Multiple Experiments and a Single Measurement: Introducing

Chemical Education Today edited by

Michelle Bushey

Multiple Experiments and a Single Measurement: Introducing Microplate Readers in the Laboratory

Department of Chemistry Trinity University San Antonio, TX 78212

by Santiago Botasini, Laura Luzuriaga, , and Eduardo Me ndez* María F. Cerda Laboratorio de Biomateriales, Instituto de Química Biol ogica, Facultad de Ciencias, Universidad de la Rep ublica, 11400 Montevideo, Uruguay *[email protected] by Gerardo Ferrer-Sueta and Ana Denicola Laboratorio de Fisicoquímica Biol ogica, Instituto de Química Biol ogica, Facultad de Ciencias, Universidad de la Rep ublica, 11400 Montevideo, Uruguay

Microplate readers are well-known instruments in biochemistry laboratories (Figure 1). This laboratory instrument is designed to detect biological, chemical, or physical events of samples in microtiter plates. The most common format is a 96well plate; however, some microplates have 6-1536 wells, with volumes in the range of 5-200 μL. Common detection modes include absorbance, fluorescence intensity, and luminescence. As the cost of microplate readers falls, they are likely to be incorporated into general chemistry laboratories. Their adoption will challenge chemistry departments to both adapt and improve on existing laboratory practices. In this article, we will show how microplate readers can become a fundamental tool in general, analytical, physical, and biological chemistry laboratories. Microplate readers allow students to gather more data in less time than many instruments current used, allowing for more time to perform data treatment. It is a common challenge to introduce data treatment as an inherent part of chemical experimentation. Computers, data loggers, and analog devices are now common in general university laboratories; their presence makes data processing fairly simple and quick. For this reason, the slowest step in the lab is not treatment of data but obtaining them. Additional concerns in today's lab include the reduction of consumables and waste materials aligned with the concepts of microscale and green chemistry. Thus, reducing sample volume without sacrificing detection represents a major advantage for the teaching lab.

one can devise a lab exercise that uses microplate readers to gather data on the iron(III)-thiocyanate system (5, 6). Such a lab could even use the Job method to determine the stoichiometry of the iron(III)-thiocynate complex (7). In biochemistry, a single experiment could gather a complete set of measurements for studying the thermodynamic stability of proteins. (8) Such an experiment would include the construction of calibration curves and the measurement of samples subject to different conditions of denaturing agents concentration such as urea and guanidinium chloride. Because the experiments mentioned above involve considerable data, the new laboratory exercises might be accompanied by a thorough data treatment, including average- and standarddeviation calculations, model fittings, and statistical tests. Electronic spreadsheets are highly recommended for this purpose. Are Microplate Readers Costly? A Brief Cost-Benefit Analysis Recently, a desktop scanner and digital image analysis was proposed (9) to analyze data from 96-well microplates. This

From Industrial and Clinical Labs to the University Microplate readers are well-established in industrial and clinical settings. This fact alone is enough to consider including them in university teaching laboratories. But a survey of educational journals shows that the number of proposed laboratory exercises that use microplate readers is limited; rather, they are mainly used in biochemical applications such as protein assay (1), drug discovery (2), high-throughput screening (3), and enzyme kinetics (1, 4). It is easy to imagine developing new exercises or adapting existing ones for microplate readers. For example, the study of chemical equilibrium could be carried out by including the calibration curve of the additive property, and the assessment of the effect of concentration and ionic strength. For example,

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Figure 1. Student measuring chemical induction of protein denaturation using a microplate reader (UT-2100C from MRC, Ltd.). (Photograph by one of the authors, María F. Cerd a.)

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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 87 No. 10 October 2010 10.1021/ed100789j Published on Web 08/12/2010

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_ 30 s for a 96-well plate http://www.biotek.com

Read time

Company URL (all accessed Jul 2010)

http://www.mrclab.com

5-15 s for a 96-well plate

5 s for a 96-well plate http://www.perbio.com

96 well

(2% from 0-2 Abs

(1% from 0-2 Abs

0.001 Abs

0-3.500 Abs

Substitute filters between 400-700 nm available upon request

96 well

(2% from 0-2 Abs

MRC, Ltd./ UT-2100C Standard filters: 405, 450, 492, and 630 nm

Estimated prices range widely: 2500-13,500 (in 2010 U.S. dollars). Consult the companies for more details.

