•Combining microfluidics and photochemistry solves the TBL multiplexing challenge•Organic surface kinetics derived in a single experiment•Ability to explore interfacial organic chemistry reactivity•Synergistic combination of surface chemistry and instrumentation There is a pressing desire to reduce feature diameters in biological arrays, which would increase the number of probes on the surface and reduce the sample volume required for analysis. The challenge that precludes miniaturization in biochips is that most nanolithography methods require high-energy beams that would denature or destroy soft matter. Alternatively, tip-based lithography (TBL) uses arrays of nanoscopic tips to pattern reagents on a surface nondestructively, but no versatile multiplexing strategy has been developed. Here, we solve this long-standing problem in TBL by combining massively parallel beam pen arrays, microfluidics, and organic surface chemistry to create multiplexed arrays whose features have a diameter of ∼1 μm. The applications of this printing platform include the preparation of gene, protein, and glycan chips that can rapidly advance “omics” research as well as provide a new tool for probing interfacial reactivity. Multiplexed microarrays—where different biological probes are spatially encoded onto a surface into spots with micrometer-scale diameters—have facilitated the rapid advancement of “omics” research. Further miniaturization of feature diameters could increase the number of probes in a microarray, reduce the sample required for analysis, and decrease costs. Tip-based lithography (TBL) has gained popularity for patterning delicate, biologically active materials, but no versatile TBL-based multiplexing strategy has been devised. Here, we combine microfluidics, beam pen lithography, and photochemical surface reactions to create multiplexed arrays. For proof of concept, the thiol-ene reaction was optimized, and the reaction kinetics were analyzed. Subsequently, we created several patterns containing multiple fluorescent alkenes, where each pattern was designed to demonstrate a different capability of this instrument. This patterning strategy is a powerful approach to studying and optimizing organic reactions on surfaces and creating massively multiplexed arrays and, as such, could provide an entirely new approach for miniaturizing biochips or understanding interfacial reactivity. Multiplexed microarrays—where different biological probes are spatially encoded onto a surface into spots with micrometer-scale diameters—have facilitated the rapid advancement of “omics” research. Further miniaturization of feature diameters could increase the number of probes in a microarray, reduce the sample required for analysis, and decrease costs. Tip-based lithography (TBL) has gained popularity for patterning delicate, biologically active materials, but no versatile TBL-based multiplexing strategy has been devised. Here, we combine microfluidics, beam pen lithography, and photochemical surface reactions to create multiplexed arrays. For proof of concept, the thiol-ene reaction was optimized, and the reaction kinetics were analyzed. Subsequently, we created several patterns containing multiple fluorescent alkenes, where each pattern was designed to demonstrate a different capability of this instrument. This patterning strategy is a powerful approach to studying and optimizing organic reactions on surfaces and creating massively multiplexed arrays and, as such, could provide an entirely new approach for miniaturizing biochips or understanding interfacial reactivity. Massively parallel tip-based lithography (TBL) uses elastomeric arrays possessing up to 107 nanoscale pyramidal tips mounted onto the piezoactuators of an atomic force microscope (AFM) and is emerging as one of the most promising strategies for patterning delicate organic and biologically active materials with sub-1 μm feature diameters.1Huo F. Zheng Z. Zheng G. Giam L.R. Zhang H. Mirkin C.A. Polymer pen lithography.Science. 2008; 321: 1658-1660Crossref PubMed Scopus (442) Google Scholar, 2Salaita K. Wang Y. Mirkin C.A. Applications of dip-pen nanolithography.Nat. Nanotechnol. 