Exploiting Femtoliter Microwells for the Sensitive Measurement of

Article pubs.acs.org/Langmuir

Exploiting Femtoliter Microwells for the Sensitive Measurement of Protein Adsorption Gary M. Nishioka,*,† Reka Geczy,†,‡ Kimberly S. Huggler,‡ Tram N. Dao,‡ and Charles W. Sokolik‡ †

H & N Instruments, Inc., PO Box 4338, Newark, Ohio 43058-4338, United States Department of Chemistry and Biochemistry, Denison University, Granville, Ohio 43023, United States

‡

S Supporting Information *

ABSTRACT: A method is described for the sensitive measurement of adsorbed proteins using femtoliter microwells. Quantitative measurement of adsorbed protein is demonstrated at surface densities from 10 fg/cm2 to 3 pg/cm2. Determination of the efficacy of barrier coatings is also demonstrated using femtoliter microwells. Adsorption at low surface densities is measured, indicating the highest affinity sites on the surface and therefore the initial stages of adsorption. The femtoliter microwell method is shown to be useful in detecting differences between effective protective coatings.

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INTRODUCTION Protein adsorption is important in many biomedical applications, such as diagnostic arrays, biosensors, implants, in vivo applications of nanomaterials, drug discovery screening, filtration membranes, and pharmaceutical processes. Consequently, there are numerous methods that have been used to measure protein adsorption on various surfaces. These include solution depletion measurements, in which the decrease in concentration of a protein solution is measured upon exposure to a surface. These methods generally study high surface area powders and achieve sensitivities down to 100 ng/ cm2.1 Another method involves the measurement of protein adsorption by I125 radiolabeling. A significant disadvantage of this technique is the adsorption of free I125 which is continuously generated from proteins in solution. In a study of adsorption on contact lenses, this interference was as high as 0.3 μg/lens.2 Ellipsometry measures the film thickness of adsorbed protein on a reflective surface from the change in polarization of elliptically polarized light. It is sensitive in the ng/cm2 range. Interpretation of measurements is straightforward for surfaces that are flat, isotropic, and uniform, but complex optical models are required for surfaces with varied optical constants and topographies.3,4 Quartz crystal microbalance methods detect protein adsorption on a piezoelectric sensor from the change occurring in its resonant frequency, yielding the mass of the adsorbed protein. It is a nanogram sensitive technique and can measure adsorption on coatings of interest on the quartz crystal. One complication is that this method also includes water coupled to the adsorbed layer.4,5 © XXXX American Chemical Society

Optical waveguide lightmode spectroscopy and other attenuated total reflection methods analyze light from an optical waveguide. The evanescent wave at the interface extends into the adjacent air or liquid to around 200 nm from the surface and is therefore modified by adsorbed proteins. These methods study the near-surface, have a sensitivity to 500 pg/ cm2, and require transparent surfaces.4 Surface plasmon resonance (SPR) measures the change in the evanescent wave caused by adsorption of proteins on a metal surface. SPR is capable of detecting protein surface concentrations of 500 pg/cm2. It requires a metal surface and cannot distinguish between specific and nonspecific interactions with the surface. It is mass sensitive and therefore less sensitive to the binding of lower molecular weight proteins.4,6 Detection of the lowest surface concentrations indicates the highest affinity sites for adsorption; this is particularly important in passivation studies that seek to prevent the initial stages of protein adsorption. We propose that an alternate and sensitive measurement of protein adsorption can be obtained by using femtoliter microwells. The area scale of several micrometers in a microwell is representative of a macroscopic surface, yet microwells enclose volumes on the order of tens of femtoliterssufficiently small for single molecule detection. Microwells have been exploited for the increase in sensitivity that is gained in their small volumes. One popular use of such volumes is the study of the kinetics of single enzyme molecules.7 When a single enzyme molecule is isolated in a sufficiently small droplet or microwell containing a fluorogenic Received: March 12, 2017 Revised: June 27, 2017 Published: June 30, 2017 A

