Quantifying Protein Concentrations Using Smartphone Colorimetry: A

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Quantifying Protein Concentrations Using Smartphone Colorimetry: A New Method for an Established Test Clifford T. Gee,† Eric Kehoe,‡ William C. K. Pomerantz,*,† and R. Lee Penn*,† †

Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States Janesville Waldorf Pemberton High School, Janesville, Minnesota 56048, United States

‡

S Supporting Information *

ABSTRACT: Proteins are involved in nearly every biological process, which makes them of interest to a range of scientists. Previous work has shown that hand-held cameras can be used to determine the concentration of colored analytes in solution, and this paper extends the approach to reactions involving a color change in order to quantify protein concentration (e.g., green to blue). Herein, we describe the successful use of smartphone colorimetry to quantify protein concentration using two common colorimetric biochemical methods, the Bradford and biuret assays. The ease of the experimental setup makes these lab experiments accessible to a wide range of students and can be used as both high school and college level laboratory experiments. KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Analytical Chemistry, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Proteins/Peptides, Quantitative Analysis, UV−Vis Spectroscopy

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These methods are especially useful when the protein concentration in solution cannot be determined by other techniques e.g., when the protein extinction coefficient is unknown or is too low for direct determination of the concentration based on the protein solution’s absorbance at 280 nm.4 Colorimetric assays are typically performed using a UV−vis spectrophotometer. In the presence of varying concentrations of protein, absorbance values at 595 and 540 nm are monitored in the Bradford and biuret assays, respectively. However, these specialized instruments are expensive and may not be available in all laboratories such as high school teaching laboratories. To mitigate this challenge, recent reports have demonstrated the efficacy of hand-held cameras for quantitative colorimetry, making colorimetric experiments accessible to a wider range of students and researchers.5 Smartphone colorimetry has been used in kinetics experiments with crystal violet,6 quantification of gold nanoparticles present in dietary supplements,7 and a variety of other experiments.8−12 These examples involve a single color with a colorless blank. The use of camera phone colorimetry with a color change has yet to be explored. Here, we extend quantitative colorimetry to protein assays that involve color changes and its application to quantify protein concentration. Bradford assays are commonly employed in laboratory experiments for introductory biochemistry courses. The

roteins are large biomacromolecules that play necessary roles in nearly all biological processes, including cell growth, metabolism, and differentiation. Consequently, significant research efforts are dedicated to studying the biological function of proteins and designing synthetic molecules to modulate the function of proteins in the context of disease. High school students encounter proteins in both the life sciences as well as in their chemistry courses [e.g., Next Generation Science Standards (NGSS)1]. One example of an interesting protein is the bromodomain containing protein 4, (Brd4). When functioning properly, Brd4 is a necessary protein for regulating gene transcription and cell growth, but when functioning improperly, Brd4 has been linked to cancer,2 a topic covered in life sciences classes and a reality that has impacted many people personally. Accurate quantification of protein concentration is crucial to studying the roles proteins play in biological processes. Concentrations are particularly important in biochemical experiments (e.g., circular dichroism and enzymatic assays) for quantitative characterization of protein structure and function. There are several different methods for protein quantification. Some methods are colorimetric assays that involve the formation of a colored complex. Quantitation is accomplished by determining visible light absorption and using Beer’s Law to convert absorbance to concentration by comparison to a standard calibration curve. Two such colorimetric assays are the Bradford and biuret assays.3 Using known standards within concentration ranges that obey Beer’s law, one can generate a linear calibration curve that can be used to estimate a protein concentration of an unknown sample. © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: September 6, 2016 Revised: April 6, 2017

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DOI: 10.1021/acs.jchemed.6b00676 J. Chem. Educ. XXXX, XXX, XXX−XXX

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Figure 1. Chemical scheme and sample images for protein colorimetric assays. (A) Chemical scheme for Coomassie Blue G-250 which becomes fully deprotonated in the presence of a protein, yielding a blue color. (B) Sample image of Bradford reagent plus water (left) and Bradford reagent plus 0.04 mg/mL bovine serum albumin (BSA) (right). (C) Chemical scheme for complexation of cupric ions in the presence of peptides and proteins forming a complex with the amide backbone, resulting in a color change from blue to violet. (D) Sample image of biuret reagent plus water (left) and biuret reagent plus 0.75 mg/mL BSA (right).

