Technology Report pubs.acs.org/jchemeduc
Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
Plate Reader Compatible 3D-Printed Device for Teaching Equilibrium Dialysis Binding Assays Cody W. Pinger,‡,§ Andre Castiaux,‡,§ Savannah Speed,‡,§ and Dana M. Spence*,†,‡,§ †
Department of Biomedical Engineering, ‡Department of Chemistry, and §Institute for Quantitative Health Sciences & Engineering, Michigan State University, East Lansing, Michigan 48824, United States
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S Supporting Information *
ABSTRACT: Plasma protein binding measurements are an important aspect of pharmacology and drug development. Therefore, performing these measurements can provide a valuable and highly practical learning experience for students across many scientific disciplines. Here, we describe the design and characterization of a 3D-printed device capable of performing equilibrium dialysis in order to measure the binding affinity between a fluorescent ligand and a common plasma protein. The device is designed for laboratory automation, having dimensions identical to those of a standard 96-well plate, allowing it to be placed into a plate reader for direct fluorescence quantitation of free ligand. Traditional equilibrium dialysis experiments initially require 5−14 h of user attention; however, a “hands-free” approach is enabled by the technology in this paper, allowing the user to set up the device in a 1 h time period and return the next day for data analysis. Here, we include instructions for measuring and calculating the ligand−receptor binding affinity of fluorescein to albumin with comparisons to values previously reported in the literature. While the described experiment is used for a protein/ligand binding event, the device can also be easily adapted to a simple small molecule diffusion study. As such, the experiment includes aspects of importance for engineering (design), chemistry (diffusion), and biochemistry (binding) students. The design files (.stl files) for the device are included in the Supporting Information so that others may use or modify them. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Graduate Education/Research, Interdisciplinary/Multidisciplinary, Analytical Chemistry, Biochemistry, Hands-On Learning/Manipulatives, Laboratory Equipment/Apparatus
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INTRODUCTION The curriculum of undergraduate science programs is becoming increasingly multidisciplinary; therefore, hands-on learning experiences need to be developed accordingly. Today’s programs require students to take classes spanning a broad range of such fields as chemistry, engineering, biology, materials, and computer science. Learning activities should be tailored to cross these disciplines to train students to think through problems holistically. The technology described here was used to train an undergraduate student interested in biomedical engineering research to measure the binding affinity of a small molecule to a large protein, an important measurement in the life sciences. In both pharmacology and biochemistry, it is fundamental to understand that the total bloodstream concentration of a drug, hormone, or small molecule consists of a fraction bound to plasma proteins and an unbound, or free, fraction.1 Importantly, it is commonly believed that only this free fraction is bioavailable to cells and tissues.2−4 Measuring the free fraction of a species under physiological conditions is often performed by equilibrium dialysis, ultrafiltration, or ultracentrifugation; however, rapid new analytical techniques have recently been developed, such as ultrafast affinity extraction chromatography.2,5 Once the free fraction of ligand is measured, an equilibrium binding constant can be calculated, as described in the Supporting Information. © XXXX American Chemical Society and Division of Chemical Education, Inc.
Measuring and calculating this binding constant is important because it provides pharmacokinetic and pharmacodynamic (PK/PD) information for the ligand.6 In equilibrium dialysis, a ligand of interest is combined with a plasma protein, such as serum albumin, and placed in a primary compartment on one side of a size-exclusion porous membrane. Subsequently, an equal volume of buffer is placed in a secondary compartment on the adjacent side of the membrane, and the free ligand is allowed to diffuse through the membrane pores and into the secondary buffer compartment, separating it from the larger protein.1,7,8 Lab experiments for undergraduate students featuring equilibrium dialysis to measure protein binding of ligands have been reported previously in this Journal.9,10 However, the technology reported here is unique in that the measurement device is engineered to allow direct quantitation of the free ligand with the use of a plate reader, shown in Figure 1. The direct quantitation feature of the device allows the typical time-consuming experiment (approximately 14 h) to be set up in a single 1 h time period and left unattended until the next day. Received: March 26, 2018 Revised: July 9, 2018
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DOI: 10.1021/acs.jchemed.8b00215 J. Chem. Educ. XXXX, XXX, XXX−XXX
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MATERIALS AND METHODS
3D-Printed Device: Design and Fabrication
In part one of this project, CAD software (AutoDesk Inventor Professional, 2016) was used to create the drawing of the device seen in Figure 2
Figure 1. Illustration of the principle of equilibrium dialysis with direct fluorescence quantitation of analyte ligand. The gray ovals represent protein (albumin), and the blue triangles represent the analyte ligand (fluorescein).