DOI: 10.1021/acs.langmuir.7b00819 Langmuir XXXX, XXX, XXX−XXX

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Langmuir substrate, then its fluorescent product grows to a concentration that is easily detected. Single molecule enzyme studies have revealed a distribution of catalytic rates within an enzyme population and varying substrate turnover rates over time. Enzyme subpopulations therefore exist, hitherto invisible in bulk experiments. A second example that exploits the sensitivity gained in microwells is an immunoassay capable of measuring very low concentrations of a biomarker.8 The method works by confining micrometer sized beads containing a typical antibody/biomarker/antibody−enzyme immunocomplex in microwells. After substrate addition single molecule detection is obtained since a high concentration of fluorescent product is generated within individual microwells. In this investigation we exploit the sensitivity gained by femtoliter microwells to study the adsorption of enzymelabeled proteins onto a surface containing an array of microwells. To minimize their volume, we use microwells in the shape of inverted pyramids, which are easily fabricated by alkaline etching of the silicon ⟨100⟩ surface. Silicon ⟨100⟩ etches anisotropically along the ⟨111⟩ crystal plane, at a 54.74° angle. Thus, a pyramid-shaped well with an opening of 5 × 5 μm is etched to a depth of 3.53 μm. This microwell encloses a volume of 29.4 fL and has a surface area of 43.26 μm2. The addition of a fluorogenic substrate following adsorption allows for the detection of as little as a single adsorbed molecule within a well. A single molecule adsorbed in a well is at a surface density of 3.8 amol/cm2. If quantifiable measurements of fluorescence down to 1% of the microwell array are possible, then surface densities down to 0.04 amol/cm2 can be detected. For a protein of molecular weight 250 kDa this is equivalent to 10 fg/cm2. We first determine if counting individual fluorescent wells does indeed count single molecules. This has been assumed but not verified.8 We accomplish this by measurement of arrays that are generated with very low surface concentrations (1) of adsorbed molecules in individual wells can be determined by the fluorescence intensity of each well. This involves some assumptions concerning the increase in microwell fluorescence as molecules adsorb, as described in the Experimental Section. If the number of adsorbed molecules is obtainable from the fluorescence intensity of each well, then higher surface densities could be measured by the described method. Finally, we demonstrate how microwell arrays can be used to evaluate surface protection strategies. Heat cleaned microwell chips undergo various surface treatments, and the adsorption of two model proteins is measured. The sensitivity of the microwell measurement allows discrimination between generally effective surface protection coatings.

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EXPERIMENTAL SECTION

Silicon Femtoliter Microwell Array Chips. Silicon chips are fabricated by a commercial foundry (Nanostructures, Inc., Santa Clara, CA). Chips contain 36 microwell arrays consisting of 5 × 5 × 3.1 μm inverted pyramids; each microwell array consists of 6000 wells in a 60 × 100 pattern, for a total of 216 000 microwells on the chip. Microwells are separated by 3 μm, and the area within the microwells is 33.4% of the total area of the top surface. Chips are heat cleaned at 500 °C for 15 h to remove residual contaminants and ensure a uniform oxide layer on the surface. Various surface treatments proceed immediately after heat cleaning; these are accomplished as follows. Microwell Array Chips with a Known and Very Low APSA Surface Density. The microwell chip surface is first biotinylated by immersion in a 100 μg/mL solution of polylysine−poly(ethylene glycol)−biotin (PK-PEG-Bt, SuSoS) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.4. The poly-L-lysine (PK) segment is highly cationic at physiological pH and adsorbs strongly to the negatively charged silicon oxide surface. The grafted poly(ethylene glycol) segment (PEG) prevents nonspecific protein adsorption by steric repulsion and excluded volume effects. The attached biotin functions to bind specifically to APSA. The immersed chip is degassed under vacuum to ensure penetration of solution into all microwells. The chip is incubated for 1 h at room temperature and then washed five times in HEPES buffer. The resulting chip is hydrophilic, coated with a PK-PEG-Bt layer. A known number of active APSA units are then added to chips. First, we measure the concentration of a stock solution of APSA (Sigma-Aldrich) as units of activity per microliter (U/μL). One unit of APSA activity hydrolyzes 1.0 μmol of p-nitrophenyl phosphate per minute at pH = 9.8; we perform these measurements on bulk solutions by standard spectrophotometry at room temperature. The stock solution (0.082 U/μL) is then serially diluted so that tens to hundreds of nanounits (nU) of activity are added per array. The strong and irreversible binding of streptavidin to biotin ensures that 100% of added APSA is adsorbed on the surface. The resulting chips are characterized by a solution derived surface density of APSA in U/array (enzyme activity per array). Microwell Array Chips with Different Surface Protections. The heat-cleaned microwell chip surface is protected from adsorption by immersion in 0.1% bovine serum albumin (BSA) blocking solution in B