mL 85% (w/v) phosphoric acid. (3) Add this mixture slowly to 850 mL of water. (4) Store away from light at 4 °C. Because Flinn is a common vendor for high school laboratories, the biuret reagent was prepared using the Flinn Scientific specifications [0.2% (w/v) CuSO4, 30% (w/v) NaOH] for this reagent. This solution can be made by mixing 100 mg of CuSO4·5H2O (dissolved in 10 mL of water) and 40 mL of 37.5% (w/v) NaOH in water. It is important to note that biuret from Flinn contains 30% sodium hydroxide while many other vendors and recipes employ only 3% (w/v) sodium hydroxide with the addition of 0.6% (w/v) potassium sodium tartrate.13 Both solutions will provide adequate results, but the tartratecontaining solution performs significantly better than the solution without it at protein concentrations above 0.5 mg/ mL because it prevents protein precipitation. If tartrate is not available, citrate may also be used as a substitute to prevent turbidity (see Supporting Information). While the Bradford reagent is more common in biochemistry laboratories for its greater sensitivity to a wider protein concentration range, biuret is roughly 10% of the cost of Bradford, making it more accessible for a high school or a low-budget laboratory. Bovine serum albumin (BSA) for the calibration curve was purchased from RPI and used as-is. Brd4 was expressed from recombinant DNA using standard protein expression protocols in BL21 (DE3) E. coli cells.14

protein-responsive molecule in the Bradford solution, Coomassie Blue G-250, is an environmentally sensitive triphenylmethane dye compound that exists in varying charge states depending on the surrounding solution conditions. The hydrophobic interactions provided by the presence of a protein stabilizes the anionic form of the dye, resulting in color change from a brownish green, with a maximum absorption at 470 nm (cationic form), to a blue color, with a maximum absorption at 595 nm (anionic form) (Figure 1A,B). While the Bradford assay is a dye-based assay, the biuret assay involves the formation of a Cu(II) complex with the amide backbone present in peptides and proteins. This coordination complex results in a color change from blue (free Cu2+), with a maximum absorption at 650 nm, to violet (HN···Cu2+···NH), with a maximum absorption at 540 nm (Figure 1C,D). The bicinchoninic acid (BCA) and Lowry assays are examples of other copper-based assays that are commercially available. Herein we report the first application of smartphone colorimetry for protein quantification using two well-known protein assays that involve changes in color as a function of protein concentration. The ubiquity of the Bradford assay as a standard laboratory experiment in biochemistry and the cheap and accessible nature of the biuret assay combined with the clear color change that occurs upon complexing with protein made these two assays ideal for use with smartphone colorimetry.

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Preparation of Calibration and “Unknown” Solutions

Standard Bradford or biuret solutions for the calibration curve were prepared by diluting 0.10 mg/mL BSA (for Bradford assay) or 3−10 mg/mL BSA (for biuret assay) with 1.5 mL of Bradford or biuret solution and water. For preparation of the calibration standards, 1.5 mL of the Bradford or biuret solutions; 0.1, 0.2, 0.3, 0.4, and 0.5 mL of appropriate protein stock solution; and enough water to yield a final volume of 2.0 mL were added to individual vials. The blank sample was prepared by mixing 1.5 mL of the Bradford or biuret and 0.5

MATERIALS AND METHODS

Preparation of Chemicals

The Bradford reagent was prepared with Coomassie Blue G250 (Sigma), methanol (Sigma), phosphoric acid (Macron), and water according to the following protocol: (1) Dissolve 50 mg Coomassie Blue G-250 in 50 mL methanol. (2) Add 100 B

DOI: 10.1021/acs.jchemed.6b00676 J. Chem. Educ. XXXX, XXX, XXX−XXX

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Figure 2. Photographs of calibration samples plus an unknown sample for smartphone colorimetry. (A) Biuret standards for calibration and “unknown” sample in front of a white background. (B) Biuret standards for calibration and “unknown” sample in front of a green (129, 255, 0) background. (C) Biuret standards for calibration and “unknown” sample in front of a yellow-orange (255, 207, 0) background.