An important application of equilibrium dialysis is the measurement of the binding of fluorescein to albumin.11,12 Fluorescein, a small molecule dye found commonly in analytical chemistry laboratories, is a strongly fluorescent molecule that is often used as a tracer molecule in vivo to investigate the breakdown of the blood−retinal barrier and the blood−brain barrier under a variety of disease states.13,14 These studies are strongly influenced by fluorescein’s affinity for albumin, the most common bloodstream serum protein. The use of equilibrium dialysis to measure the fraction of fluorescein bound to albumin under physiological conditions is, therefore, important in interpreting the results of such in vivo studies. Learning to make this measurement empowers the user with a foundation of knowledge to set up and execute binding experiments with any macromolecule and smaller ligand. Recently, protein binding experiments have been optimized by the use of 3D printing.6,8 3D printing, also known as additive manufacturing, is a technique revolutionizing laboratories and experiments by allowing scientists to create devices to fit their specific research needs.15 The impact of 3D printing on today’s research is so substantial that the undergraduate student should be well versed on the technique.16 The purpose of the development and use of this technology is to foster a learning environment in which a student interested in research gains hands-on experience from design and fabrication of a 3D-printed measurement device, to subsequent application of the device to an important measurement in life science. Prior to this technology, this measurement was not able to be accurately performed in an appropriate time frame. Here, an automated equilibrium dialysis device was developed and successfully implemented to train undergraduate research assistants in a biomedical engineering research laboratory. All of the reported measurements were performed by an undergraduate student researcher in training. Recommendations for incorporating the device into a laboratory classroom are provided in the Supporting Information, including details on device fabrication (attached .stl files of the devices are provided), sample calculations, sample questions, and experimental design.
Figure 2. Diagram showing the dimensions (millimeters) of the 3Dprinted device which can fit directly into a plate reader.
This software is free for students or anyone with a “.edu” email address. It includes online tutorials and can be downloaded at www.autodesk.com. The CAD drawing is saved as an .stl file and submitted to the 3D printer (Objet Connex 350) for printing. The .stl files of the devices used in this paper are included in the Supporting Information to encourage readers to print our version of the device for example use. The dimensions of this device correspond to the exact size and shape, featuring 12 different wells for use in equilibrium dialysis. Each of the 12 wells features two compartments, a sample compartment and a buffer compartment, divided by a void where a membrane insert will be placed. The device is designed so that each sample compartment is precisely aligned with the location of a measurement well in a standard 96-well plate. This allows the device to fit into a standard plate reader, and the optics of the instrument will automatically align with the sample compartment of the device. Details for the design of the device are included in the Supporting Information. The final product is the device seen in Figure 3.
Figure 3. Left: Final assembled CAD drawing of the device before it is submitted as an .stl file to the 3D printer. Right: Photograph of the printed device.
Membrane Integration
3D-printed membrane-holding inserts were designed as windows for a tight, leak-free fit into the void between compartments in each well. A Print−Pause−Print technique was used to incorporate size-exclusion dialysis membranes into B
DOI: 10.1021/acs.jchemed.8b00215 J. Chem. Educ. XXXX, XXX, XXX−XXX
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the membrane insert, as previously reported.8 The exact dimensions of the insets are provided in Figure 4.
Figure 5. Schematic diagram showing how the device is prepared. The wells in the black box contain 2.0 μM fluorescein in buffer without BSA in the sample compartment, while the wells in the red box contain 2.0 μM fluorescein in buffer with 42 mg/mL BSA in the sample compartment. The wells in the blue box contain standards. The white compartment represents a “buffer compartment”, while the green compartment represents a “sample compartment”. The black rectangles show where membrane-holding inserts are placed.