DOI: 10.1021/acs.langmuir.7b00819 Langmuir XXXX, XXX, XXX−XXX

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We test the simple assumption that fluorescence of microwells increases linearly with the number of adsorbed molecules, with a peak width = 3. Microwells with fluorescence intensities from 6 to 8 are assigned one molecule, from 9 to 11 two molecules, and so on.

pH 7.4 HEPES buffer, evacuation to ensure well penetration, 24 h incubation, and storage in HEPES buffer. Other chips undergo similar treatment using a commercial blocking buffer (reagent diluent solution (RD), Biotechne). Chips are also protected with a dodecane self-assembled monolayer (C12 SAM) by immersion of heat-cleaned chips in a 1% (v/v) solution of dodecyltrichlorosilane in dicylclohexyl for 5 min, followed by washes in hexane and acetone.9,10 Finally, chips with a dense PEG surface layer are created by 24 h immersion in 100 μg/mL PK-PEG at 22 and 60 °C following the procedure described previously. Note that these surfaces are not biotinylated. Poly(ethylene glycol) Composite Covers. Covers to seal chips consist of a glass slide supporting a thin film of an elastomeric PEG composite. This composite is transparent and was developed as a mold for high-resolution soft lithography.11 The monomer solution is prepared by first combining poly(ethylene glycol) diacrylate with 2hydroxy-2-methylpropiophenone (both from Aldrich) in a 19:1 v/v ratio. This solution is then combined with an adhesive photopolymer (NOA-63, Norland Optical Adhesives) in a 1:4 weight ratio. The solution is then centrifuged to remove bubbles. To ensure a smooth top surface, the monomer solution is cast against a smooth, polished silicon wafer surface. To ensure easy release of the polymer from the silicon, a self-assembled close-packed dodecane monolayer is applied to the polished silicon surface following the procedure described previously. Covers are prepared by adding monomer solution to the silicon surface and then placing a glass window on the solution, sandwiching the solution between the glass and silicon. The solution is cured 1 h under UV (Blak-Ray longwave 100 W lamp). The silicon wafer is subsequently removed, resulting in a smooth, elastomeric PEG composite layer on glass. Sectioning and microscopic examination of these windows after use indicates composite layer thicknesses between 15 and 30 μm. Adsorption Measurements. Figure 1 illustrates the procedure for using femtoliter microwells to study protein adsorption. The top

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RESULTS AND DISCUSSION Single Molecules Are Detected in Microwells.12 Figure 2 plots the number of fluorescent wells observed after

Figure 2. Microwell fluorescence after adsorption from highly dilute solutions of alkaline−phosphatase−streptavidin (APSA). The solution characterized quantity of applied APSA is listed as nanounits (nU) of enzyme activity per microarray. The data are fit to a two-parameter exponential rise to a maximum, yielding an initial slope of 258 wells/ nU, or 258 molecules/nU: equivalent to 0.43 pmol/U. This is in rough agreement with the solution characterized activity of 0.20−0.54 pmol/ U, demonstrating that the assumption that a fluorescent well can indicate a single molecule is valid.

adsorption of APSA from highly dilute solutions. The solution characterized quantity of applied APSA is plotted as nanounits of activity per array. We assume quantitative adsorption of APSA onto the biotinylated surface and uniform distribution over the available surface. This means that 33.4% of applied APSA adsorbs within microwells, with the remaining APSA adsorbing onto the top surface and border areas. Figure 2 reveals that the number of fluorescent wells increases as the quantity of applied APSA increases. Presumably this is initially caused by single APSA molecules filling microwells. At higher quantities of applied APSA the number of adsorbed molecules saturates the array, equaling the number of available microwells of 6000 per array. Initially, single molecules fill wells, and this is represented by the initial points in Figure 2. The initial slope measures the change in the number of occupied wells as the applied quantity approaches zero. This slope represents the first molecule entering the array, giving the best measure of the activity of a well containing a single molecule. To obtain the initial slope of the data, we fit it to a two-parameter exponential rise to a maximum. No mechanistic justification is sought for this equation; it is merely used for convenience. We should note that typical adsorption models such as Langmuir, or chemisorption, and BET or other physisorption models are not appropriate for this process. In these models as molecules occupy the surface the number of available adsorption sites

