Compound Library), including 1053 natural compounds from Traditional Chinese Medicine (most of them from herb), 1527 drugs approved by Food and Drug Administration (FDA) and 795 known inhibitors, was prepared as follows. Each compound was dissolved in DMSO to a concentration of 10 mM and was printed in duplicate on both phenyl-isocyanate functionalized slides and hexyl-isocyanate functionalized glass slides. Biotin-BSA at a concentration of 7600 nM in 1ˆ phosphate-buffered saline (PBS) and biotin-(PEG)2 -NH2 at a concentration of 5 mM in DMSO were also printed as the inner and the outer borders of SMMs to serve as position markers and positive controls. There were 148 columns and 64 rows in each SMM over an area of 37 ˆ 16 mm2 . The SMM was dried at 45 ˝ C for 24 h and were then stored in a ´20 ˝ C freezer until binding reactions with proteins. 2.4. Detection of SMMs with a Label-Free Ellipsometry-Based Scanning Microscope Binding reactions of protein probes with SMMs were monitored in situ using an optical biosensor, scanning oblique-incidence reflectivity difference (OI-RD) microscope, whose working principle has been reported previously [33–36]. In essence, the phase change of the reflected optical beam (i.e., the OI-RD signal) from the printed region is measured by the microscope and such a change is proportional to the surface mass density of protein probes that are captured by immobilized small molecules [37]. For optical detection, biotinylated compound microarrays and SMMs of 3375 bioactive compounds were assembled into respective fluidic cartridges. The microarrays were (1) washed in situ with a flow of 1ˆ PBS to remove excess unbound small molecules; (2) exposed to 7600 nM BSA in 1ˆ PBS for 30 min to block unprinted isocyanate functionalized surface; (3) exposed to anti-biotin antibody at a concentration of 86 nM for 2 h or to SAVD at a concentration of 154 nM for 1 h. The surface mass density changes in both printed and unprinted regions were determined from the optical phase difference images of the SMMs. 3. Results 3.1. Immobilization Efficiencies of Compounds with Different Nucleophilic Residues Printed on Hexyl-Isocyanate Functionalized Slides Hexyl-isocyanate functionalized surfaces activated with pyridine vapor at room temperature can covalently capture a variety of compounds with nucleophilic residues [25,27]. We quantified the immobilization efficiency for five biotinylated compounds on this surface, each having a distinct nucleophilic residue that reacts with the isocyanate group with different affinities. Printed microarrays of biotinylated primary amine (Biotin-1), biotinylated nitrophenyl ester (Biotin-2), biotinylated hydrazide (Biotin-3), biotinylated primary hydroxyl (Biotin-4), and D-biotin (Biotin-5) on hexyl-isocyanate functionalized slide were catalyzed with pyridine vapor at room temperature. To determine immobilization efficiencies of these compounds, the microarray was subsequently reacted with unlabeled mouse anti-biotin antibodies (with a molecular weight of 150 kDa) and the surface mass density of the antibodies captured by the immobilized compounds was measured with the OI-RD scanning microscope. Figure 2a shows that all five biotinylated compounds are indeed immobilized on the hexyl-isocyanate functionalized surface, albeit with different immobilization efficiencies as the amounts of captures mouse anti-biotin antibodies differ. From surface mass densities of the captured

is close to unity as the concentration of anti-biotin antibody [c] is 86 nM. As a result, the coverage of anti-biotin antibody can be used as the measure of the immobilization efficiency of biotinylated compounds on hexyl-isocyanate functionalized surface. Figure 2b displays the immobilization efficiencies for five printed biotinylated compounds. The immobilization efficiency for compounds with primary Sensors 2016, 16, 378 amine residues is about 80%; the efficiency for compounds with hydrazide and 5 of 15 primary hydroxyl residues is about 30%; and for compounds with carboxylic acid residues, the immobilization efficiency drops to about 8%, consistent with the previous reports [23]. The anti-biotin antibodies by the is biotinylated printed from solutions inofmixtures of DMSO immobilization efficiency essentially compounds unchanged when the concentration compounds for with ddH2 Ovaries and by the same from solutions in no pure DMSO, of wecaptured can conclude that printing from 1 mMcompounds to 10 mM. printed As expected, we found evidence mouse antibodies on the negative controls. theanti-biotin hexyl-isocyanate functionalized surface is not sensitive to the moisture in the printing solution.

Figure 2. (a) After reaction in OI-RD OI-RDimage image(differential (differential OI-RD Figure 2. (a) After reactionwith withanti-biotin anti-biotinantibody, antibody, the the change change in OI-RD image) of a biotinylated compound microarray on hexyl-isocyanate functionalized slide with (PEG) image) of a biotinylated compound microarray on hexyl-isocyanate functionalized slide with (PEG)6 6 andand catalyzed in pyridine vapor at room temperature; (b)(b) Extracted immobilization efficiencies of of five catalyzed in pyridine vapor at room temperature; Extracted immobilization efficiencies five biotinylated compounds with different nucleophilic residues available for surface anchoring. biotinylated compounds with different nucleophilic residues available for surface anchoring.

resultsimmobilization displayed in Figure 2 show that small with molecules with nucleophilic To The quantify efficiencies of printing compounds different nucleophilicresidues residues, on hexyl-isocyanate functionalized surface followed by catalyzation in pyridine vapor atcaptured room we calculated the coverage of antibodies as the ratio of the surface mass density of antibodies temperature produces useful small-molecule microarrays. However, immobilization efficiencies for by the compounds to that of one monolayer of antibodies in upright or “end-on” configuration. small molecules with carboxylic acid residues and even hydrazide and primary hydroxyl residues The latter is roughly 1.3 ˆ 10´6 g/cm2 , assuming that one IgG molecule measures 4.4 nm ˆ 4.4 nm ˆ are low, rendering subsequent binding reactions with protein probes more susceptible to 23.5 nm [38,39]. The coverage of captured antibodies is proportional to the amount of immobilized background noises and drifts in label-free detection systems. small molecule available to bind the antibodies. It is also proportional to the factor of [c]/([c] + KD ), where constant between protein and small molecule [c] is the concentration D is the dissociation 3.2. K Immobilization Efficiencies and Spot Morphology of Printed Compounds vs.and the Length of Spacer Molecule of protein. Since K between unlabeled mouse anti-biotin antibody and biotin is ~1 nM [40], [c]/([c] + KD ) D on Hexyl-Isocyanate Functionalized Surface is close to unity as the concentration of anti-biotin antibody [c] is 86 nM. As a result, the coverage of Polyethylene glycol (PEG)n is used as a spacer to separate the isocyanate residue from the solid anti-biotin antibody can be used as the measure of the immobilization efficiency of biotinylated surface so that the effect of the surface on the reactivity of subsequently immobilized compounds compounds on hexyl-isocyanate functionalized surface. Figure 2b displays the immobilization efficiencies for five printed biotinylated compounds. The immobilization efficiency for compounds with primary amine residues is about 80%; the efficiency for compounds with hydrazide and primary hydroxyl residues is about 30%; and for compounds with carboxylic acid residues, the immobilization efficiency drops to about 8%, consistent with the previous reports [23]. The immobilization efficiency is essentially unchanged when the concentration of compounds for printing varies from 1 mM to 10 mM. As expected, we found no evidence of captured mouse anti-biotin antibodies on the negative controls. The results displayed in Figure 2 show that printing small molecules with nucleophilic residues on hexyl-isocyanate functionalized surface followed by catalyzation in pyridine vapor at room temperature produces useful small-molecule microarrays. However, immobilization efficiencies for small molecules with carboxylic acid residues and even hydrazide and primary hydroxyl residues are low, rendering subsequent binding reactions with protein probes more susceptible to background noises and drifts in label-free detection systems.