Figure 3. Comparison of colorimetric assays by UV−vis spectrophotometry and smartphone colorimetry. (A) Photograph of Bradford assay cuvettes in front of 595 nm (RGB: 255, 207, 0) colored background. (B) Graph of absorbance (via spectrophotometry) versus protein concentration. (C) Graph of absorbance (via smartphone colorimetry following R values) versus protein concentration. (D) Photograph of biuret assay cuvettes in front of 540 nm (RGB: 129,255,0) colored background. (E) Graph of absorbance (via spectrophotometry) versus protein concentration. (F) Graph of absorbance (via smartphone colorimetry following G values) versus protein concentration. In all plots, blue diamonds refer to protein calibration, and the red circle indicates the “unknown” sample at its expected concentration.

equilibration time is important for the color to fully develop. Insufficient equilibration time will often lead to poorer results. An example of this time-dependent effect is shown in the teacher’s guide in the Supporting Information.

mL of water. Experiments can all be performed with regular tap water. As noted in the sample student guide (Supporting Information), these low volumes of protein solution can be delivered into the individual square plastic cuvettes (1 cm path length) by counting drops of the solution and weighing the sample using a balance after each addition to account for the actual amounts added. This approach works well since the densities of each solution and water are all nearly identical. Further, if students do not have access to a balance, counting drops and converting volume to mass can also provide adequate data, as noted in the Results section. Absorbance measurements were performed 40 min after mixing for the Bradford and 20 min after mixing for the biuret samples in order to ensure full development of the color change. The “unknown” protein concentration sample was prepared in a similar manner, with the estimated concentration falling within the linear range of the colorimetric agents. The “unknown” protein concentration in the sample Bradford assays was 0.014 mg/mL Brd4 as determined by UV absorption (ε280: 26,930 M−1 cm−1) and 1.25 mg/mL BSA by mass in the sample biuret assay. Because the change in color is a time-dependent process, the

Instrumentation and Setup

Cuvettes containing the standard, blank, and “unknown” samples were analyzed using both a standard UV−vis spectrophotometer and smartphone colorimetry. For absorption measurements, samples were analyzed at 595 nm for Bradford and 540 nm for biuret using a Beckman Coulter DU 720 UV−vis spectrophotometer. Acquiring Images

For smartphone colorimetry, cuvettes were lined up in front of a computer monitor, and images were acquired using the back facing lens of varying smartphones (Samsung Galaxy S5, iPhone 6). Cuvettes were lined up in front of a computer monitor with varying background colors matching the wavelengths measured for absorption in addition to a white background (Figure 2). For Bradford samples, an RGB code of (255, 207, 0) was used, while (129, 255, 0) was used for the C

DOI: 10.1021/acs.jchemed.6b00676 J. Chem. Educ. XXXX, XXX, XXX−XXX

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estimating a given protein concentration using Beer’s Law and a standard calibration curve. Smartphone colorimetry data were comparable to data collected using the spectrophotometer (Figure 3B,C). In the example of the Bradford assay above (Figure 3A−C), the “unknown” concentration was 0.014 mg/mL, and the UV−vis absorption measurements yielded an estimated concentration of 0.011 mg/mL while a concentration of 0.0090 mg/mL was estimated via camera phone colorimetry. In the example of the biuret assay (using the tartrate-containing solution) (Figure 3D−F), the “unknown” concentration was 1.25 mg/mL, and the UV−vis absorption measurements yielded an estimated concentration of 1.32 mg/mL while a concentration of 1.28 mg/mL was estimated via camera phone colorimetry. Images acquired in front of backgrounds displaying the color matching the maximum absorption wavelength of the color agent yielded more accurate results as compared to images of the samples arranged in front of a white display. However, if a backlit background is not available, a plain white background is suitable for collecting quantitative measurements. Kuntzleman and Jacobson also reported good success using colored construction paper in lieu of a colored backlit screen for similar types of measurements.11 The utility of this method lies not only in its efficacy for determining protein concentration via the measurement of a color change but also in its robustness to a range of factors, including lighting conditions and picture quality, which can vary dramatically from one classroom to another. Given the camera quality found in the average smartphone, this type of protein quantification colorimetry experiment is now more widely accessible to students who may have previously been unable to perform such an experiment. As a final assessment of the suitability of the experiment for a high school classroom as well as the clarity of the instructions for the experiment, the method was tested with a group of tenth grade high school chemistry students. The lab was conducted with two different class sizes of 25 and 15 students, respectively, and run over three 50 min class periods. The first class period was used to introduce the background for the lab and demonstrate the experimental procedure which can be found in the Supporting Information. The second period was used to prepare the solutions and capture the image using smartphones. During the last day, the images were analyzed and the results discussed. In order to ensure the method is easily reproducible in any high school setting, a few changes were made from the original protocol. For high school implementation we used the biuret and albumin as they are available from Flinn Scientific. Also, because many laboratories may not have access to protein expression facilities, using an unknown concentration of albumin, or other commercially available proteins (e.g., lysozyme or cytochrome C), is a more practical alternative than Brd4. For this study, six groups had access to electronic balances with centigram precision. In order to balance time efficiency and reagents, the students worked in groups of five. The two groups without access to balances were provided with a conversion from drops of solution to mass for each of the solutions. Analysis of the students’ data pointed to several areas for further clarification in the protocol. At this point, all students had demonstrated a strong grasp of Beer’s law; however, analysis of the results show that only four of the eight groups produced an unknown concentration that agreed within 10% of