Figure 4. Left: Diagram showing the dimensions (millimeters) of the membrane-holding inserts. Right: Final assembled CAD drawing of the inset.
A detailed description of how to fabricate the membraneholding inserts is included in the Supporting Information. Reagents
Bovine serum albumin (BSA, lyophilized powder) was purchased from Sigma (St. Louis, MO). Fluorescein sodium salt was also purchased from Sigma and diluted into distilled and deionized H2O (DDW) to prepare a stock solution 300 μM in water.
7.5, 8, 8.5, and 9 h. This was accomplished by measuring the fluorescence (excitation wavelength, 485 nm; emission wavelength, 512 nm; cutoff, 495 nm) in the “buffer compartment” of each sample well, including the wells containing standards. The data was then exported to a separate spreadsheet (Microsoft Excel) for data analysis.
Sample Preparation
“Hands-Free” Equilibrium Dialysis Experiments
A stock solution of BSA was prepared daily by diluting the lyophilized BSA with dialysis buffer (200 mM phosphate buffer in DDW 7.40, sterile filtered through 0.22 μm filters from Sigma) to make a working solution of 50 mg/mL BSA. From this stock solution, samples were prepared (10 mL total) to contain 42 mg/mL BSA and 2.0 μM fluorescein sodium salt. A separate sample containing no BSA was prepared in the exact same manner, while containing 2.0 μM fluorescein sodium salt in dialysis buffer. Standards (3 mL total) containing known concentrations of fluorescein were prepared in buffer, ranging in concentration from 0.125 to 1.0 μM, including a blank. All of the sample preparation was performed by an undergraduate student in approximately 30−45 min. Characterizing Incubation Time-Equilibrium Dialysis Experiments. The cleaned membrane holders were inserted into the device, as shown in Figure 3. Then, a 1.1 mL aliquot of sample containing 42 mg/mL BSA and 2 μM fluorescein was pipetted into the sample compartment of the first 3 wells, followed by 1.1 mL of dialysis buffer into the corresponding buffer wells on the adjacent side of the membrane. This was repeated in the next 3 wells with sample containing 0 mg/mL BSA and 2 μM fluorescein. Five of the other wells contained standards in both the sample and buffer compartments. For instance, 1.1 mL of 1.0 μM fluorescein standard was pipetted into both the sample and buffer compartments of the same well, and this was repeated for the other four standards in four other wells. A layout of the samples and standards in the device can be seen in Figure 5. The entire device was agitated at 220 rpm at 37 °C on an orbital shaker (Talboys) to provide constant mixing at a physiological temperature. The device was removed from the shaker and placed directly into a plate reader (FlexStation 3, Molecular Devices) for free ligand quantitation at 2, 4, 5.5, 6.5,
In this experiment, the device was set up with samples and standards exactly as described in the previous section and shown in Figure 5. Instead of placing the device onto an orbital shaker after addition of samples and standards, the device was immediately placed into the plate reader. Using the settings in the instrument software, the instrument was programmed to measure the fluorescence of the samples (as described previously) every 5 min for the next 14 h, while shaking for one full minute prior to the reading. The settings can be seen in Figure S1 in the Supporting Information. Once the user clicked “start”, they were able to leave until the next day to export, analyze, and interpret the data. Calculations and Data Analysis
The fluorescence intensity values measured on the device by the plate reader were exported to a Microsoft Excel spreadsheet for data analysis. The fluorescence intensities of the standards were used to make an external standards calibration curve (Figure 6), which was used to quantify the concentration of free fluorescein in the buffer compartments of the sample wells (slope = 2.15 × 104, y-intercept = 1.83 × 103, R2 = 0.998). The free concentration was then used in the equations in Supporting Information to calculate the percent protein bound and global equilibrium constant nKa of the system. Sample calculations can be found in the Supporting Information.