3.2. Immobilization Efficiencies and Spot Morphology of Printed Compounds vs. the Length of Spacer Molecule on Hexyl-Isocyanate Functionalized Surface Polyethylene glycol (PEG)n is used as a spacer to separate the isocyanate residue from the solid surface so that the effect of the surface on the reactivity of subsequently immobilized compounds with

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protein probes in solution is reduced or minimized [41,42]. We studied the compound immobilization with protein probes in solution is reduced or minimized [41,42]. We studied the compound efficiency as a function of the length of (PEG)n (n = 1, 2, 6, 12, 24, 36) (Figure 3). Clearly, the immobilization efficiency as a function of the length of (PEG)n (n = 1, 2, 6, 12, 24, 36) (Figure Sensors 2016, 16, 378 6 of 15 3). immobilization efficiencies are lessened with short PEG spacers (n = 1, 2) and we observed significant Clearly, the immobilization efficiencies are lessened with short PEG spacers (n = 1, 2) and we non-specific binding of protein on these surfaces. The surfaces with We longer spacer = 6, 12, 24, 36) with protein probes in solution is reduced or minimized [41,42]. studied the(ncompound observed significant non-specific binding of protein on these surfaces. The surfaces with longer both improve the immobilization efficiency and reduce non-specific binding of subsequent protein immobilization efficiency a function the length of (PEG) n (n = 1, 2, 6, 12, non-specific 24, 36) (Figure 3). spacer (n = 6, 12, 24, 36) bothasimprove the of immobilization efficiency and reduce binding probes. However, whenprobes. the spacer length increases from nshort = 12increases to n spacers = 36, the the protein immobilization efficiencies are lessened with PEG (nnspot 1, morphology 2) n and wethe of of Clearly, subsequent However, when the spacer length from == 12 to = 36, observed significant non-specific binding of protein on these surfaces. The surfaces with longer printed compounds becomes more varied in terms of displacement of the spot centroid from spot morphology of printed compounds becomes more varied in terms of displacement of the spotthe spacer (n = 6,the 12, 24, 36) both improve thethe immobilization efficiency and reduce non-specific binding printed location, shape and spreading of spot. As a result, surface (PEG) thewith spacer 6 as centroid from printed location, shape and spreading of thethe spot. As a with result, the surface of subsequent protein probes. However, when the spacer length increases from n = 12 to n = 36, the and gives the best result terms of efficiency, binding, (PEG) 6 as theimmobilization spacer gives the bestinimmobilization resultreduction in terms of of non-specific efficiency, reduction of spot morphology of printed compounds becomes more varied in terms of displacement of the spot repeatability and uniformity of spot morphology. non-specific binding, and repeatability and uniformity of spot morphology. centroid from the printed location, shape and spreading of the spot. As a result, the surface with (PEG)6 as the spacer gives the best immobilization result in terms of efficiency, reduction of non-specific binding, and repeatability and uniformity of spot morphology.

Figure 3. Immobilization efficiencies of biotinylated compounds on hexyl-isocyanate functionalized Figure 3. Immobilization efficiencies of biotinylated compounds on hexyl-isocyanate functionalized slide with spacers (PEG)n of varying lengths (n = 1, 2, 6, 12, 24, 36). slideFigure with spacers (PEG)n ofefficiencies varying lengths (n = 1, 2,compounds 6, 12, 24, 36). 3. Immobilization of biotinylated on hexyl-isocyanate functionalized slide with spacers (PEG)n of varying lengths (n = 1, 2, 6, 12, 24, 36). Using a contact-angle measurement system (OCA15, DataPhysics Instruments GmbH, Using a contact-angle Instruments GmbH, Filderstadt, Filderstadt, Germany), wemeasurement measured thesystem contact(OCA15, angles ofDataPhysics DMSO on hexyl-isocyanate functionalized Using a contact-angle measurement system (OCA15, DataPhysics Instruments GmbH, Germany), we measured angles of DMSO surfaces surfaces as a function ofthe the contact PEG spacer length (Figureon 4).hexyl-isocyanate The contact anglefunctionalized decreases as the length as Filderstadt, Germany), we measured the contact angles of DMSO on hexyl-isocyanate functionalized a function ofas the PEG spacer length (Figure 4). The contact angle decreases as the length of the PEG of surfaces the PEG spacer increases, indicating that the wettability increases with the spacer length. Printed a function of the PEG spacer length (Figure 4). The contact angle decreases as the length droplets on a spacer highly hydrophilic arethe expected towith form irregular because of droplets significant spacer indicating thatindicating thesurface wettability increases the spacer length. Printed of increases, the PEG increases, that wettability increases with spots the spacer length. Printed on a spreading and inhomogeneous wettability across the surface. This explains inconsistent spot highly hydrophilic surface are expected to form irregular spots because of significant spreading and droplets on a highly hydrophilic surface are expected to form irregular spots because of significant morphology found on surface with long PEG spacers (n = 12, 24, 36). Consistent spot morphology is inhomogeneous wettability across the surface. This explains inconsistent spot morphology found spreading and inhomogeneous wettability across the surface. This explains inconsistent spot on obtained with contact angle of (n DMSO on isocyanate functionalized larger than 35.with contact morphology found surface with long (n = 12, 24, morphology 36).surface Consistent morphology is surface with long PEGon spacers = 12, 24,PEG 36).spacers Consistent spot isspot obtained ˝ with angle functionalized of DMSO on isocyanate larger than 35. angleobtained of DMSO oncontact isocyanate surfacefunctionalized larger than 35surface .

