Letter pubs.acs.org/Langmuir
Lysozyme Pattern Formation in Evaporating Drops Heather Meloy Gorr,* Joshua M. Zueger, and John A. Barnard Department of Mechanical Engineering and Materials Science, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States ABSTRACT: Liquid droplets containing suspended particles deposited on a solid, flat surface generally form ringlike structures due to the redistribution of solute during evaporation (the “coffee ring effect”). The forms of the deposited patterns depend on interactions between solute(s), solvent, and substrate. In this study, deposition patterns from droplets of a simplified model biological fluid (DI water + lysozyme) are examined by scanning probe and optical microscopy. The overall lysozyme residue morphology is complex (with both a perimeter “rim” and undulating interior) but varies little with concentration. However, the final packing of lysozyme molecules is strongly dependent on initial concentration.
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INTRODUCTION When a sessile droplet of a colloidal solution evaporates from a flat solid substrate, solute particles generally accumulate on the substrate nonuniformly, forming various patterns. For pinned droplets, the constant contact diameter acts as a geometric constraint and outward radial flows carry solute to the perimeter, where the evaporative flux is the highest. Solute particles accumulate along the contact line as solvent removed by evaporation at the edge of the drop is replaced by solvent flowing from the center.1−3 This so-called “coffee ring effect” has generated interest because of its relevance to a wide range of technologies and processes including inkjet printing4,5 and lab-on-a-chip applications.6 Interest has also emerged in interpreting patterns of evaporated biological fluid droplets for medical diagnostic purposes. Several groups7−9 have demonstrated that patterns in the dried residue of human biological fluids exhibit distinct characteristics reflecting the health of a patient. The multicomponent flows and accompanying phase transitions in these complex biofluid systems are believed to be similar to those of sessile drops of aqueous colloidal suspensions.10 Model colloidal solutions of biological relevance are thus a good starting point for understanding more complicated biological systems. Since the work of Deegan et al., other researchers have examined the mechanisms of droplet evaporation11−15 and various combinations of solute and solvent.16−21 Interesting gel or glassy transitions have also been observed in polymer solutions15−17 and recently in drops of whole blood.9 These studies generally use macroscopic droplets, although several do consider micro- and nanoscale drops.5,19,20 In the present work, pattern formation from sessile droplet evaporation of dilute aqueous solutions of lysozyme, a simplified model biological fluid, is studied. AFM and optical microscopy are used to study the morphology of dried sessile droplets as a function of concentration and droplet size, focusing on diameters from ∼15 to 50 μm. © 2012 American Chemical Society
EXPERIMENTAL DETAILS
Lysozyme. Lysozyme is a small, compact, globular protein with a roughly ellipsoidal shape, found in high concentration in human mucosal secretions including tears22 and saliva.23 Lysozyme is well studied and has been the subject of protein crystallization24,25 and adsorption studies26,27 The lysozyme molecule has approximate dimensions of 3.0 nm × 3.0 nm × 4.5 nm,28 molecular mass of 14 kDa, isoelectric point of pH 11.1, and carries a net positive charge at physiological pH. Solutions. Lysozyme solutions were prepared from a single batch of high purity commercial lysozyme powder (Sigma Aldrich, L6876). As-received powder was dissolved in deionized water (Millipore, resistivity of 18.2 MΩ·cm) at 35 °C to concentrations (ρ) 0.1, 0.25, 0.5, 0.75, and 1.0 g/100 mL. Solutions were prepared at room temperature, stored at 2 °C, brought to room temperature prior to deposition, and used within 4 weeks. Substrates and Deposition Techniques. Silicon wafer substrates were ultrasonically cleaned in isopropyl alcohol, then acetone, and finally rinsed with