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RESULTS AND DISCUSSION The device is engineered so that the free ligand in the sample compartment is able to diffuse through the pores of the membrane and into the buffer compartment, where it can be quantified separately from the ligand−protein complex. C
DOI: 10.1021/acs.jchemed.8b00215 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 7. (A) Samples contain 2.0 μM fluorescein in buffer and no BSA. The concentration of fluorescein in the buffer compartment of the well is monitored periodically. The concentration at 6.5 h is not statistically different than at 9 h (∼1 μM). In part B, samples contain 42 mg/mL BSA and 2.0 μM fluorescein. The concentration of fluorescein in the buffer compartment becomes at 6.5 h (0.16 ± 0.02 μM) is equal to that at 9 h. n = 3 The asterisk indicates that the difference is not significant p > 0.05 (error = sd).
Figure 6. External standards calibration curve comparing the fluorescence intensity of fluorescein standards to their known concentrations. The calibration curve was generated by placing the device containing the standards directly into the plate reader and measuring the fluorescence intensity of the buffer compartments. (error = sd).
corresponding buffer compartments. The free fluorescein in the buffer compartment reached a steady concentration after 6.5 h (0.16 ± 0.02 μM), indicated by a concentration not statistically different from that at 9 h (0.18 ± 0.03 μM, n = 3, p = 0.5), as displayed in Figure 7B. Using equations in the Supporting Information, we can calculate the percentage of fluorescein bound to protein to be 81 ± 3%, which is equal to values reported in the literature for albumin (human) binding to fluorescein (53−83%).11 Then, using other equations in Supporting Information, we can calculate an approximate global equilibrium constant, or nKa, to be 1.5 ± 0.3 × 104 M−1, where n is equal to the number of fluorescein molecules bound per molecule of albumin. This can be compared to values reported in the literature for BSA binding to fluorescein (Ka = 2.8 × 104 M−1 or nKa = 8.4 × 104 M−1; nKa = 1.45 × 105 M−1, no error was reported for either value).12,17 Full step-by-step sample calculations for calculating % protein bound and nKa are included in the Supporting Information for ease of use and instruction. A 9 h incubation requiring periodic attention from the user is not practical for any undergraduate laboratory classroom; therefore, a “hands-free” equilibrium dialysis technique was developed so that the device could be set up in under 1 h and placed into the plate reader to run automatically overnight. The plate reader was simply programmed to make fluorescence measurements of the buffer compartments every 5 min for 14 h, with shaking for one full minute prior to each reading. The binding system reached equilibrium at approximately 12 h, as indicated by the free fluorescein concentration at 12 h (0.16 ± 0.01 μM, n = 3) being statistically equal to that at 14 h (0.17 ± 0.01 μM, n = 3, p = 0.42). This technique required a longer incubation time because shaking was not constant, as it was in the previous experiment shown in Figure 7. The average concentration, including standard deviation, of the three wells was plotted in Figure 8 The data from this experiment agrees well with the previous experiment and the values reported in the literature, confirming its ability to measure the binding affinity between small fluorescent molecules and large plasma proteins, in a time-efficient manner. Importantly, an undergraduate research assistant in training was able to independently prepare all of the samples and set up the device in under 1 h. The student then returned the following day to export and analyze the data.