Figure 4. 4. Contact angles of DMSO on hexyl-isocyanate functionalizedsurface surfacewith with spacers (PEG) n of Figure Contact angles DMSOon onhexyl-isocyanate hexyl-isocyanate functionalized spacers (PEG) n of Figure 4. Contact angles of of DMSO functionalized surface with spacers (PEG) n of varying lengths (n = 1, 2, 6, 12, 24, 36). varying lengths 1, 6, 2, 6, 24,36). 36). varying lengths (n =(n1,= 2, 12,12,24,

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3.3. Dependence of Immobilization Efficiency on the Penultimate Chemical Group to the Isocyanate Residue 3.3. Dependence of Immobilization Efficiency on the Penultimate Chemical Group to the Isocyanate Residue and and Post-Printing Treatment Post-Printing Treatment Figure 5 shows immobilization efficiencies of biotinylated compounds on hexyl-isocyanate Figure 5 shows immobilization efficiencies of biotinylated compounds on hexyl-isocyanate functionalized and phenyl-isocyanate functionalized surfaces, after catalyzation in pyridine vapor at functionalized and phenyl-isocyanate functionalized surfaces, after catalyzation in pyridine vapor room temperature in both cases. The efficiencies are improved for all compounds on a at room temperature in both cases. The efficiencies are improved for all compounds on a phenyl-isocyanate functionalized surface when compared to a hexyl-isocyanate functionalized phenyl-isocyanate functionalized surface when compared to a hexyl-isocyanate functionalized surface. surface. In particular, the immobilization efficiency for compounds with carboxylic acid residues is In particular, the immobilization efficiency for compounds with carboxylic acid residues is improved by improved by a factor of 1.7. Changing the penultimate group from hexyl to phenyl clearly enhances a factor of 1.7. Changing the penultimate group from hexyl to phenyl clearly enhances the attachment the attachment of nucleophiles. The presence of water in printing solutions does not affect of nucleophiles. The presence of water in printing solutions does not affect immobilization efficiency immobilization efficiency on a phenyl-isocyanate surface, as on a hexyl-isocyanate surface. on a phenyl-isocyanate surface, as on a hexyl-isocyanate surface.

Figure 5. 5. Immobilization Immobilization efficiencies efficiencies of of five five biotinylated biotinylated compounds compounds with with different different nucleophilic nucleophilic Figure residues printed on hexyl-isocyanate surface vs. those printed on phenyl-isocyanate surface. The residues printed on hexyl-isocyanate surface vs. those printed on phenyl-isocyanate surface. The compound microarrays were both treated with pyridine vapor catalyzation at room temperature. compound microarrays were both treated with pyridine vapor catalyzation at room temperature.

We We further examined the effects of post-printing treatments of compound compound microarrays microarrays on on immobilization immobilization efficiency. efficiency. In In addition addition to to catalyzation catalyzation in in pyridine pyridine vapor vapor at at room room temperature, temperature, we we explored threeother othertreatment treatment conditions: (1) drying printed microarrays attemperature room temperature explored three conditions: (1) drying printed microarrays at room without ˝ without catalyzation in pyridine vapor; (2) annealing printed microarrays to 45 °C without catalyzation in pyridine vapor; (2) annealing printed microarrays to 45 C without catalyzation in catalyzation in pyridine vapor; (3) of printed microarrays in pyridine vapor at6).45The °C pyridine vapor; (3) catalyzation of catalyzation printed microarrays in pyridine vapor at 45 ˝ C (Figure (Figure 6). The efficiencies immobilization efficiencieswith of compounds ester, hydrazide, and immobilization of compounds nitrophenylwith ester,nitrophenyl hydrazide, and primary hydroxyl primary hydroxyl are improved byroom simply drying at room temperature instead of catalyzation are improved by simply drying at temperature instead of catalyzation in pyridine vapor in at pyridine vapor at room temperature. In addition, annealing the at printed microarrays improves at 45 °C room temperature. In addition, annealing the printed microarrays 45 ˝ C significantly significantly improves the immobilization efficiency for compounds with carboxylic acid residues. the immobilization efficiency for compounds with carboxylic acid residues. On the hexyl-isocyanate ˝ C alone On the hexyl-isocyanate we observed the same thermal trend, i.e., post-printing thermal surface, we observed thesurface, same trend, i.e., post-printing annealing treatment at 45annealing treatment at 45 °C alone yields the highest immobilization efficiency among all four treatment yields the highest immobilization efficiency among all four treatment conditions. conditions. Compared with immobilization on a hexyl-isocyanate functionalized surface followed by Compared with immobilization a hexyl-isocyanate functionalized surface followed are by catalyzation in pyridine vapor at roomon temperature, immobilization efficiencies of compounds catalyzation in pyridineonvapor at room temperature, immobilization efficiencies compounds are improved significantly a phenyl-isocyanate functionalized surface followed by of thermal annealing improved significantly phenyl-isocyanate functionalized by thermal annealing at 45 ˝ C alone, as shownoninaFigure 7, to the following extents: asurface factor offollowed 2 improvement for hydrazide; at 45 °Cofalone, as shownforinprimary Figure hydroxyl, 7, to the making following extents: a factor of 2 improvement for a factor 3 improvement it close to 60%; and a factor of 3 improvement hydrazide; a factor of 3 improvement for primary hydroxyl, making it close to 60%; and a factor of 3 for carboxylic acid, resulting in immobilization efficiency of 25%. Such improved immobilization improvement for carboxylic acid,increase resulting immobilization Such improved efficiencies should dramatically theinfraction of a largeefficiency collectionofof25%. compounds without immobilization efficiencies dramatically increase the fraction of a largesolid collection of special common handles to beshould efficiently immobilized on an isocyanate functionalized surface for compounds without special common handles to be efficiently immobilized on an isocyanate screening assays. functionalized solid surface for screening assays.

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Figure 6. 6.Immobilization biotinylated compounds compoundswith withdifferent differentnucleophilic nucleophilic Figure 6. Immobilization efficiencies of five five biotinylated compounds with different nucleophilic Figure Immobilization efficiencies efficiencies of biotinylated residues printed on a phenyl-isocyanate functionalized surface as functions of post-printing residues printed on aon phenyl-isocyanate functionalized surface as functions of post-printing treatment. residues printed a phenyl-isocyanate functionalized surface as functions of post-printing treatment. treatment.