DI water and blown dry using compressed air prior to deposition. Droplets were deposited under ambient conditions with a Preval aerosol spray system (AnalTech, Inc.). Characterization. A Keyence digital optical microscope (VHX600) was used to examine the deposits and record the real-time evaporation process at 28 fps. Color images were converted to grayscale. Drops with diameters < 50 μm were characterized in air by tapping mode atomic force microscopy (AFM; Digital Instruments, D3100) with a standard tip. AFM data was analyzed with Gwyddion SPM software to measure the geometry and rms roughness of the dehydrated residues. A drop shape analysis (DSA) system (Kruss, D100) was used to measure contact angles, drop diameters, and drop volumes for macroscopic drops. The initial contact angle of lysozyme solutions on silicon wafers was ∼55°; little dependence on lysozyme concentration was found. Received: October 4, 2011 Revised: February 11, 2012 Published: February 16, 2012 4039
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RESULTS
five concentrations in the size range studied. Surprisingly, even 10-fold changes in solution concentration lead to minimal changes in residue profile. To further quantify the deposit geometry, the critical dimensions, rms roughness, and volume of the deposit were extracted from the AFM data. The outer drop diameter, D, and the inner diameter between the two minima, Di, were measured as illustrated in Figure 1d. These values were used to calculate the width of the ring, w = 1/2(D − Di). The width was found to scale with drop diameter for drops from all five concentrations; that is, the relative size of the ring, w/D, is independent of concentration. The rms roughness of the deposits measured in the 5 μm × 5 μm region of the drop interior indicated in Figure 1c is ∼3 nm, consistent with the finite size of the lysozyme molecules. The volume of the deposit after evaporation, VD, was determined for each drop. Assuming spherical cap geometry for the initial drop profile, the drop volume is proportional to D3. Our expectation was that the deposit residue volume would also be proportional to D3. ln VD versus ln D is plotted in Figure 2 for all five concentrations. The linear fit is the average
The generally observed morphology of the dried drop residues is documented in Figure 1.
Figure 2. ln−ln plot of deposited volume (VD) versus drop diameter (D) for all five lysozyme concentrations (ρ). The slope of the average best-fit line is 2.99 (R2 = 0.99).
of the slopes of the best fit lines for the five different concentrations. As expected, the slope is ∼3, indicating that VD ∝ D3 for all concentrations of lysozyme. The error bars in the insert of Figure 2 reflect the standard deviation in measurements as well as AFM measurement uncertainty. If the deposit densities were independent of initial solution concentration, one would expect separate but parallel lines of slope 3 for each concentration with differences in the yintercepts. On the contrary, the variation in y-intercept of the linear fits for the different concentrations is negligible. The surprising uniformity of the deposit geometry despite the 10fold concentration difference leads to consideration of lysozyme packing in the deposit. The volume of lysozyme in the liquid drop was estimated from the initial liquid drop volume, the solution concentration, and the volume of one lysozyme molecule, assuming a prolate spheroid form. From the volume of lysozyme in solution, VS, volume of the deposit, VD, and conservation of lysozyme molecules during evaporation, the volume fraction of lysozyme in the deposit, φ, is determined by φ = VS/VD. The mean volume fraction of lysozyme in the deposit, φ, is plotted in Figure 3 versus concentration. The error bars correspond to the standard deviation in the data.
Figure 1. Representative images of a single evaporated lysozyme drop deposit (ρ = 1.0; D = 32.6 μm). (a) Optical microscopy image; (b) 3D view of AFM topographical data; (c) 2-D AFM topographic map with 5 μm × 5 μm area used in roughness measurement indicated; (d) cross-sectional height profile illustrating D and Di measurements. The shaded area indicates the “ring”.