In order to characterize the ability of the device to be used for quantitative measurements, standards of known analyte concentration were pipetted into separate wells of the device (in both the sample and buffer compartments), and then the device was placed directly into the plate reader for fluorescence intensity measurements of the buffer compartments. An external standards calibration curve was created from the data (Figure 6), and an R2 value of 0.998 was measured, demonstrating the capability to perform quantitative measurements of analyte in the buffer compartments. The standards were removed from the device, rearranged in to different wells, and measured again to ensure there was no bias or error associated with different wells in the device. Nonspecific binding of analyte ligand to the membrane is a major criticism of equilibrium dialysis, as this would lead to the calculation of a deceptively large binding constant. Therefore, nonspecific binding was analyzed in the first experiment by pipetting 1.1 mL of 2.0 μM fluorescein (no albumin) in the sample compartment of the first three wells, followed by adding an equal volume (1.1 mL) of buffer to the respective buffer compartments, and measuring the concentration of free fluorescein that diffused into the buffer compartment periodically over the course of 9 h. Shown in Figure 7A, the concentration of fluorescein in the buffer compartment at 6.5 h (1.04 ± 0.05 μM) was not statistically different from that measured at 9 h (1.08 ± 0.04 μM, n = 3, p = 0.3); therefore, the fluorescein concentration had equilibrated, and 50% of the original 2.0 μM sample was in each compartment. This result suggests that there is negligible nonspecific binding of fluorescein to the membrane. For equilibrium dialysis binding measurements to be accurate, samples must be incubated long enough for the free ligand concentration to equilibrate between the two compartments. An inadequate incubation time will lead to a deceptively high Ka approximation. An experiment was designed to evaluate the appropriate incubation time for the system, as indicated by the time when the concentration of ligand in the buffer compartment ceases increasing. A 1.1 mL sample containing a physiological bloodstream concentration of albumin (42 mg/mL, or ∼632 μM) and 2.0 μM fluorescein was pipetted into the sample compartment of three separate wells, followed by 1.1 mL of dialysis buffer into the D
DOI: 10.1021/acs.jchemed.8b00215 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Device CAD file (ZIP) Membrane insert CAD file (ZIP)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Dana M. Spence: 0000-0002-4754-6671 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support for this project is from the National Institutes of Health (DK110665 and GM110406).
Figure 8. For the “hands-free” equilibrium dialysis technique, the concentration of fluorescein in the buffer compartment at 12 h was not statistically different than at 14 h (0.17 ± 0.01 μM, n = 3, p = 0.42) (error = sd).
The same device has been used several times per week for the past 5 months without failure, while only changing out the membrane inserts for each use, therefore demonstrating the integrity of the device design.
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CONCLUSION Equilibrium dialysis is a straightforward approach for performing binding measurements and is commonly used in undergraduate biochemistry laboratories; however, the timeconsuming nature of the technique and high error involved when improperly performed can make it troublesome to perform in a normal lab period. A 3D-printed device was designed and fabricated for the purpose of making accurate equilibrium dialysis binding measurements between a small fluorescent molecule and a plasma protein directly on a plate reader in a time-efficient manner. Customizable measurement devices made amenable to plate reader technology have been used many times throughout the literature over the past seven years to study bioanalytical chemistry;18−20 therefore, the technique is mature enough to incorporate into undergraduate education. All of the measurements described here were executed by an undergraduate research assistant in order to train them to perform binding measurements. Therefore, the experiment could be readily implemented into a variety of laboratory settings in bioengineering, analytical chemistry, biochemistry, pharmacology, and more. Instructors are encouraged to utilize the high-throughput and automated nature of the device to design experiments that enforce specific concepts based on curriculum needs, such as investigating the effects of pH and temperature changes on macromolecular binding affinity, competitive binding, the effects of membrane pore size, and viscosity on diffusion rates, or the molecular-weight dependency of diffusion coefficients.