Figure Immobilization efficiencies efficiencies of of five five biotinylated biotinylated compounds Figure 7. 7.Immobilization compoundswith withdifferent differentnucleophilic nucleophilic Figure 7. Immobilization efficiencies of functionalized five biotinylated compounds with differentinnucleophilic residues printed on a hexyl-isocyanate slide followed by catalyzation pyridine residues printed on a hexyl-isocyanate functionalized slide followed by catalyzation in pyridine vapor residues printed on a hexyl-isocyanate functionalized slide followed by catalyzation in pyridine vapor at room temperature vs. those printed on phenyl-isocyanate functionalized slide followed by at room temperature vs. those printed on phenyl-isocyanate functionalized slide followed by thermal vapor at room temperature vs. those printed on phenyl-isocyanate functionalized slide followed by thermal annealing at 45 °C for 24 h. annealing at 45 ˝ C for 24 h. thermal annealing at 45 °C for 24 h.

3.4. Stability of Phenyl-Isocyanate Functionalized Slides in Storage 3.4. Stability of Phenyl-Isocyanate Functionalized Slides in Storage 3.4. Stability of Phenyl-Isocyanate Functionalized Slides in Storage The stability of a functionalized glass slide is important for producing reproducible binding The stability of a functionalized glass slide is important for producing reproducible binding The stability of a functionalized glassimmediately slide is important for producing assays as one may not use the slide after fabrication. To reproducible explore this binding issue assays as one may we notmeasured use the slide immediatelyefficiencies after fabrication. To explore this issuefabricated experimentally, experimentally, immobilization of compound microarrays assays as one may not use the slide immediately after fabrication. To explore this on issue wephenyl-isocyanate measured immobilization efficiencies compound fabricated on phenyl-isocyanate functionalized slides of that have beenmicroarrays stored for various lengths of time before on experimentally, we measured immobilization efficiencies of compound microarrays fabricated functionalized that haveon been stored for various On lengths timewe before printinga the compounds printing theslides compounds the slide Dayof 0, fabricated number of phenyl-isocyanate functionalized slides thatsurface. have been stored for various lengths of time before onphenyl-isocyanate the slide surface.functionalized On Day 0, weslides. fabricated a number of phenyl-isocyanate functionalized slides. These slides were stored in a −20 C freezer. On the day as printing the compounds on the˝ slide surface. On Day 0, we fabricated a number of These slideson were in a ´20 Ccompounds freezer. On theprinted day ason indicated on the axis, the biotinylated indicated the xstored axis, biotinylated were a in slide (taken outx from freezer phenyl-isocyanate functionalized slides. These slides were stored a −20 C freezer. On the day as compounds were printed on a slideand (taken out from the freezer and thawed to room temperature) and thawed to room temperature) annealed at 45 °C for 24 h. Immobilization efficiencies offreezer the indicated on the x axis, biotinylated compounds were printed on a slide (taken out from the ˝ C for 24 h. Immobilization efficiencies of the printed compounds as functions of and annealed at 45 printed compounds as functions of slide storage at time (Figure S1) that the functionality and thawed to room temperature) and annealed 45 °C for 24 h.shows Immobilization efficienciesof ofathe phenyl-isocyanate functionalized slide decreases graduallyofover time. For example,functionalized the efficiency for slide storage time (Figure S1) shows that the functionality a phenyl-isocyanate printed compounds as functions of slide storage time (Figure S1) shows that the functionalityslide of a

phenyl-isocyanate functionalized slide decreases gradually over time. For example, the efficiency for

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decreases gradually over time. For example, the efficiency for the compounds with carboxylic acid residues drops by 50% on Day 20 from the efficiency on Day 0, and disappears completely on Day 55. 2016, 16, 378 9 of 15 lost On aSensors hexyl-isocyanate functionalized slide, its capability to immobilize carboxylic acids is also after Day 55.

the compounds with carboxylic acid residues drops by 50% on Day 20 from the efficiency on Day 0, and disappears completely on Day 55. On a hexyl-isocyanate functionalized slide, its capability to 3.5. Immobilization of a Large Collection of Bioactive Compounds on Isocyanate Functionalized Glass immobilize carboxylic is also lost after Day 55. Slides—Confirmation of theacids Benefit of the Optimal Procedures for SMM Fabrication

3.5. Immobilization of a Largecompounds Collection of Bioactive Compounds on Isocyanate Functionalized Glass glass slides SMMs of 3375 bioactive are prepared on hexyl-isocyanate functionalized ˝ Slides—Confirmation of the Benefit of the Optimal Procedures for SMM Fabrication and on phenyl-isocyanate functionalized slides followed by thermal annealing at 45 C for 24 h. From