2007; 2: 145-155Crossref PubMed Scopus (747) Google Scholar, 3Garcia R. Knoll A.W. Riedo E. Advanced scanning probe lithography.Nat. Nanotechnol. 2014; 9: 577-587Crossref PubMed Scopus (468) Google Scholar, 4Giam L.R. Mirkin C.A. Cantilever-free scanning probe molecular printing.Angew. Chem. Int. Ed. 2011; 50: 7482-7485Crossref PubMed Scopus (45) Google Scholar The primary benefits of TBL are that it obtains nanoscale dimensions while eschewing denaturing or destructive radiation and that it can print arbitrary patterns over large (>cm2) areas5Braunschweig A.B. Huo F. Mirkin C.A. Molecular printing.Nat. Chem. 2009; 1: 353-358Crossref PubMed Scopus (151) Google Scholar while circumventing expensive mask fabrication for each new pattern. In beam-pen lithography (BPL), the pyramids are coated with Au, and an aperture is etched into the apex, enabling mask-free photolithography.6Huo F. Zheng G. Liao X. Giam L.R. Chai J. Chen X. Shim W. Mirkin C.A. Beam pen lithography.Nat. Nanotechnol. 2010; 5: 637-640Crossref PubMed Scopus (153) Google Scholar When BPL is combined with a digital micromirror device, each tip in the array creates an independent pattern, so that truly arbitrary patterns can be printed over cm2 areas while nanoscale feature diameters are maintained.7Liao X. Brown K.A. Schmucker A.L. Liu G. He S. Shim W. Mirkin C.A. Desktop nanofabrication with massively multiplexed beam pen lithography.Nat. Commun. 2013; 4: 2103Crossref PubMed Scopus (82) Google Scholar Because of its versatility, TBL is being used for investigating diverse scientific challenges, including understanding stem cell differentiation,8Cabezas M.D. Eichelsdoerfer D.J. Brown K.A. Mrksich M. Mirkin C.A. Combinatorial screening of mesenchymal stem cell adhesion and differentiation using polymer pen lithography.Methods Cell Biol. 2014; 119: 261-276Crossref PubMed Scopus (12) Google Scholar studying the movement of lipids,9Brinkmann F. Hirtz M. Greiner A.M. Weschenfelder M. Waterkotte B. Bastmeyer M. Fuchs H. Interdigitated multicolored bioink micropatterns by multiplexed polymer pen lithography.Small. 2013; 9: 3266-3275Crossref PubMed Google Scholar patterning brush polymers,10Xie Z. Chen C. Zhou X. Gao T. Liu D. Miao Q. Zheng Z. Massively parallel patterning of complex 2D and 3D functional polymer brushes by polymer pen lithography.ACS Appl. Mater. Interfaces. 2014; 6: 11955-11964Crossref PubMed Scopus (53) Google Scholar and constructing new photovoltaic architectures.11Aizawa M. Buriak J.M. Block copolymer templated chemistry for the formation of metallic nanoparticle arrays on semiconductor surfaces.Chem. Mater. 2007; 19: 5090-5101Crossref Scopus (183) Google Scholar Our group has focused on using massively parallel TBL to study organic surface chemistry.12Liu X. Carbonell C. Braunschweig A.B. Towards scanning probe lithography-based 4D nanoprinting by advancing surface chemistry, nanopatterning strategies, and characterization protocols.Chem. Soc. Rev. 2016; 45: 6289-6310Crossref PubMed Google Scholar, 13Carbonell C. Braunschweig A.B. Toward 4D nanoprinting with tip-induced organic surface reactions.Acc. Chem. Res. 2017; 50: 190-198Crossref PubMed Scopus (27) Google Scholar These efforts have resulted in new methods of preparing glycan arrays,14Bian S. He J. Schesing K.B. Braunschweig A.B. Polymer pen lithography (PPL)-induced site-specific click chemistry for the formation of functional glycan arrays.Small. 2012; 8: 2000-2005Crossref PubMed Scopus (44) Google Scholar, 15Bian S. Zieba S.B. Morris W. Han X. Richter D.C. Brown K.A. Mirkin C.A. Braunschweig A.B. Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays.Chem. Sci. 2014; 5: 2023-2030Crossref Scopus (59) Google Scholar creating grafted-from brush polymer patterns,15Bian S. Zieba S.B. Morris W. Han X. Richter D.C. Brown K.A. Mirkin C.A. Braunschweig A.B. Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays.Chem. Sci. 2014; 5: 2023-2030Crossref Scopus (59) Google Scholar functionalizing the basal plane of graphene,16Bian S. Scott A.M. Cao Y. Liang Y. Osuna S. Houk K.N. Braunschweig A.B. Covalently patterned graphene surfaces by a force-accelerated Diels-Alder reaction.J. Am. Chem. Soc. 2013; 135: 9240-9243Crossref PubMed Scopus (108) Google Scholar and deriving quantitative models of how force accelerates surface reactions.17Han X. Bian S. Liang Y. Houk K.N. Braunschweig A.B. Reactions in elastomeric nanoreactors reveal the role of force on the kinetics of the Huisgen reaction on surfaces.J. Am. Chem. Soc. 2014; 136: 10553-10556Crossref PubMed Scopus (30) Google Scholar An unaddressed challenge that continues to preclude the wider adoption of TBL is the difficulty involved in creating multiplexed patterns, where different inks are printed in close proximity with ∼1-μm-scale spot diameters. Multiplexed patterns are critical for many applications, particularly in “omics” research18Barbulovic-Nad I. Lucente M. Sun Y. Zhang M.J. Wheeler A.R. Bussmann M. Bio-microarray fabrication techniques - a review.Crit. Rev. Biotechnol. 2006; 26: 237-259Crossref PubMed Scopus (304) Google Scholar, 19Park S. Gildersleeve J.C. Blixt O. Shin I. Carbohydrate microarrays.Chem. Soc. Rev. 2013; 42: 4310-4326Crossref PubMed Scopus (210) Google Scholar where they are used in gene, protein, or glycan chips to study biological recognition in parallel and under identical experimental conditions. These substrates are most commonly prepared by pin printing, which rarely produces feature diameters below 75 μm.18Barbulovic-Nad I. Lucente M. Sun Y. Zhang M.J. Wheeler A.R. Bussmann M. Bio-microarray fabrication techniques - a review.Crit. Rev. Biotechnol. 2006; 26: 237-259Crossref PubMed Scopus (304) Google Scholar, 20Heller M.J. DNA microarray technology: devices, systems, and applications.Annu. Rev. Biomed. Eng. 2002; 4: 129-153Crossref PubMed Scopus (932) Google Scholar There is a compelling desire to continue miniaturization in biochips21Romanov V. Davidoff S.N. Miles A.R. Grainger D.W. Gale B.K. Brooks B.D. A critical comparison of protein microarray fabrication technologies.Analyst. 2014; 139: 1303-1326Crossref PubMed Google Scholar because reducing feature diameters would decrease the quantity of material needed for printing and analysis, and the increased number of features that fit in a given area could increase throughput substantially. Strategies for creating multiplexed arrays with TBL have been developed, but each has strict limitations on the patterns that can be printed, and as such, no flexible TBL-based multiplexing approach has yet been developed. For example, TBL multiplexing was accomplished by using inkwells to load different inks onto a polymer pen array,22Zheng Z. Daniel W.L. Giam L.R. Huo F. Senesi A.J. Zheng G. Mirkin C.A. Multiplexed protein arrays enabled by polymer pen lithography: addressing the inking challenge.Angew. Chem. Int. Ed. 2009; 48: 7626-7629Crossref PubMed Scopus (101) Google Scholar, 23Arrabito G. Schroeder H. Schröder K. Filips C. Marggraf U. Dopp C. Venkatachalapathy M. Dehmelt L. Bastiaens P.I.H. Neyer A. et al.Configurable low-cost plotter device for fabrication of multi-color sub-cellular scale microarrays.Small. 2014; 10: 2870-2876Crossref PubMed Scopus (33) Google Scholar but the drawback was that the pattern created by any pen contained only a single ink. Other strategies are based on depositing inks onto pads to dip the arrays9Brinkmann F. Hirtz M. Greiner A.M. Weschenfelder M. Waterkotte B. Bastmeyer M. Fuchs H. Interdigitated multicolored bioink micropatterns by multiplexed polymer pen lithography.Small. 2013; 9: 3266-3275Crossref PubMed Google Scholar or depositing directly onto the arrays via pipetting6Huo F. Zheng G. Liao X. Giam L.R. Chai J. Chen X. Shim W. Mirkin C.A. Beam pen lithography.Nat. Nanotechnol. 2010; 5: 637-640Crossref PubMed Scopus (153) Google Scholar and spin coating of the inks,24Kumar R. Weigel S. Meyer R. Niemeyer C.M. Fuchs H. Hirtz M. Multi-color polymer pen lithography for oligonucleotide arrays.Chem. Commun. 