6, 12, 24, 48, and 96 well

Plate types

a

(1% from 0-2 Abs

(1% from 0-2 Abs

(1% from 0-2 Abs

Accuracy

Linearity

0.001 Abs

0-3.500 Abs

0-3.000 Abs 0.001 Abs


Absorbance range


Wavelength range

Standard filters: 405, 450, and 620 nm

Thermo Scientific/ Multiskan EX

Resolution

4 standard filters (not specified)

Biotek Instruments/ELx800

Wavelength selection

Features

http://www.daigger.com

30 s for a 96-well plate

24, 48, and 96 well

(1% from 0-2.000 Abs at 405 mm

(1%; ( 0.010 Abs from 0-2.000 Abs at 405 mm

0.001 Abs

http://www.midsci.com/

Reads and prints absorbances of 96 wells in ∼2 min

96 well

Not published

1% or better

Not published

-0.20-3.00 Abs

Substitute filters between 340-630 nm available

400-750 nm

0.000-3.000 Abs

Standard filters: 405, 450, 492, and 630 nm

Midwest Scientific/ Stat Fax 2100 Microplate

Standard filters: 405, 450, 490, and 630 nm

Daigger/Automated Microplate Reader

Manufacturer/Model of Instrument

Table 1. Comparison of Some Technical Specifications of Commercially Available Microplate Readersa

Chemical Education Today

r 2010 American Chemical Society and Division of Chemical Education, Inc.

Chemical Education Today Table 2. Common Supplies and Cost Estimates for Working with Microplate Readers Varieties (96-Well Options)

Item Microplates

Micropipets

Microplate washer

Approximate Cost (U.S. Dollars)/Units

Transparent polypropylene

340/100

Transparent polyprostyrene

200/100

UV transparent

900/100

Transparent PVC

150-200/100

Black (for fluorescence)

350/100

Single channel

180-300

8 Channel

800

12 Channel

1000

;

1850

proposal argued that the costs associated with microplate readers might be too high to allow for widespread use in university laboratories, based on basic unit costs of around $10,000. (See Table 1 for model comparisons and company Web site URLs.) However, simple reasoning lets us reach the opposite conclusion: How many spectrophotometers are needed for a 20-student group to perform 96 measurements? A microplate reader can gather that number of measurements in less than 1 min. Hence, perhaps it is unnecessary for a lab to have as many pieces of equipment as are usually needed when using common spectrometers (a student basic unit typically costs about $3000). Microplate readers are advantageous in other ways. For example, their use can greatly reduce reagent consumption. This in turn can improve lab safety, reduce waste, and allow for the incorporation of green chemistry and microscale concepts in the teaching activities. Models and Availability The most basic microplate readers consist of a simple filter colorimeter capable of measuring several fixed wavelengths between 400 and 700 nm (Table 1). These apparatuses were originally designed for the routine testing of samples by standard color reactions, so the preset wavelengths may not be suitable for every experiment. That said, many readers do allow for the use of custom filters. Typically, standalone microplate readers provide either digital data output via a USB port or printer output. Common upgrades to the basic setup include temperature control, shaking capabilities, and time-programmed measurements for kinetic experiments. For more versatile experiments, users may choose a reader equipped with either a UV lamp (caution: UV-transparent plates are needed!) or a monochromator rather than using filters. Moreover, users can choose alternative detection modes such as fluorescence and luminescence. In fact, fluorescence plate readers are excellent substitutes or complements for spectrofluorometers in the teaching lab. For instance, we have devised two experimental exercises (10, 11), each for a 3-h lab session. In these exercises, considerable time is spent waiting for measurements at the only spectrofluorometer available in the lab; what's more, the spectrofluorometer measures the emissions from just one sample at a time. By using a microplate reader, waiting and measuring time practically vanish. The students can devote more time to preparing the samples, treating the data, and discussing the results.