OI-RD images microarrays beforeare washing (Figure S2), 2982 compounds are successfully SMMs of of printed 3375 bioactive compounds prepared on hexyl-isocyanate functionalized glass transferred (not necessarily immobilized via nucleophile-isocyanate to45the slides and on phenyl-isocyanate functionalized slides followed by thermal reaction) annealing at C surface. for 24 h. The From OI-RD images of are printed microarrays to before washingdue (Figure S2),wetting 2982 compounds remaining 393 compounds not transferred the surface, to poor propertiesare of their successfully transferred (not necessarily immobilized via nucleophile-isocyanate reaction)with to the printing solutions on the isocyanate-functionalized surface. After the slides are washed 1ˆ PBS surface.we Thetake remaining 393 compounds are not transferred to the surface, due (Figure to poor S3b) wetting and dried, auto-fluorescence images (Figure S3a) and OI-RD images of these properties of their printing solutions on the isocyanate-functionalized surface. After the slides are slides to identify compounds that are successfully immobilized through the nucleophile-isocyanate washed with 1× PBS and dried, we take auto-fluorescence images (Figure S3a) and OI-RD images reaction. In the auto-fluorescence image, the immobilized compounds that are auto-fluorescent appear (Figure S3b) of these slides to identify compounds that are successfully immobilized through the as bright doublets; those that are immobilized and yet notimage, auto-fluorescent appear as dark that doublets nucleophile-isocyanate reaction. In the auto-fluorescence the immobilized compounds if excess materials from neighboring auto-fluorescent compounds are washed over them during are auto-fluorescent appear as bright doublets; those that are immobilized and yet not the washing step and the unprinted regionifbecomes fluorescent. immobilized compounds auto-fluorescent appear as dark doublets excess materials from Those neighboring auto-fluorescent that compounds are not auto-fluorescent andthem haveduring no excess auto-fluorescent materials washed them can are washed over the washing step and the unprinted regionover becomes fluorescent. are if notthey auto-fluorescent and have no excess be identified as Those brightimmobilized doublets incompounds the OI-RDthat image are immobilized enough for OI-RD auto-fluorescent materialsofwashed overimmobilized them can be compounds identified as on bright doublets in dried the OI-RD detection. For the purpose counting a washed and slide, we image if they are immobilized enough for OI-RD detection. For the purpose of counting immobilized combine the auto-fluorescent image and the properly scaled corresponding OI-RD image of the slide. compounds on a washed and dried slide, we combine the auto-fluorescent image and the properly Figure 8a shows the combined auto-fluorescence/OI-RD image of a printed compound microarray scaled corresponding OI-RD image of the slide. Figure 8a shows the combined on a hexyl-isocyanate functionalized slide. By counting bright and dark doublets in the image, we auto-fluorescence/OI-RD image of a printed compound microarray on a hexyl-isocyanate find functionalized that 1746 compounds, out of 2982 printed, are immobilized with an overall slide. By counting bright and dark doublets in the image, we findimmobilization that 1746 percentage of 59%. Figure 8b shows theancombined auto-fluorescence/OI-RD image of compounds, outIn of comparison, 2982 printed, are immobilized with overall immobilization percentage of 59%. a printed compound microarray on a phenyl-isocyanate functionalized slide. One can immediately In comparison, Figure 8b shows the combined auto-fluorescence/OI-RD image of a printed see that there microarray are many on more bright and dark doublets. slide. We identified 2165 compounds, compound a phenyl-isocyanate functionalized One can immediately see that out thereprinted, are manythat moreare bright and dark doublets. We identified compounds, out of 2982 printed, of 2982 immobilized successfully with a2165 markedly improved immobilization that areof immobilized successfully a markedly image improved percentage of 73%. percentage 73%. Considering thatwith the combined willimmobilization miss those immobilized compounds Considering that the combined image will miss those immobilized compounds (doublets) that (i) are over (doublets) that (i) are not auto-fluorescent, (ii) have no excess auto-fluorescent materials washed not auto-fluorescent, (ii) have no excess auto-fluorescent materials washed over them, and (iii) are them, and (iii) are too small to be detected in our current OI-RD scanning microscope, the actual too small to be detected in our current OI-RD scanning microscope, the actual immobilization immobilization percentage is expected to be higher than 73%. In addition, three compounds with percentage is expected to be higher than 73%. In addition, three compounds with different different nucleophilic functional (i.e., amine, phenol, carboxylic are highlighted in color nucleophilic functional groupsgroups (i.e., amine, phenol, carboxylic acid) acid) are highlighted in color rectangles in Figure 8b and listed in Table 1, indicating effective immobilization of compounds rectangles in Figure 8b and listed in Table 1, indicating effective immobilization of compounds with with nucleophilic groups on on a phenyl-isocyanate surface. nucleophilic groups a phenyl-isocyanate functionalized functionalized surface.

Figure 8. Cont.

Figure 8. Cont.

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2016, 2016, 16, 16, 16, 16, xx

10 10 of of 15 15

Figure 8. Combined auto-fluorescence/OI-RD image compound microarray Figure 8. Combined auto-fluorescence/OI-RD imageofofa aprinted-and-washed printed-and-washed compound microarray Figure 8. Combined auto-fluorescence/OI-RD image of a printed-and-washed compound microarray (a) on a hexyl-isocyanate functionalized slide and (b) on a phenyl-isocyanate functionalized slide.slide. Figure 8. Combined auto-fluorescence/OI-RD image of a printed-and-washed compound microarray (a) on a hexyl-isocyanate functionalized slide and (b) on a phenyl-isocyanate functionalized (a) (a) on on aa hexyl-isocyanate hexyl-isocyanate functionalized functionalized slide slide and and (b) (b) on on aa phenyl-isocyanate phenyl-isocyanate functionalized functionalized slide. slide.

Table 1. Structures of three light spots in Figure 8b highlighted by color rectangles.

Table three in 8b Figure 8b highlighted by color rectangles. Table Structures three light spots in highlighted by Table1.1. 1. Structures Structures of of of three lightlight spots spots in Figure Figure 8b highlighted by color color rectangles. rectangles.

Compound Names Compound Names Compound Names

Compound Structures Rectangular Color Compound Structures Color Color Compound Structures Rectangular Rectangular

NVP-AEW541 NVP-AEW541 NVP-AEW541

WY-14643

Arbidol HCl WY-14643 WY-14643

red red

red

green

blue green green

3.6. Screening Microarrays of~3000 Compounds for Protein Ligands—Further Confirmation of Improved Immobilization Efficiencies in Terms of Identified Ligands To examine whether the improvement in immobilization efficiency of a compound collection Arbidol HCl blue blue Arbidol HCl indeed leads to a corresponding increase in the number of protein ligand discovered on the SMM platform, we use streptavidin as a protein probe and incubate microarrays of 3375 compounds printed on hexyl- and phenyl-isocyanate functionalized slides in the solution of streptavidin at 154 nM for 1 h. Both microarrays are treated with thermal annealing at 45 C for 24 h before the reaction. 3.6. Screening Microarrays of~3000 Compounds for Protein Ligands—Further Confirmation of Improved From changes in OI-RD image (Figure S4), 15for compounds on the hexyl-isocyanate slide reacted with 3.6. Screening Microarrays Compounds Immobilization Efficiencies inof~3000 Terms of Identified Ligands Protein Ligands—Further Confirmation of Improved streptavidin. On the phenyl-isocyanate slide, in addition to these 15 compounds, 7 additional Immobilization Efficiencies in Terms of Identified Ligands To examine thereact improvement in immobilization efficiency compound collection compounds arewhether found to with streptavidin. Based on Figuresof7aand 8, this is expected as more indeed leads to whether a corresponding increase in the number of protein ligandefficiency discovered of on athe SMM To examine the improvement in immobilization compound collection compounds from the collection of 3375 compounds are immobilized on the phenyl-isocyanate slide. platform, we use streptavidin as a protein probe and incubate microarrays of 3375 compounds indeed to a corresponding increase in the number of in protein ligand discovered theofSMM All leads 22 hit compounds (i.e., ligands of streptavidin) are listed Table 2, each having at leaston one printed on hexyl- and phenyl-isocyanate functionalized slides in the solution of streptavidin at 154 nM the following nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine, platform, we use streptavidin as a protein probe and incubate microarrays of 3375 compounds printed for 1 h. Both microarrays are treated with thermal annealing at 45 C for 24 h before the reaction. secondary amine, phenol, and carboxylic acid. Among the sevenofadditional compounds From changes in OI-RD image (Figure S4), 15 compounds on the hexyl-isocyanate slide reactedhit with on hexyland phenyl-isocyanate functionalized slides in the solution streptavidin at 154 nM for 1 h. ˝has discovered on the phenyl-isocyanate slide, a carboxylic for immobilization, Onare thetreated phenyl-isocyanate slide, inGW4064 addition to 15 compounds, 7 additional Bothstreptavidin. microarrays with thermal annealing atonly 45these C for 24 h beforeacid the reaction. From changes compounds are found to react with streptavidin. Based on Figures 7 and 8, this is expected of as small more molecules confirming that the phenyl-isocyanate surface is indeed better for immobilization in OI-RD image (Figure S4), 15 compounds on the hexyl-isocyanate slide reacted with streptavidin. On compounds from the collection of 3375 compounds are immobilized on the phenyl-isocyanate slide. with low isocyanate reactivity.