The deposits have very smooth surfaces and are radially symmetric with a single, well-defined “coffee ring” present at the perimeter. The typical radial thickness variation in the residues is illustrated in the AFM images (b and c). The ring height is much greater than the drop interior, suggesting that the majority of the solute collects at the pinned periphery during evaporation due to radial flows. Within the perimeter ring, the deposit thickness falls rapidly to a minimum and then rises gradually to a central mound with a small depression at the very center. This morphology is observed consistently for all 4040
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Similar gel transitions have been observed in related studies on the evaporation of sessile droplets of polymer solutions15−17 and biological fluids8,9 due to the effective increase in concentration during evaporation of the solvent. In these systems of interacting particles, a glassy or gel-like “skin” is formed near the air−water interface and the final deposits are a result of the competition between gelation and desiccation kinetics. Recently, phase transitions in drops of whole blood have been studied, where a gel-like skin forms at the periphery of the drop while the “coffee ring” develops and the gelation front moves inward as evaporation proceeds. This is followed by rapid gelation of the central area and then a decrease in evaporation rate while the remaining liquid evaporates through the porous gel matrix.9 A similar line of reasoning is used to describe the phase change in the aqueous lysozyme drop system. Millimeter scale drops of the same lysozyme/DI water solutions were used to document the morphological evolution during evaporation. A sequence of frames illustrating the drying process is shown in Figure 4. Radial flows carry solute to the perimeter of the drop as the skin forms, effectively increasing the concentration in the ring, which begins to gel (Figure 4a, b). Gelation continues toward the still liquid center of the drop (Figure 4c, d). Once the gel has formed at the periphery, creating the rim, and proceeded toward the central area of the drop forming the typical craterlike shape, the remaining liquid depins (Figure 4e) and rapidly recedes (Figure 4f, g). The residual lysozyme in this liquid collects in a mound shape on top of the well-defined craterlike shape. Finally, as the remaining liquid evaporates through the gel, the general shape is evident (Figure 4h). Periodic surface cracks (not found in small drops) also appear in the final stages of evaporation. Optical surface profiler measurements (Veeco/Wyko NT 1100) confirm that the overall shape of the large and small deposits is similar and thus the evaporation process is assumed to be similar in the two size ranges.
Figure 3. Mean volume fraction of the deposit (φ) vs concentration (ρ). The linear fit through the data points has a slope of 0.29 (R2 = 0.99). The error bars correspond to the standard deviation in the data.
The data is linear, indicating that while the total volume of the deposit is not concentration dependent and scales with diameter, the volume fraction of lysozyme within the deposited volume (its apparent density) increases linearly with concentration. The highest φ observed is ∼0.31 (approximately 70% of the deposit is air or water). As φ decreases, the fraction of open volume in the deposit increases correspondingly, leading to extremely porous deposits from the lowest concentration solution.
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DISCUSSION
It is reasonable to propose that the lysozyme solution drops undergo a gel transition during evaporation, and that this gel is packed more densely with increasing solution concentration. The gelation of lysozyme29,30 can be brought about by changes in concentration, temperature, pH, and ionic strength, as well as the addition of a solvent. A transparent, viscoelastic gel matrix is often observed, with increasing bond character with increasing initial concentration.
Figure 4. Sequential images during the drying process of millimeter-scale lysozyme solution droplet (ρ = 2.0 g/100 mL) over time (a) τ ∼ 1, (b) 3, (c) 4, (d) 4.5, (e) 5, (f) 5.5, (g) 5.75, and (f) 6 min. The radius of the droplet remains unchanged for the majority of the drying process, until the coffee-ring structure is formed and the remaining liquid depins and begins to recede in (e). 4041