2016; 52: 12310-12313Crossref PubMed Google Scholar where different inks are loaded in different regions of the pen array, up to a maximum of nine inks. These approaches, however, place strict limitations on the patterns that can be printed, and the majority of the surface is sacrificial. Ideally, a multiplexing strategy should allow an arbitrary number of different inks to be patterned on a surface and have no restrictions on how the different inks are arranged. One aspect of this inability to multiplex with TBL, and more generally reduce feature diameters in biological arrays, is the need to develop and optimize surface reactions that are compatible with both the biological probe and the printing protocol. The current state of TBL-based multiplexing calls for an entirely new approach that considers how instrumentation and surface chemistry can be designed synergistically to pattern surfaces with multiple inks into arbitrary patterns and with ∼1 μm feature diameters. Recently, we reported an attempt to address the TBL multiplexing challenge by combining a fluid cell, transparent polymer pen arrays, and photochemical organic surface reactions.25Liu X. Zheng Y. Peurifoy S.R. Kothari E.A. Braunschweig A.B. Optimization of 4D polymer printing within a massively parallel flow-through photochemical microreactor.Polymer Chem. 2016; 7: 3229-3235Crossref Google Scholar Polymer pen arrays were embedded within a microfluidic cell, and fluorescent inks were introduced below the pen arrays in solution. Light was channeled through the arrays to locally induce a photochemical reaction between thiol groups on the surface and dye-functionalized methacrylate inks, resulting in grafted-from fluorescent brush polymers. This approach—using transient light rather than direct material deposition by the tips to immobilize molecules into a spot—provides a straightforward route to switching the molecules that are immobilized by simply introducing new solutions into the cell after each spot is printed. In addition, because reactions are carried out in solution, rather than in a polymer matrix,26Huang L. Braunschweig A.B. Shim W. Qin L. Lim J.K. Hurst S.J. Huo F. Xue C. Jang J.-W. Mirkin C.A. Matrix-assisted dip-pen nanolithography (MA-DPN) and polymer pen lithography (MA-PPL).Small. 2010; 6: 1077-1081Crossref PubMed Scopus (71) Google Scholar the kinetics and environment of the reaction are more familiar and easier to optimize. Using this fluid cell, we successfully created a pattern, where each tip printed two spots of methacrylate brush polymers that were side-chain functionalized with different dyes. Although an important proof of concept, we could not print more complex patterns because the fluid cells were cumbersome and unreliable, the background signal was too high because of light leaking through the transparent polymer pen arrays, and the inability to efficiently change the inks could not be overcome. As a result, this previous work failed to address the multiplexing challenge but provided a starting point from which we build in the work described herein. In the current approach, we report three important innovations that together enable the versatile printing of multiplexed patterns by TBL. Specifically, these are that (1) polymer pen arrays are replaced with beam pen arrays, which focuses light and significantly reduces the background signal; (2) the fluid cell is removed, and instead reactions occur within a droplet pulled below the array by capillary forces; and (3) reactant solutions are mixed in a microfluidic cell upstream of the droplet, enabling the efficient switching of inks. Here, we describe the instrument design, demonstrate the optimization of the thiol-ene photochemical click reaction27Hoyle C.E. Bowman C.N. Thiol-ene click chemistry.Angew. Chem. Int. Ed. 2010; 49: 1540-1573Crossref PubMed Scopus (2979) Google Scholar within the droplet, and use this strategy to print a series of multiplexed patterns. The notable components that enable the instrument’s multiplexed lithography are the microfluidic system, the optics (including the beam pen array), and the motorized stage. A scheme of the printer is provided in Figure 1 and in Figures S16 and S17. The microfluidic system was composed of three syringe pumps that control the flow rates of different ink solutions into a polydimethylsiloxane microfluidic mixer. This mixer incorporates chaotic ridges28Stroock A.D. Dertinger S.K.W. Ajdari A. Mezić I. Stone H.A. Whitesides G.M. Chaotic mixer for microchannels.Science. 