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REFERENCES
(1) Du Souich, P.; Verges, J.; Erill, S. Plasma protein binding and pharmacological response. Clin. Pharmacokinet. 1993, 24 (6), 435− 440. (2) Oravcová, J.; Böhs, B.; Lindner, W. Drug-protein binding studies new trends in analytical and experimental methodology. J. Chromatogr., Biomed. Appl. 1996, 677 (1), 1−28. (3) Koch-Weser, J.; Sellers, E. M. Binding of Drugs to Serum Albumin. N. Engl. J. Med. 1976, 294 (6), 311−316. (4) Hage, D. S.; Jackson, A.; Sobansky, M. R.; Schiel, J. E.; Yoo, M. J.; Joseph, K. Characterization of drug−protein interactions in blood using high-performance affinity chromatography. J. Sep. Sci. 2009, 32 (5−6), 835−853. (5) Mallik, R.; Yoo, M. J.; Briscoe, C. J.; Hage, D. S. Analysis of drug−protein binding by ultrafast affinity chromatography using immobilized human serum albumin. J. Chromatogr. A 2010, 1217, 2796−2803. (6) Heller, A.; Lockwood, S.; Janes, T.; Spence, D. Technologies for Measuring Pharmacokinetic Profiles. Annu. Rev. Anal. Chem. 2018, 11, 79−100. (7) Zamek-Gliszczynski, M. J.; Ruterbories, K. J.; Ajamie, R. T.; Wickremsinhe, E. R.; Pothuri, L.; Rao, M. V. S.; Basavanakatti, V. N.; Pinjari, J.; Ramanathan, V. K.; Chaudhary, A. K. Validation of 96-well Equilibrium Dialysis with Non-radiolabeled Drug for Definitive Measurement of Protein Binding and Application to Clinical Development of Highly-Bound Drugs. J. Pharm. Sci. 2011, 100 (6), 2498−2507. (8) Pinger, C. W.; Heller, A. A.; Spence, D. M. A Printed Equilibrium Dialysis Device with Integrated Membranes for Improved Binding Affinity Measurements. Anal. Chem. 2017, 89 (14), 7302− 7306. (9) Katz, S. A.; Paritt, C.; Purdy, R. Equilibrium dialysis: A laboratory experiment. J. Chem. Educ. 1970, 47 (10), 721. (10) Sohl, J. L.; Splittgerber, A. G. The binding of Coomassie brilliant blue to Bovine Serum Albumin: A physical biochemistry experiment. J. Chem. Educ. 1991, 68 (3), 262. (11) Li, W.; Rockey, J. H. Fluorescein binding to normal human serum proteins demonstrated by equilibrium dialysis. Arch. Ophthalmol. 1982, 100 (3), 484−487. (12) Andersson, L. O.; Rehnström, A.; Eaker, D. L. Studies on “Nonspecific” binding. Eur. J. Biochem. 1971, 20 (3), 371−380. (13) Hawkins, B. T.; Egleton, R. D. Fluorescence imaging of blood− brain barrier disruption. J. Neurosci. Methods 2006, 151 (2), 262−267. (14) Nagataki, S.; Matsunaga, I. Binding of fluorescein monoglucuronide to human serum albumin. Investigative ophthalmology & visual science 1985, 26 (8), 1175−1178. (15) Gross, B. C.; Erkal, J. L.; Lockwood, S. Y.; Chen, C.; Spence, D. M. Evaluation of 3D printing and its potential impact on biotechnology and the chemical sciences. Anal. Chem. 2014, 86, 3240−3253.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00215. Full details on how to design and fabricate the device, along with detailed information on equilibrium dialysis, including necessary equations and sample problems, sample prelab questions with answers, and device and prelab information (PDF, DOCX) E
DOI: 10.1021/acs.jchemed.8b00215 J. Chem. Educ. XXXX, XXX, XXX−XXX
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(16) Bharti, N.; Singh, S. Three-Dimensional (3D) Printers in Libraries: Perspective and Preliminary Safety Analysis. J. Chem. Educ. 2017, 94 (7), 879−885. (17) Laurence, D. A study of the adsorption of dyes on bovine serum albumin by the method of polarization of fluorescence. Biochem. J. 1952, 51 (2), 168. (18) Halpin, S. T.; Spence, D. M. Direct Plate-Reader Measurement of Nitric Oxide Released from Hypoxic Erythrocytes Flowing through a Microfluidic Device. Anal. Chem. 2010, 82 (17), 7492−7497. (19) Chen, C.; Wang, Y.; Lockwood, S. Y.; Spence, D. M. 3Dprinted fluidic devices enable quantitative evaluation of blood components in modified storage solutions for use in transfusion medicine. Analyst 2014, 139, 3219−3226. (20) Liu, Y.; Chen, C.; Summers, S.; Medawala, W.; Spence, D. M. C-peptide and zinc delivery to erythrocytes requires the presence of albumin: implications in diabetes explored with a 3D-printed fluidic device. Integrative Biology 2015, 7 (5), 534−543.
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DOI: 10.1021/acs.jchemed.8b00215 J. Chem. Educ. XXXX, XXX, XXX−XXX