the phenyl-isocyanate slide, in addition to these 15 compounds, 7 additional compounds are found All 22 hit compounds (i.e., ligands of streptavidin) are listed in Table 2, each having at least one of to react with streptavidin. Based on Figures 7 and 8 this is expected as more compounds from the collection of 3375 compounds are immobilized on the phenyl-isocyanate slide. All 22 hit compounds (i.e., ligands of streptavidin) are listed in Table 2, each having at least one of the following nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine, secondary amine, phenol, and carboxylic acid. Among the seven additional hit compounds discovered on the phenyl-isocyanate slide, GW4064 only has a carboxylic acid for immobilization, confirming that the phenyl-isocyanate surface is indeed better for immobilization of small molecules with low isocyanate reactivity.

2016, 16,that 16, x the phenyl-isocyanate surface is indeed better for immobilization of11 of 15 molecules confirming small 2016, 16, 16, x with low isocyanate reactivity. 2016, 16, 16, 16, 16, xx 11 of of11 15of 15 2016, 11 15 the following nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine,

the 2016,following 16, 16, 16, x nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine, 11 of of 15 15 2016, 16, 11 2016, 16, 16, xx 11 of 15 secondary amine, phenol, and carboxylic acid. Among the seven additional hit compounds secondary amine, phenol, and carboxylic acid. Among the seven additional hit compounds the following nucleophilic residues different isocyanate reactivity: primary amine, amine, the following following nucleophilic residues withwith different isocyanate reactivity: primary amine, arylaryl amine, the nucleophilic residues with different isocyanate reactivity: amine, aryl Table 2. Hiton compounds and surface coverage ofonly SAVD on both aprimary phenyl-isocyanate surface and discovered the phenyl-isocyanate slide, GW4064 has aa carboxylic acid for immobilization, the following following nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine, amine, discovered on the phenyl-isocyanate slide, GW4064 only hasthe carboxylic acid for immobilization, the nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine, secondary amine, phenol, and carboxylic acid. Among the seven additional hit compounds the following nucleophilic residues with different isocyanate reactivity: primary amine, aryl amine, secondary amine, phenol, and carboxylic acid. Among seven additional hit compounds secondary amine, phenol, acid. Among the seven hit confirming the phenyl-isocyanate surface is indeed better for immobilization of molecules secondary that amine, phenol, and and carboxylic carboxylic acid. Among the seven additional additional hit compounds compounds hexyl-isocyanate surface. confirming the phenyl-isocyanate surface isGW4064 indeed better for immobilization of small small molecules secondary amine, phenol, and carboxylic acid. Among the seven additional hit compounds discovered on the phenyl-isocyanate slide, only has a carboxylic for immobilization, secondary amine, phenol, and carboxylic acid. Among the seven additional hit compounds Sensors 2016, 16, 378 that 11 of 15 discovered on the the phenyl-isocyanate slide, GW4064 only has a carboxylic carboxylic acidacid for immobilization, discovered on phenyl-isocyanate slide, GW4064 only has a acid for immobilization, with low isocyanate reactivity. discovered on the the phenyl-isocyanate phenyl-isocyanate slide, GW4064 GW4064 only only has has aa carboxylic carboxylic acid acid for for immobilization, immobilization, with low isocyanate reactivity. discovered on slide, confirming that the phenyl-isocyanate surface is indeed better for immobilization of small molecules discovered on the phenyl-isocyanate slide, GW4064 only has a carboxylic acid for immobilization, confirming that the phenyl-isocyanate surface is indeed better for immobilization of small molecules confirming molecules Coverage confirmingthat thatthe thephenyl-isocyanate phenyl-isocyanatesurface surfaceis is indeed indeedbetter betterfor forimmobilization immobilizationof ofsmall smallSAVD molecules confirming that the phenyl-isocyanate surface is indeed better for immobilization of small molecules with isocyanate reactivity. confirming that the phenyl-isocyanate surface is indeed better for immobilization molecules SAVD Coverage of onsmall with lowlow isocyanate reactivity. with low isocyanate reactivity. Table 2. Hit compounds and surface coverage of SAVD on both a phenyl-isocyanate surface and withTable low isocyanate reactivity. 2.compounds Hit compounds surface coverageofofSAVD SAVD on on both both aa phenyl-isocyanate surface andandon Compound with low isocyanate reactivity. Table 2. Hit andand surface coverage phenyl-isocyanate surface with low isocyanate reactivity. Compound Structures Phenyl-Isocyanate hexyl-isocyanate surface. hexyl-isocyanate surface. Table 2.surface. Hit compounds surface coverage of SAVD on both a phenyl-isocyanate surface Names Hexyl-Isocyanate hexyl-isocyanate Table 2. Hit Hit compounds and and surface coverage of SAVD SAVD on both both phenyl-isocyanate surface and and Table 2. compounds and surface coverage of on aa phenyl-isocyanate surface and Table 2. 2. Hit Hit compounds compounds and and surface surface coverage coverage of of SAVD SAVD on on both both a phenyl-isocyanate phenyl-isocyanate surface and and Surface SAVD Table surface hexyl-isocyanate surface. Table 2. Hit compounds and surface coverage of SAVD on both aa phenyl-isocyanate surface andSurface Coverage hexyl-isocyanate surface. SAVD Coverage hexyl-isocyanate surface. SAVD Coverage on hexyl-isocyanate surface. surface. SAVD Coverage on hexyl-isocyanate hexyl-isocyanate surface. Compound on SAVD Coverage on SAVD Coverage on Compound on SAVD Coverage Compound Structures Phenyl-Isocyanate SAVD Coverage SAVD Coverage Compound Structures Phenyl-Isocyanate SAVD Coverage on Hexyl-Isocyanate Compound Names Compound Structures Phenyl-Isocyanate Names Hexyl-Isocyanate SAVD Coverage on SAVD Coverage SAVD Coverage Names SAVD on Coverage Compound on SAVD Coverage Surface Compound SAVD Coverage on on Hexyl-Isocyanate Compound on Surface SAVD Coverage on Surface Surface Compound Structures Phenyl-Isocyanate SAVD Coverage on Surface Compound Structures Phenyl-Isocyanate Compound on Compound Structures Phenyl-Isocyanate Surface Compound on Names Hexyl-Isocyanate Compound on Names Hexyl-Isocyanate Compound Structures Structures Phenyl-Isocyanate Hexyl-Isocyanate Names Compound Phenyl-Isocyanate Surface Compound Structures Phenyl-Isocyanate Surface Names Hexyl-Isocyanate BMS 777607 0.85 Hexyl-Isocyanate 0.54 Surface Names Hexyl-Isocyanate Surface Names Surface Surface Surface Surface Surface Surface Surface Surface BMS 0.85 0.54 BMS 777607 777607 0.85 BMS 777607 0.85 0.540.54 777607 BMSBMS 777607 BMS BMS 777607 777607 BMS 777607 BMS 777607