2002; 295: 647-651Crossref PubMed Scopus (2844) Google Scholar that efficiently homogenize the solutions before entering 1.59 mm (1/16 inch) polyether ether ketone (PEEK) tubing, with the exit placed adjacent to the beam pen array (Figures S13 and S14). We drew solution between the beam pen array and the reactive surface via capillary forces—circumventing the need for a cumbersome fluid cell—and withdrew it via PEEK tubing on the opposite side of the tip array by applying negative pressure with another syringe pump. For uniform introduction and removal of inks from the reaction droplet, the withdrawal rate was always greater than the flow rate. To induce the photochemical surface reactions, we passed light to the surface through the apertures in the beam pen arrays. The light source was a 1,400 mW/cm2 365 nm collimated LED. A mirror directs the light through a GRIT UVFS ground-glass diffuser and onto the back of the tip array. The diffuser was necessary to ensure that light spread uniformly across the back of the tip array. To control the movement of surface with nanometer precision, we placed it on the piezoactuated translation stage of a Park XE-150 AFM that was also equipped with an x,y tilting stage to level the surface with respect to the tips. The 100 × 100 tip arrays were fabricated according to previously reported protocols6Huo F. Zheng G. Liao X. Giam L.R. Chai J. Chen X. Shim W. Mirkin C.A. Beam pen lithography.Nat. Nanotechnol. 2010; 5: 637-640Crossref PubMed Scopus (153) Google Scholar, 29Eichelsdoerfer D.J. Liao X. Cabezas M.D. Morris W. Radha B. Brown K.A. Giam L.R. Braunschweig A.B. Mirkin C.A. Large-area molecular patterning with polymer pen lithography.Nat. Protoc. 2013; 8: 2548-2560Crossref PubMed Scopus (77) Google Scholar with an 80 μm tip-to-tip pitch, and apertures varied from 200 nm to 10 μm (Figures S11 and S12). The beam pen arrays were mounted onto the z-piezoactuator of the Park PPL head, which controls the z height of the tips over the substrate. Two microcameras optically monitored the leveling between the tip array and the x and y axes of the reactive surface. The printer is designed to immobilize inks in the droplet onto the surface via photochemical organic surface reactions. The thiol-ene photochemical click reaction (Figure 2A) between alkene-functionalized dyes in the droplet and thiol groups on the glass slides was selected for this proof-of-concept study because it is believed to proceed rapidly and in high yield,27Hoyle C.E. Bowman C.N. Thiol-ene click chemistry.Angew. Chem. Int. Ed. 2010; 49: 1540-1573Crossref PubMed Scopus (2979) Google Scholar, 30Jonkheijm P. Weinrich D. Köhn M. Engelkamp H. Christianen P.C.M. Kuhlmann J. Maan J.C. Nüsse D. Schroeder H. Wacker R. et al.Photochemical surface patterning by the thiol-ene reaction.Angew. Chem. Int. Ed. 2008; 47: 4421-4424Crossref PubMed Scopus (171) Google Scholar, 31Wendeln C. Rinnen S. Schulz C. Arlinghaus H.F. Ravoo B.J. Photochemical microcontact printing by thiol-ene and thiol-yne click chemistry.Langmuir. 2010; 26: 15966-15971Crossref PubMed Google Scholar and previously our group15Bian S. Zieba S.B. Morris W. Han X. Richter D.C. Brown K.A. Mirkin C.A. Braunschweig A.B. Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays.Chem. Sci. 2014; 5: 2023-2030Crossref Scopus (59) Google Scholar and others32Zhou Y. Xie Z. Brown K.A. Park D.J. Zhou X. Chen P.-C. Hirtz M. Lin Q.-Y. Dravid V.P. Schatz G.C. et al.Apertureless cantilever-free pen arrays for scanning photochemical printing.Small. 2015; 11: 913-918Crossref PubMed Scopus (38) Google Scholar have used it in the context of TBL. To study the thiol-ene reaction within this printing tool, we prepared thiol-terminated glass slides according to previously published protocols from our group (Scheme S2),15Bian S. Zieba S.B. Morris W. Han X. Richter D.C. Brown K.A. Mirkin C.A. Braunschweig A.B. Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays.Chem. Sci. 2014; 5: 2023-2030Crossref Scopus (59) Google Scholar and alkene fluorophores 1, 2, and 3, which were selected for their distinct absorption and emission profiles, were prepared via organic synthesis (Figure 2B, Scheme S1). The molecules were all characterized by 1H NMR, 13C NMR, high-resolution mass spectrometry, and UV-visible (UV-vis) and fluorescence spectroscopies (Figures S1–S10). All data were consistent with the proposed molecular structures. To understand how different printing parameters affect the reaction within the droplet, we printed 2 onto thiol-terminated glass slides under a variety of different conditions. The variables to be considered for each print are light intensity (I), reaction time (t), dye concentration [2], photoinitiator (2,2-dimethoxy-2-phenylacetophenone [DMPA]) concentration [DMPA], tip array height above the surface (z height), and average diameter of the apertures for the beam pen tip arrays. A major benefit of this approach is that the immobilization chemistry can be optimized rapidly because each spot in a pattern can be printed under different conditions. As a demonstration, we printed a 5 × 5 dot pattern of 2 with each tip by using a beam pen array with an aperture diameter of 2.26 ± 1.03 μm, where each spot was printed with different I and t. Specifically, a solution containing 30 mM 2 and 30 mM DMPA was prepared in DMF:DMSO 75:25 v/v. The syringe pump allowed the solution to enter into the reaction droplet at a flow rate of 5 μL/min. The 5 × 5 array was printed under continuous solution flow (5 μL/min) with I = 20%, 40%, 60%, 80%, and 100% of the 1,400 mW/cm2 and t = 1, 2, 5, 10, and 20 min with a reference dot (t = 10 min, I = 100%) for identifying the origin of the pattern (Figure 3). After the print, the slide was rinsed with EtOH (10 mL) and sonicated in DMF (5 min) for the removal of any physisorbed dye, such that only covalently bound dye remained on the surfaces. The pattern was analyzed with fluorescence microscopy, and the fluorescence intensity and diameter of each spot was determined with ImageJ33Schneider C.A. Rasband W.S. Eliceiri K.W. NIH image to ImageJ: 25 years of image analysis.Nat. Methods. 2012; 9: 671-675Crossref PubMed Scopus (34981) Google Scholar software. We compared the effect of the different reaction conditions on the immobilization efficiency by measuring the normalized fluorescence (NF) of each spot in the pattern (Equation 1).NF=mean intensity of the featuremean intensity of thebackground.(Equation 1) NF is used because it is relatively independent of imaging parameters and microscope optics and can be used for comparing data measured on different microscopy platforms.34Bian S. Schesing K.B. Braunschweig A.B. Matrix-assisted polymer pen lithography induced Staudinger ligation.Chem. Commun. 2012; 48: 4995-4997Crossref PubMed Scopus (19) Google Scholar We used Otsu's algorithm35Sundberg M. Månsson A. Tågerud S. Contact angle measurements by confocal microscopy for non-destructive microscale surface characterization.J. Colloid Interface Sci. 2007; 313: 454-460Crossref PubMed Scopus (50) Google Scholar, 36Ghaye J. Kamat M.A. Corbino-Giunta L. Silacci P. Vergères G. De Micheli G. Carrara S. Image thresholding techniques for localization of sub-resolution fluorescent biomarkers.Cytometry A. 2013; 83: 1001-1016Crossref PubMed Scopus (9) Google Scholar to determine the NF of each feature. In brief, each feature was cropped from the image, and Otsu's threshold was applied to separate features into foreground and background (Figure S18). By dividing the mean intensity by the background, we determined the NF. To arrive at statistically significant values of NF for each print, we analyzed 30 patterns of the 10,000 printed across the surface. The analysis of the fluorescence data demonstrates that both I and t affect NF. The highest NF (I = 20%, t = 20 min) of 1.40 ± 0.04 was close to the maximum observed for a fluorophore monolayer in previous work.14Bian S. He J. Schesing K.B. Braunschweig A.B. Polymer pen lithography (PPL)-induced site-specific click chemistry for the formation of functional glycan arrays.Small. 2012; 8: 2000-2005Crossref PubMed Scopus (44) Google Scholar Interestingly, NF decreased with increasing I, and the greatest NF arose with spots printed at the lowest I. This is consistent with previous results15Bian S. Zieba S.B. Morris W. Han X. Richter D.C. Brown K.A. Mirkin C.A. Braunschweig A.B. Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays.Chem. Sci. 2014; 5: 2023-2030Crossref Scopus (59) Google Scholar, 25Liu X. Zheng Y. Peurifoy S.R. Kothari E.A. Braunschweig A.B. Optimization of 4D polymer printing within a massively parallel flow-through photochemical microreactor.Polymer Chem. 2016; 7: 3229-3235Crossref Google Scholar and suggests that complex chemical dynamics may arise from the high radical concentration at high photon flux, which may disrupt the underlying monolayer. Also, NF was observed to increase with t regardless of I. The changes in the NF of the spots with respect to t (I = 20%) were fit to a pseudo-second-order adsorption model37Largitte L. Pasquier R. A review of the kinetics adsorption models and their application to the adsorption of lead by an activated carbon.Chem. Eng. Res. Des. 2016; 109: 495-504Crossref Scopus (511) Google Scholar, 38Yousaf M.N. Chan E.W.L. Mrksich M. The kinetic order of an interfacial Diels–Alder reaction depends on the environment of the immobilized dienophile.Angew. Chem. Int. Ed. 2000; 39: 1943-1946Crossref PubMed Scopus (63) Google Scholar (Equation 2),NF=kadsNFmax−NFmin2t1+kadsNFmax−NFmint+NFmin,(Equation 2) where kads is the pseudo-second-order adsorption rate constant. We developed this kinetic model by considering the formation of the C–S bond to be irreversible. In addition, Mrksich used this model previously to determine rates of reaction for covalent bond formation on monolayers.38Yousaf M.N. Chan E.W.L. Mrksich M. The kinetic order of an interfacial Diels–Alder reaction depends on the environment of the immobilized dienophile.Angew. Chem. Int. Ed. 2000; 39: 1943-1946Crossref PubMed Scopus (63) Google Scholar From the fit of the data (R2 = 0.998), and assuming a maximum coverage of 2.7 × 1014 molecules·cm2,39Chidsey C.E.D. Bertozzi C.R. Putvinski T.M. Mujsce A.M. Coadsorption of ferrocene-terminated and unsubstituted alkanethiols on gold: electroactive self-assembled monolayers.J. Am. Chem. Soc. 1990; 112: 4301-4306Crossref Scopus (1089) Google Scholar we determined kads = 1.1 × 10−17 cm2·molecule−1·s−1 and a maximum extrapolated NF of 1.49, which matched well with the maximum NF of 1.40 ± 0.04 that we observed experimentally (Figure 3). For comparison, the data were also fit to a pseudo-first-order Langmuir isotherm, whose fit was only slightly less good (R2 = 0.993) (Equation S2). The second-order kinetics can be attributed to the two-step reaction process,38Yousaf M.N. Chan E.W.L. Mrksich M. The kinetic order of an interfacial Diels–Alder reaction depends on the environment of the immobilized dienophile.Angew. Chem. Int. Ed. 2000; 39: 1943-1946Crossref PubMed Scopus (63) Google Scholar, 40Northrop B.H. Coffey R.N. Thiol-ene click chemistry: computational and kinetic analysis of the influence of alkene functionality.J. Am. Chem. Soc. 2012; 134: 13804-13817Crossref PubMed Scopus (195) Google Scholar which involves formation of the thiol-centered radical followed by C–S bond formation. Thus, in a single pattern, we were able to print with 25 different conditions, which was sufficient to derive quantitative kinetics, thereby demonstrating how this platform can be used to accelerate reaction optimization, measure quantitative chemical kinetics, and understand interfacial reactivity. We also explored the role of z height on the resulting patterns. In brief, we printed a 3 × 3 pattern of 2 (100 mM 1:1 2:DMPA, DMF:DMSO 75:25 v/v, I = 20%, 5 μL/min, t = 10 min) by changing z heights (0, 6, and 12 μm) with a 2.26 ± 1.03 μm aperture diameter beam pen array. After washing the surface and analyzing the fluorescence images, we observed no significant difference in either the spot diameters (4.08 ± 0.42 μm) or the NF (1.15 ± 0.02) (Figure S21). This is a significant contrast to our previous work using transparent PPL arrays in fluid cells, where both NF and diameter were sensitively dependent upon z height.25Liu X. Zheng Y. Peurifoy S.R. Kothari E.A. Braunschweig A.B. Optimization of 4D polymer printing within a massively parallel flow-through photochemical microreactor.Polymer Chem. 2016; 7: 3229-3235Crossref Google Scholar We also explored the role of the concentration of 2 and DMPA by printing at five different concentrations (5, 30, 50, 70, 100, and 150 mM; 1:1 2:DMPA, DMF:DMSO 75:25 v/v, I = 25%, 5 μL/min, t = 20 m