biuret. These values can be obtained using an online wavelength to RGB conversion tool (e.g., Academo’s Wavelength to Color Relationship15). Data Analysis and Comparison

Absorption data from the spectrophotometer were plotted using spreadsheet software and fitted to a linear trendline. The generated equation was used to estimate the protein concentration. Absorbance measurements from the smartphone images were obtained by analyzing RGB (red, green, blue) values from images of each sample (intensity range 0−255). A variety of software, freeware, and web applications (e.g., Adobe Photoshop, ImageJ, Color Code Picker)5 as well as smartphone applications (e.g., Colorometer for iPhone and ColorMeter Free for Android)11 are available for this type of analysis and provide similar results. Download links can be found in the Supporting Information. RGB values can be obtained by analyzing single points or averaging multiple points over a specified area. Single point and area measurements yield nearly identical results, as noted in a previous report.5 Absorbance (A) is calculated from RGB values using the following formula: ⎛ I ⎞ A = −log⎜ n ⎟ ⎝ Iblank ⎠

(1)

Here, In corresponds to the R, G, or B value of each sample, and Iblank corresponds to the R, G, or B value for the blank. For the Bradford assay, the best results were obtained using the R values while for the biuret assay, the best results were obtained using the G values. For alternative colorimetric experiments or assays, it would be best to follow whichever channel (R, G, or B) provides the greatest dynamic range. These “absorbance” values were plotted as a function of concentration and fit to a linear trendline to estimate protein concentration of the “unknown” sample.

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HAZARDS The Bradford solution is acidic while the biuret solution is basic. Both are corrosive and should be handled with care. Both solutions should be neutralized appropriately prior to disposal down the drain with excess water. Alternatively, solutions can be disposed of in separate waste containers as appropriate. Protective gloves and eyewear (preferably splash proof goggles) in addition to standard lab-appropriate attire (long pants and closed-toed shoes) should be worn at all times while handling Bradford or biuret reagents.

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RESULTS Samples from both the Bradford and biuret assays were evaluated by a UV−vis spectrophotometer and by smartphone colorimetry to benchmark a newer technique against an established technique. Calibration curves generated R2 values greater than 0.9, and using the equation generated from fitting a linear trendline, the concentration of protein in the “unknown” sample was calculated (Figure 3).

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DISCUSSION The use of a smartphone camera is effective for quantitative colorimetry measurements. It can be used for the appearance of color, as previously reported,5−10 and also for a color change, as described here. The obtained data were adequate for correctly D

DOI: 10.1021/acs.jchemed.6b00676 J. Chem. Educ. XXXX, XXX, XXX−XXX

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by the NSF-CAREER Award CHE-1352091. C.T.G. would like to acknowledge the University of Minnesota Interdisciplinary Doctoral Fellowship.

the unknown protein concentration. The remaining four groups had considerably larger errors. It should be noted that positive results were independent of sample preparation method. In the four groups that yielded accurate results and in the four that yielded poor data, three groups prepared samples with a balance and one group counted drops. After reviewing the students’ work, it became clear that some groups whose analysis fell outside of 10% of the accepted concentration had not taken images of suitable quality for analysis. A common error was taking the picture from too far away, leaving the color intensity in the image washed out, as the area of interest was very bright relative to the background. Poor image focus also led to unreliable data. To alleviate these problems for future implementation, examples of good and poor images for students have now been added to the instructional guide which can be found in the Supporting Information. In general, the instructor felt the results were of similar quality to those from other quantitative methods carried out by high school students at this level. The use of photocolorimetry, Beer’s Law, and protein topics aligns well with courses introducing content that uses the Next Generation Science Standards, HS-LS1-1, regarding content on protein function, as well as HS-PS4-5 which describes how some technological devices use the principles of wave behavior and wave interactions with matter to transmit and capture information and energy.