0.850.85 0.85 0.85 0.85 0.85

JNJ-7706621

0.85

Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Biotin-NH2 Azelnidipine Azelnidipine

Azelnidipine Azelnidipine

Azelnidipine Azelnidipine Azelnidipine Azelnidipine Azelnidipine Azelnidipine Amfenac Amfenac sodium sodium Amfenac Amfenac Amfenac Amfenac monohydrate Amfenac Amfenac sodium monohydrate Amfenac sodium Amfenac sodium sodium sodium monohydrate sodium sodium monohydrate sodium monohydrate monohydrate monohydrate monohydrate monohydrate monohydrate Dryocrassin Dryocrassin

222

H2 N H H222N N

O

0.850.85 0.85 0.85 0.85 0.85

0.280.28 0.28 0.28 0.28 0.28

0.73 0.73 0.73

0.73

0.61 0.610.61

0.730.73 0.73 0.73 0.73 0.73

0.610.61 0.61 0.61 0.61 0.61

0.64 0.64 0.64 0.64 0.640.64 0.64 0.64 0.64 0.64

0.28 0.28 0.28 0.280.28 0.28 0.28 0.28 0.28

0.64 0.64 0.640.64 0.640.64 0.64 0.64 0.64 0.64 0.61 0.61

0.640.64 0.64 0.64 0.64 0.64 0.64

0.450.45 0.45 0.45 0.45 0.45 0.45 0.39 0.39

Magnolol Magnolol Magnolol Magnolol Magnolol Magnolol Magnolol Magnolol LDN193189 LDN193189

0.580.58 0.580.58 0.58 0.58 0.58 0.52 0.52 0.58

0.390.39 0.39 0.390.39 0.39 0.39 0.36 0.36

0.520.52 0.520.52 0.52 0.52 0.52

0.360.36 0.36 0.360.36 0.36 0.36

Manidipine Manidipine Manidipine Manidipine Manidipine Manidipine Manidipine Tyrphostin Tyrphostin AG AG 879 879 Tyrphostin Tyrphostin AG AG Manidipine Tyrphostin Tyrphostin AG AG Tyrphostin AG Tyrphostin AG 879 879 879 Tyrphostin AG 879 879 879 2016, 16, 16,879 x

0.51 0.51

0.52

0.510.51 0.51 0.51 0.51 0.510.51 0.48 0.48

0.51 0.48

0.48 0.48 0.48 0.48 0.480.48

0.45 0.45

NVP-BSK805 NVP-BSK805 NVP-BSK805

PF-04929113 PF-04929113

Flupirtine

0.64

0.45

0.39

0.36

0.450.45 0.45 0.450.45 0.45 0.45 0.48 0.48 0.480.48 0.48 0.48 0.48 0.48 0.48

0.45 12 of of 15 15 12

2016, 16, 16, x

Tyrphostin AG Honokiol Honokiol 879Honokiol

0.28

0.45 0.45

0.610.61 0.610.61 0.61 0.61 0.61 0.61 0.58 0.58

LDN193189 Manidipine Manidipine

0.61

0.64 0.64

Dryocrassin Dryocrassin Dryocrassin Dryocrassin Dryocrassin Dryocrassin Dryocrassin Dryocrassin Magnolol Magnolol

LDN193189 LDN193189 LDN193189 LDN193189 LDN193189 LDN193189

0.28

0.28 0.280.28

2

O O H2N N H2 N H 2HN HHN N 2N HN OO H H22N O O O O O O HNO HN HN O O NH HN O NH NH HN O HN O O S OS S O O NH NH NH NH NH NH O S O NH O NH NHSS O NH NH S O O SS NH NH NH

222 22

JNJ-7706621 JNJ-7706621 JNJ-7706621 JNJ-7706621 JNJ-7706621 JNJ-7706621

0.85 0.85 0.85 2

JNJ-7706621 JNJ-7706621 JNJ-7706621

0.540.54 0.54 0.54 0.54 0.54

0.45 0.45 0.45

0.39 0.39 0.39

0.33 0.33

0.48

0.360.36 0.36

0.330.33 0.33

0.27 0.27

0.48

2016, 16, 16, 2016, 16, 16, 2016, 16, 16, 2016, 16, 16, xxxx 2016, 16, 16, 2016, 16, 16, 16, 16, xx x 2016,

12 of 15 12 of 15 12 of 15 12 of 15 12 of 15 12 of of 15 15 12

2016,Honokiol 16, 16, x Honokiol

0.45 0.45 0.45 0.45 0.45 0.45

Honokiol Honokiol Honokiol Honokiol

Honokiol Sensors 2016, 16, 378

Honokiol

NVP-BSK805 NVP-BSK805 NVP-BSK805 NVP-BSK805 NVP-BSK805 NVP-BSK805 NVP-BSK805 Compound Names