Figure 1. Illustration of the measurement of protein adsorption using femtoliter microwells. Enzyme-labeled protein adsorbs onto the microwell surface, substrate is added, the wells are sealed, and microwells containing at least one protein molecule fluoresce. At low surface densities the number of adsorbed molecules is determined by counting the number of fluorescent wells. surface of the adsorbent, containing microwell arrays, is incubated with the enzyme-conjugated adsorbate of interest. For cases where adsorption is low, molecules adsorb at a low surface density, adsorbing into a fraction of the available microwells, as well as on the top surface between wells. After washing, a fluorogenic substrate is added, and the microwells are sealed by the PEG composite cover. Fluorescent product is then created in microwells containing as little as one enzyme-conjugated molecule. The intensity of each fluorescent well and their total number are then measured by fluorescence microscopy. Analysis of Fluorescent Microarrays. Fluorescent images are analyzed as described in the Supporting Information. Briefly, fluorescence intensity of each microwell is measured as a value between 0 and 255. Blank control arrays show background fluorescence at intensities between 3 and 5. Microwells with fluorescence 6 and above are considered filled. C

DOI: 10.1021/acs.langmuir.7b00819 Langmuir XXXX, XXX, XXX−XXX

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Langmuir decreases. In contrast, as molecules adsorb in microwells the number of available microwells does not decrease. The initial slope of 258 wells/nU obtained in Figure 2 represents the relation between the number of molecules and the enzyme activity, or 258 molecules/nU. This is equivalent to 0.43 pmol/U. This value for the activity of APSA can be compared on a molar basis to the activity of the enzyme alone, since APSA contains one alkaline phosphatase segment. Highly purified alkaline phosphatase is reported to have an activity of ≥7500 U/mg.13 This is equivalent to ≤0.89 pmol/U. These results indicate that when we add a unit of APSA activity we are detecting at least 50% of the solution characterized expected number of molecules. However, this discrepancy probably results from errors in the solution characterization, not the fluorescence measurement. This discrepancy is reduced if the actual molecular activity of alkaline phosphatase is in fact greater than 7500 U/mg. A greater source of this discrepancy arises since extremely dilute APSA solutions are prepared, so losses from adsorption during solution preparation are probable. To minimize these effects, we used LoBind tips and microcentrifuge tubes (Eppendorf) and prepared solutions immediately before use. We flushed pipet tips three times before use, but we did not attempt to presaturate the tubes because of possible desorption effects. Protein loss from 1 μg/mL solutions in LoBind tubes is less than 5% after 1 h of storage;14 however, significantly greater loss was recently measured for more dilute 1.5 nM protein solutions. In a study of five proteins, adsorptive loss in LoBind microplates ranged from 23% to 61%.15 If we take into account adsorptive loss for our solutions as being in this range, then the expected activity based on solution addition is 0.20−0.54 pmol/U, in agreement with the measured activity of 0.43 pmol/ U. Therefore, the assumption that a fluorescent well can indicate a single molecule is shown to be valid, and adsorbed single molecules can indeed be counted in microwell arrays. This makes possible the measurement of very low adsorbed surface densities. Increases in Fluorescence Intensity of Limited Use in Measuring Number of Adsorbed Molecules. As adsorption proceeds, the growth in the number of fluorescent wells decreases, but the fluorescence intensity of wells increases. Figure 3 results from assuming a linear increase in fluorescence intensity with the number of adsorbed molecules, as described previously. This appears to be a valid assumption up to three molecules per well. The slope of the line in this region agrees within 10% of the initial slope from Figure 2. At higher densities (not shown) well fluorescence increases more slowly and nonlinearly, presumably from intrawell interference between molecules. For our particular chip the upper limit for quantitative adsorption measurements is

Figure 3. Estimated number of adsorbed APSA molecules per array. Above an average of three molecules per well (18 000 molecules/ array), the fluorescence of wells does not increase linearly. The slope of the line = 234 molecules/nU.