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Finally, top-of-the-line equipment includes several detection modes, automated monochromators for spectral data acquisition, automated liquid handling, and delivery. Such equipment is of course beyond the means of some chemistry laboratories, especially in a climate of scarce departmental resources. Prices range from less than $3000 for a basic standalone unit that measures absorbance at preset wavelengths to tens of thousands of dollars for advanced research apparatuses. Important Facts To Consider When Using Microplate Readers If a lab does try to incorporate microplate readers into their practice, some caveats should be kept in mind. First, remember that in these spectrophotometers the optic path is not fixed but instead is determined by the volume of sample added to each well. For this reason, when calculating concentrations based on the absorbance measurements, you should run along a calibration curve with a standard of the chromophore. If you want to use the reported extinction coefficient, ελ (M-1 cm-1), remember to determine the pathlength correction factor for the microplate you use. Each well on a plate should have the same volume in order to compare the value of the physical property measured. The problem of volume can be circumvented by (i) using goodquality multichannel pipettes with the appropriate tips and (ii) always checking micropipet reproducibility. This is a particularly important point to consider, as students may be unfamiliar with pipetting or using multichannel pipettes. To reduce pipetting errors, it is advisable to run many replications. Another issue to consider is that thermal equilibrium within the plastic plate is slower than within glass materials (12). This is not a problem so long as enough time is allowed for reaching thermal equilibration. But be careful with long incubation times at relatively high temperatures: Such conditions could differently evaporate the well content and produce artifactual changes in absorbance. Additional care should be exercised when purchasing microplates. Disposable plastic microplates come in several types (Table 2). The surface of the wells can be coated with a substance favoring adsorption of protein for enzyme-linked immunosorbent assay (ELISA) or cell culture (not recommended for simple absorbance readings), or could be plain plastic. There is also diversity in the geometry of the well. Flatbottomed, U-bottomed, and V-bottomed microplate wells are common, and they provide different advantages and challenges in regard to the path length-to-volume ratio. Material is also important, the most usual being polystyrene and PVC. Polypropylene is also available, as well as specialized plates for UV that have an acrylic copolymer bottom window. Even quartz microplates are commercially available (though very expensive!). If fluorescence or luminescence will be used as detection modes (in suitable equipment), then opaque plates have the advantage of reducing optical interference among the wells. At our institution, many of the teaching laboratories are introducing or adapting most of their experimental classes to the microplate readers, owing not only to a significant reduction of reagent use (and thus, cost), waste management, and safety, but also for improving the quality of the exercises, favoring individual work, and showing to students the relevance of data treatment. These laboratories include analytical chemistry, physical chemistry, biochemistry, and enzymology, thus, demonstrating the wide applicability of this tool in the experimental teaching.


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Literature Cited 1. Bevilacqua, V. L. H.; Powers, J. L.; Vogelien, D. L.; Rascati, R. J.; Hall, M.; Diehl, K.; Tran, C. M.; Jain, S. S.; Chabayta, R. J. Chem. Educ. 2002, 79, 1311. 2. Wentland, M. P.; Raza, S.; Gao, Y. J. Chem. Educ. 2004, 81, 398. 3. Taylor, A. T. S. Biochem. Mol. Biol. Educ. 2005, 33, 16. 4. Powers, J. L.; Kiesman, N. E.; Tran, C. M.; Brown, J. H.; Bevilacqua, V. L. H. Biochem. Mol. Biol. Educ. 2007, 35, 287. 5. Cobb, C. L.; Love, G. A. J. Chem. Educ. 1998, 75, 90.

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6. Lewin, S. Z.; Wagner, R. S. J. Chem. Educ. 1953, 30, 445. 7. Hill, Z. D.; MacCarthy, P. J. Chem. Educ. 1986, 63, 162. 8. Walters, J.; Milam, S. L.; Clark, A. C. Methods Enzymol. 2009, 455, 1. 9. Soldat, D. J.; Barak, P.; Lepore, B. J. J. Chem. Educ. 2009, 86, 617. 10. Moller, M.; Denicola, A. Biochem. Mol. Biol. Educ. 2002, 30, 175. 11. Moller, M.; Denicola, A. Biochem. Mol. Biol. Educ. 2002, 30, 309. 12. Curbelo, E.; Cerda, M. F.; Mendez, E. J. Chem. Educ. 2007, 84, 1326.


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Multiple Experiments and a Single Measurement: Introducing

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