PF-04929113 PF-04929113 PF-04929113 PF-04929113 PF-04929113 PF-04929113

PF-04929113 PF-04929113

0.45 Table 2. Cont.

Compound Structures

0.36 0.36 0.3612 of 15 0.36 0.36 0.36 12 of 15 0.36

0.39 0.39 0.39 0.39 0.39 0.39

SAVD Coverage 0.39 on Phenyl-Isocyanate Surface

0.33 0.33 0.33 0.33 0.33 0.33

0.33 0.33 0.33 0.33 0.33 0.33 SAVD 0.33Coverage on Hexyl-Isocyanate Surface

0.27 0.27 0.27 0.27 0.27 0.27

0.33 0.33

0.270.27

Flupirtine Flupirtine Flupirtine Flupirtine Flupirtine Flupirtine maleate maleate maleate maleate Flupirtine maleate Flupirtine maleatemaleate

0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30

0.27 0.27 0.27 0.27 0.27 0.27 0.270.27

Linifanib Linifanib Linifanib Linifanib Linifanib Linifanib Linifanib (ABT-869) (ABT-869) (ABT-869) (ABT-869) Linifanib (ABT-869) (ABT-869) (ABT-869) (ABT-869)

0.27 0.27 0.270.27

maleate

0.27 0.27 0.27 0.27

Sciadopitysin Sciadopitysin Sciadopitysin Sciadopitysin Sciadopitysin Sciadopitysin Sciadopitysin Sciadopitysin

0.36 0.36 0.36 0.36 0.36 0.360.36

00

ABT-263 ABT-263 ABT-263 ABT-263 ABT-263 ABT-263 (Navitoclax) ABT-263 ABT-263 (Navitoclax) (Navitoclax) (Navitoclax) (Navitoclax) (Navitoclax) (Navitoclax) (Navitoclax)

0.33 0.33 0.33 0.33 0.330.33 0.33

0

0.29 0.29 0.29 0.29 0.290.29 0.29

0

0.27

0

GW4064

GW4064 GW4064 GW4064 GW4064 GW4064 GW4064 GW4064

MLN8054

MLN8054 MLN8054 MLN8054 MLN8054 MLN8054 MLN8054 MLN8054 Flunarizine dihydrochlor

Flunarizine Flunarizine Flunarizine Flunarizine Flunarizine Flunarizine Flunarizine dihydrochlor dihydrochlor dihydrochlor dihydrochlor dihydrochlor dihydrochlor dihydrochlor Nomilin

Nomilin Nomilin Nomilin Nomilin Nomilin Nomilin Nomilin Liothyronine sodium

Liothyronine Liothyronine sodium sodium sodium sodium sodium sodium

Liothyronine Liothyronine Liothyronine Liothyronine Liothyronine sodium

0.36

0.18 0.18 0.18 0.18

0.18 0.180.18 0.18

0.33

0.29 0.29

0.27 0.27

0.27 0.27 0.270.27 0.27 0.26

0.26 0.26

0.25 0.25

0.18 0.180.18 0.18 0.18 0.18 0.18

00000 0

0

00000 0

0

00000 0

0

00000 0

0

00000 0

0

0.25 0.25 0.25 0.250.25 0.25 0.18

0

0

0.26 0.26 0.260.26 0.26 0.25

00000 0

0

0

00000 0

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4. Discussion We investigated several surface chemistry parameters in fabrication of SMMs on isocyanate functionalized glass slides for optimal immobilization efficiency, spot morphology and reproducibility. We find that both the penultimate group next to the isocyanate reside and post-printing treatment have significant impacts on immobilization efficiencies of small molecules, particularly those having residues with inherently low reactivity with isocyanate. Both presumably facilitate covalent attachment of nucleophilic groups to the isocyanate residues on the functionalized surface. In particular, we find that a phenyl-isocyanate functionalized surface is more efficient for small molecule immobilization than a hexyl-isocyanate functionalized surface by almost a factor of two. We further find that thermal annealing of printed small molecule microarrays at 45 ˝ C yields higher immobilization efficiency by almost another factor of two than the method of catalyzation in pyridine vapor at room temperature [25,27]. As a result, a combination of phenyl-isocyanate functionalized surface and annealing fabricated microarrays at 45 ˝ C dramatically enhances immobilization efficiency, particularly for compounds with nucleophilic residues such as hydrazide, primary hydroxyl, and carboxylic acid. The overall improvement of immobilization efficiency and spot morphology are confirmed with SMMs of 3375 bioactive compounds printed on both phenyl-isocyanate and hexyl-isocyanate surfaces. In addition we find that a (PEG)n spacer with n = 6 is optimal in yielding high-quality SMM spot morphology while minimizing non-specific protein binding to the surface. Furthermore, we find that isocyanate functionalized surfaces degrade after storage in ´20 ˝ C freezer for a period of time. Ambient moisture reacts with surface isocyanate residue slowly and causes the surface density of the isocyanate residues to diminish in time. It is therefore important to prepare isocyanate functionalized glass slides and use them immediately. On the other hand, water present in the printing solution has little effect on the immobilization efficiency of small molecules, presumably due to rapid evaporation of deposited solution droplets. Insensitivity of immobilization efficiency to water in a printing solution is a pre-requisite for fabrication of SMMs, as ambient moisture is easily absorbed into DMSO during long printing runs. In conclusion, we find that in combination with post-printing thermal annealing treatment at 45 ˝ C, a phenyl-isocyanate functionalized glass surface with (PEG)6 as the spacer between the phenyl-isocyanate residues and the solid surface is optimal for fabrication of SMMs. The high immobilization percentage (over 73%) and spot morphology of SMMs on such surfaces should improve the quality and the range of SMM applications in high-throughput drug lead discovery. Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1424-8220/16/ 3/378/s1. Acknowledgments: This work is supported by National Natural Science Foundation of China (Grant No. 61505032 and No. 11574056), Shanghai Pujiang Program (Grant No. 13PJ1400300), Natural Science Foundation of Jiangsu Higher Education Institutions of China (Grant No. BK20130116) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education of the People's Republic of China. We would like to thank Professor Shengce Tao from Shanghai Jiao Tong University and Professor Qing Mu from Fudan University for their assistance in this work. Author Contributions: Chenggang Zhu carried out the experiments. Xiangdong Zhu and James Landry provided data acquisition and analysis tools. Xiangdong Zhu also contributed to revision and editing of the manuscript. Zhaomeng Cui, Quanfu Li and Yongjun Dang contributed compound library. Fengyun Zheng contributed printing tools. Yiyan Fei and Lan Mi wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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