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CONCLUSION Smartphones and other portable cameras were successfully used to determine the concentration of a protein sample using assays that yield a color change as a function of protein concentration. This new method for measuring protein concentrations allows for greater accessibility to such colorimetric assays for introductory laboratories and laboratories lacking expensive equipment like spectrophotometers. This type of experiment also fits in well with the Science and Engineering Practices from the HS-LS1 section of the NGSS.1 A detailed step-by-step procedure for the experiment suitable for use in high school or college settings is provided in the Supporting Information.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00676. Sample student guide, providing step-by-step instructions, as well as some instructor notes (PDF, DOCX)

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REFERENCES

(1) Next Generation Science Standards. http://www.nextgenscience. org/ (accessed Mar. 2017). (2) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.; West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E. Selective inhibition of BET bromodomains. Nature 2010, 468 (7327), 1067−73. (3) Thermo Scientific Pierce Protein Assay Technical Handbook, 2nd ed.; Thermo Fisher Scientific: Waltham, MA, 2010. (4) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to Measure and Predict the Molar Absorption-Coefficient of a protein. Protein Sci. 1995, 4 (11), 2411−2423. (5) Kehoe, E.; Penn, R. L. Introducing Colorimetric Analysis with Camera Phones and Digital Cameras: An Activity for High School or General Chemistry. J. Chem. Educ. 2013, 90 (9), 1191−1195. (6) Knutson, T. R.; Knutson, C. M.; Mozzetti, A. R.; Campos, A. R.; Haynes, C. L.; Penn, R. L. A Fresh Look at the Crystal Violet Lab with Handheld Camera Colorimetry. J. Chem. Educ. 2015, 92 (10), 1692− 1695. (7) Campos, A. R.; Knutson, C. M.; Knutson, T. R.; Mozzetti, A. R.; Haynes, C. L.; Penn, R. L. Quantifying Gold Nanoparticle Concentration in a Dietary Supplement Using Smartphone Colorimetry and Google Applications. J. Chem. Educ. 2016, 93 (2), 318− 321. (8) Montangero, M. Determining the Amount of Copper(II) Ions in a Solution Using a Smartphone. J. Chem. Educ. 2015, 92 (10), 1759− 1762. (9) de Morais, C. d. L. M.; Silva, S. R. B.; Vieira, D. S.; Lima, K. M. G. Integrating a Smartphone and Molecular Modeling for Determining the Binding Constant and Stoichiometry Ratio of the Iron(II)− Phenanthroline Complex: An Activity for Analytical and Physical Chemistry Laboratories. J. Chem. Educ. 2016, 93 (10), 1760−1765. (10) Moraes, E. P.; da Silva, N. S. A.; de Morais, C. d. L. M.; Neves, L. S. d.; Lima, K. M. G. d. Low-Cost Method for Quantifying Sodium in Coconut Water and Seawater for the Undergraduate Analytical Chemistry Laboratory: Flame Test, a Mobile Phone Camera, and Image Processing. J. Chem. Educ. 2014, 91 (11), 1958−1960. (11) Kuntzleman, T. S.; Jacobson, E. C. Teaching Beer’s Law and Absorption Spectrophotometry with a Smart Phone: A Substantially Simplified Protocol. J. Chem. Educ. 2016, 93 (7), 1249−1252. (12) Moraes, E. P.; Confessor, M. R.; Gasparotto, L. H. S. Integrating Mobile Phones into Science Teaching To Help Students Develop a Procedure To Evaluate the Corrosion Rate of Iron in Simulated Seawater. J. Chem. Educ. 2015, 92 (10), 1696−1699. (13) Gornall, A. G.; Bardawill, C. J.; David, M. M. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 1949, 177 (2), 751−766. (14) Mishra, N. K.; Urick, A. K.; Ember, S. W.; Schonbrunn, E.; Pomerantz, W. C. Fluorinated Aromatic Amino Acids Are Sensitive F NMR Probes for Bromodomain-Ligand Interactions. ACS Chem. Biol. 2014, 9 (12), 2755−2760. (15) Wavelength to Colour Relationship. https://academo.org/demos/ wavelength-to-colour-relationship/ (accessed Mar. 2017).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

William C. K. Pomerantz: 0000-0002-0163-4078 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Alex Ayoub for his suggestion of substituting potassium sodium tartrate with sodium citrate as a cheaper alternative reagent. This project was funded in part E

DOI: 10.1021/acs.jchemed.6b00676 J. Chem. Educ. XXXX, XXX, XXX−XXX