Microwell Adsorption Measurements Distinguish between Protected Surfaces. The sensitivity of this measurement makes possible comparisons between lowadsorbing surfaces. As a demonstration of the usefulness of this technique, microwell chips underwent various blocking treatments to reduce protein adsorption. After incubation in dilute solutions of APSA (10−13 M) followed by washing, the amount of adsorbed APSA is measured. As seen in Figure 4, all treatments were effective in reducing APSA adsorption by 95− 99% compared to untreated chips. The BSA coating was most effective, followed by treatment with PK-PEG. In a second demonstration utilizing a different and more concentrated protein solution, coated chips are incubated in a 6 × 10−10 M solution of an alkaline-phosphatase antibody (antiCD9) conjugate. Wells coated with dodecyl SAM or commercial blocking buffer (reagent diluent (RD); Biotechne) are all occupied with this protein, and no difference relative to the untreated surface is discerned. It should be noted that a significant reduction in adsorption probably occurs, but above the limit of 3 pg/cm2 for this method. Reduced adsorption is observed for the remaining coatings, with the PK-PEG coatings exhibiting the most effective protection. Ogaki and co-workers investigated PK-PEG coatings on titanium-coated silicon. These coatings were created by adsorption of PK-PEG from 100 μg/mL solutions at temperatures ranging between 20 and 80 °C. They concluded that an increase in PK-PEG surface density occurred with increasing deposition temperature. A concomitant decrease in protein adsorption was observed as surface densities increased.16 In contrast, Figure 4 reveals no significant decrease in adsorption for surfaces coated by PK-PEG deposition at an elevated temperature. Although we duplicated the adsorption conditions as described, our surface differed physically by not being flat but consisting of microwells and also chemically by consisting of silicon dioxide rather than titanium dioxide. In addition, our adsorbed protein densities of up to 3 pg/cm2 are in a range significantly lower than they measured. Therefore, we conclude that no additional benefit is conferred using elevated temperatures for PK-PEG treatment under our specific

3 molecules/well × 6000 wells/array × 36 arrays/0.334 = 1.9 × 106 molecules

The equivalent concentration in a volume of 10 μL = 3 × 10−13 mmol/mL. Since the surface area of each microwell is 43.26 μm2, the corresponding upper limit of surface density is 3 pg/ cm2. More sophisticated models could increase the upper limit for surface concentration measurement. At the lower limit, 1% of wells filled corresponds to a surface density of 10 fg/cm2. D

DOI: 10.1021/acs.langmuir.7b00819 Langmuir XXXX, XXX, XXX−XXX

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therefore prove useful in detecting differences between highly effective protective coatings. The described method provides a sensitive method for the detection of adsorbed proteins3 to 4 orders of magnitude more sensitive than other methods. It could prove useful as an additional technique in studies of protein adsorption.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b00819. Figures A and B (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (G.M.N.). ORCID

Gary M. Nishioka: 0000-0002-7638-4851

Figure 4. Effect of various surface treatments on the adsorption from dilute solutions (8.8 × 10−14 mmol/mL) of alkaline phosphatase− streptavidin (black column, APSA) and less dilute solutions (6.5 × 10−10 mmol/mL) of alkaline−phosphatase−anti-CD9 conjugate (brown column, AP-antibody), relative to an untreated heat-cleaned chip. Surface treatments are PKPEG coated from a 0.01 μg/mL solution at 22 and 60 °C, BSA from a 0.1% solution, dodecane (C12) self-assembled monolayer from a 1% silane solution, and coating from a commercial blocking buffer (RD).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Trent Edmunds for preparing Figure 1,. This work was supported in part by NIH Grant 1R43TR001010-01, the Denison University Research Foundation, the Denison University Anderson Research Assistantship, and the Denison University Miller Summer Research Assistantship.

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conditions. Additional adsorption experiments are planned to determine if higher treatment temperatures with PK-PEG provide superior protection under other conditions. Limitations. There are a number of limitations in using microwell arrays to study adsorption. First, the adsorbent of interest needs to contain microwells or be coated onto a support consisting of microwells. SPR also involves coating adsorbents, in this case onto a metal surface. Since SPR has been successfully used to study numerous materials, this coating requirement is readily circumvented for many materials. A second limitation is that the adsorbent must not interfere with the enzyme product signal, i.e., will not fluoresce. This limitation can be circumvented by the use of a different enzyme or substrate to create product that fluoresces or absorbs at a different wavelength. Finally, the microwell method requires an enzyme-labeled adsorbate. This could affect adsorption characteristics. One potential solution is to first adsorb the protein of interest and then detect adsorbed protein using an enzyme conjugated antibody. A second potential solution is to attach enzymes at different sites on the protein adsorbate; this would yield information on the effect of the label on adsorption.

ABBREVIATIONS APSA, alkaline phosphatase streptavidin; BSA, bovine serum albumin; C12, dodecane self-assembled monolayer; HEPES, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PEG, poly(ethylene glycol); PK-PEG, polylysine−poly(ethylene glycol); RD, reagent diluent: a commercial blocking buffer; SAM, selfassembled monolayer; SPR, surface plasmon resonance.