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(10) Sefiane, K. On the Formation of Regular Patterns From Drying Droplets and Their Potential Use for Bio-Medical Applications. J. Bionic Eng. 2010, 7, S82−S93. (11) Fischer, B. J. Particle Convection in an Evaporating Colloidal Droplet. Langmuir 2002, 18, 60−67. (12) Hu, H.; Larson, R. G. Marangoni Effect Reverses Coffee-Ring Depositions. J. Phys. Chem. B 2006, 110, 7090−7094. (13) Girard, F.; Antoni, M.; Sefiane, K. On the Effect of Marangoni Flow on Evaporation Rates of Heated Water Drops. Langmuir 2008, 24, 9207−9210. (14) Bhardwaj, R.; Fang, X.; Attinger, D. Pattern Formation During the Evaporation of a Colloidal Nanoliter Drop: A Numerical and Experimental Study. New J. Phys. 2009, 11, 075020. (15) Okuzono, T.; Kobayashi, M.; Doi, M. Final Shape of a Drying Thin Film. Phys. Rev. E 2009, 80, 021603. (16) Pauchard, L.; Allain, C. Stable and Unstable Surface Evolution During the Drying of a Polymer Solution Drop. Phys. Rev. E 2003, 68, 052801. (17) Gorand, Y.; Pauchard, L.; Calligari, G.; Hulin, J. P.; Allain, C. Mechanical Instability Induced by the Desiccation of Sessile Drops. Langmuir 2004, 20, 5138−5140. (18) Wang, J.; Evans, J. R. G. Drying Behaviour of Droplets of Mixed Powder Suspensions. J. Eur. Ceram. Soc. 2006, 26, 3123−3131. (19) Li, F.-I.; Leo, P. H.; Barnard, J. A. Dendrimer Pattern Formation in Evaporating Drops: Solvent, Size, and Concentration Effects. J. Phys. Chem. C 2008, 112, 14266−14273. (20) Shen, X.; Ho, C.-M.; Wong, T.-S. Minimal Size of Coffee Ring Structure. J. Phys. Chem. B 2010, 114, 5269−5274. (21) Yunker, P. J.; Still, T.; Lohr, M. A.; Yodh, A. G. Suppression of the Coffee-Ring Effect by Shape-Dependent Capillary Interactions. Nature 2011, 476, 308−311. (22) Ronen, D.; Eylan, E.; Romano, A.; Stein, R.; Modan, M. A Spectrophotometric Method for Quantitative Determination of Lysozyme in Human Tears: Description and Evaluation of the Method and Screening of 60 Healthy Subjects. Invest. Ophthalmol. 1975, 14, 479−484. (23) Yeh, C.-K.; Dodds, M. W. J.; Zuo, P.; Johnson, D. A. Lysozyme Concentrations and Candidal Counts. Arch. Oral Biol. 1997, 42, 25− 31. (24) Pusey, M. L.; Snyder, R. S.; Naumann, R. Protein Crystal Growth: Growth Kinetics for Tetragonal Lysozyme Crystals. J. Biol. Chem. 1986, 261, 6524−6529. (25) Liu, Y.; Wang, X.; Ching, C. B. Toward Further Understanding of Lysozyme Crystallization: Phase Diagram, Protein-Protein Interaction, Nucleation Kinetics, and Growth Kinetics. Cryst. Growth Des. 2010, 10, 548−558. (26) Kim, D. T.; Blanch, H. W.; Radke, C. J. Direct Imaging of Lysozyme Adsorption onto Mica by Atomic Force Microscopy. Langmuir 2002, 18, 5841−5850. (27) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Aggregation of Lysozyme and of Poly(ethylene glycol)-Modified Lysozyme After Adsorption to Silica. Colloids Surf., B 2007, 57, 81−88. (28) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Structure of Hen Egg-white Lysozyme: A Three-dimensional Fourier Synthesis at 2 Angstrom Resolution. Nature 1965, 206, 757−761. (29) da Silva, M. A.; Areas, E. P. G. Solvent-Induced Lysozyme Gels: Rheology, Fractal Analysis, and Sol-Gel Kinetics. J. Colloid Interface Sci. 2005, 289, 394−401. (30) Yan, H.; Frielinghaus, H.; Nykanen, A.; Ruokolainen, J.; Saiani, A.; Miller, A. F. Thermoreversible Lysozyme Hydrogels: Properties and an Insight Into the Gelation Pathway. Soft Matter 2008, 4, 1313− 1325.
Unlike the flow-dominated coffee ring shapes of noninteracting particle systems, the shape of lysozyme deposits is dictated by the formation of the skin at the free surface. The initial shape of the skin depends only on the wetting diameter and the contact angle, which was equivalent for all concentrations in this study. Recently, the shape of desiccated polymer solution deposits was predicted numerically based on the ratio of the initial and gelation concentrations.15 Higher ratios of initial to gelation concentration resulted in domelike shapes while lower ratios resulted in craterlike shapes similar to those in this study. These shapes are attributed to skin formation and suppression of the radial flow when the concentration approaches the gelation concentration, or the concentration at which the viscosity increases sharply. The patterns that remain on the substrate show negligible concentration dependence in the width and height of the ring and total volume of the deposit. However, the volume fraction of lysozyme molecules in the deposit increases linearly with concentration. Thus, drops with higher lysozyme concentration in solution produce a denser, much more tightly packed lysozyme deposit, although the overall morphology is similar to that of the lower solution concentrations. This result is due to formation of a loosely packed gel-like structure of lysozyme molecules at the air−water interface which maintains the geometric shape observed in the deposit.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the facilities, scientific, and technical assistance of the Materials Micro-Characterization Laboratory of the Department of Mechanical Engineering and Materials Science, Swanson School of Engineering, University of Pittsburgh.
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REFERENCES
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