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REFERENCES

(1) Koltisko, B.; Walton, A. Chromatographic Analysis of Protein Adsorption. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum: New York, 1985; Chapter 6. (2) Hall, B.; Heynen, M.; Jones, L. W.; Forrest, J. A. Analysis of Using I125 Radiolabeling for Quantifying Protein on Contact Lenses. Curr. Eye Res. 2016, 41 (4), 456−465. (3) Mora, M. F.; Wehmeyer, J. L.; Synowicki, R.; Garcia, C. D. Investigating Protein Adsorption via Spectroscopic Ellipsometry. In Biological Interactions on Materials Surfaces Understanding and Controlling Protein, Cell, and Tissue Responses; Puleo, D. A., Bizios, R., Eds.; Springer Science: Berlin, 2009; Vol. XX, Chapter 2, pp 19− 41. (4) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. A Comparative Study of Protein Adsorption on Titanium Oxide Surfaces Using in situ Ellipsometry, Optical Waveguide Lightmode Spectroscopy, and Quartz Crystal Microbalance/Dissipation. Colloids Surf., B 2002, 24, 155−170. (5) Dixon, M. C. Quartz Crystal Microbalance with Dissipation Monitoring: Enabling Real-Time Characterization of Biological Materials and Their Interactions. J. Biomol. Tech. 2008, 19, 151−158. (6) Nguyen, H. H.; Park, J.; Kang, S.; Kim, M. Surface Plasmon Resonance: A Versatile Technique for Biosensor Applications. Sensors 2015, 15, 10481−10510. (7) Liebherr, R. B.; Gorris, H. H. Enzyme Molecules in Solitary Confinement. Molecules 2014, 19, 14417−14445.

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CONCLUSIONS Adsorbed single molecules can be detected in microwell arrays, making possible the measurement of very low surface densities. The solution derived applied quantity of active enzyme is in reasonable agreement with the measured number of adsorbed molecules. Adsorption is measured quantitatively from 10 fg/ cm2 to 3 pg/cm2. Differences in the efficacy of barrier coatings can be determined using microwell arrays. Since low surface densities of the adsorbate are measured, the highest affinity sites and the initial stages of adsorption are determined. This method could E

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Langmuir (8) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 2010, 28 (6), 595−599. (9) Kim, B. J.; Liebau, M.; Huskens, J.; Reinhoudt, D. N.; Brugger, J. A Self-Assembled Monolayer-Assisted Surface Microfabrication and Release Technique. Microelectron. Eng. 2001, 57−58, 755−760. (10) Kim, G. M.; Kim, B.; Liebau, M.; Huskens, J.; Reinhoudt, D. N.; Brugger, J. Surface Modification With Self-Assembled Monolayers for Nanoscale Replication of Photoplastic MEMS. J. Microelectromech. Syst. 2002, 11 (3), 175−181. (11) Lee, N. Y.; Lim, J. R.; Lee, M. J.; Kim, J. B.; Jo, S. J.; Baik, H. K.; Kim, Y. S. Hydrophilic Composite Elastomeric Mold for HighResolution Soft Lithography. Langmuir 2006, 22, 9018. (12) It should be noted that the cover must segregate the fluorescent product in microwells. We initially used covers coated with a thin coating of a silicone polymer (Sylgard 184). Fluorescent product diffused through the silicone polymer, resulting in its dilution. The average fluorescence of this diluted solution was too low to be measured at low adsorption concentrations. (13) Sigma-Aldrich, BioUltra Phosphatase, Alkaline, #P0114. (14) Eppendorf LoBind: Evaluation of Protein Recovery in Eppendorf Protein LoBind Tubes and Plates. Eppendorf Technical Report #180, 2010. (15) Weikart, C. M.; Klibanov, A. M.; Breeland, A. P.; Taha, A. H.; Maurer, B. R.; Martin, S. P. Plasma-Treated Microplates with Enhanced Protein Recoveries and Minimized Extractables. SLAS Technology 2017, 22 (1), 98−105. (16) Ogaki, R.; Andersen, O. Z.; Jensen, G. V.; Kolind, K.; Kraft, D. C. E.; Pedersen, J. S.; Foss, M. Temperature-Induced Ultradense PEG Polyelectrolyte Surface Grafting Provides Effective Long-Term Bioresistance Against Mammalian Cells, Serum, and Whole Blood. Biomacromolecules 2012, 13, 3668−3677.

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DOI: 10.1021/acs.langmuir.7b00819 Langmuir XXXX, XXX, XXX−XXX