In the Laboratory
Assembly of a Modular Fluorimeter and Associated Software: Using LabVIEW in an Advanced Undergraduate Analytical Chemistry Laboratory W. Russ Algar, Melissa Massey, and Ulrich J. Krull* Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto–Mississauga, Mississauga, ON, L5L 1C6, Canada; *
[email protected] computer NI USB-6008 GND
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Figure 1. Schematic of the modular fluorimeter. Key components are the LED light source, colored glass filter, photodiode and amplifier, DAQ module (NI USB-6008), and a computer with LabVIEW installed.
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Although LabVIEW is more novice-friendly than line code, it is sufficiently powerful for use by experts in instrumentation and interfacing. In an educational setting, LabVIEW has been used to teach chemistry students the key concepts of instrumentation and interfacing through electrochemistry experiments (5), UV–visible spectroscopy (6), and calorimetry and chromatography (7). In a third-year instrumental analysis course at the University of Toronto, we have developed a laboratory activity that teaches LabVIEW and interfacing skills by building a simple fluorimeter. Students work in groups of two or three and the activity typically requires 16–20 hours of laboratory time. This includes roughly eight hours of learning and applying basic LabVIEW skills. Students focus on the concepts of interfacing, although the lab also serves as a basic introduction to fluorimetry. Instrument and Software Design The modular fluorimeter is intended to be simple in design. This keeps the focus on LabVIEW and interfacing, while allowing the most efficient use of lab time. Figure 1 shows a schematic diagram of a typical setup. The light source is an array of four blue LEDs and a silicon photodiode is used as the detector. A colored glass long-pass filter is used to block scattered sourcelight from reaching the detector. The LED emission spectrum and absorption spectrum of the colored glass filter are shown in Figure 2. Fluorescein is a very bright fluorophore that works well
Normalized Absorbance or Emission
Computer-controlled instruments are ubiquitous in modern chemistry. Desktop computers have revolutionized the way scientists perform experiments, allowing them to automate tasks for greater speed, precision, and multitasking. Computers are able to control instruments and dramatically increase the efficiency in which data are recorded, processed, and displayed. Chemists must now be able to operate, maintain, and even develop instrumentation. Therefore, teaching the concepts of instrumentation and instrument control is essential in training new chemists. We have integrated LabVIEW into teaching instrumentation to undergraduate students. The LabVIEW programming language from National Instruments (Austin, TX) is the interfacing tool of choice in both academia and industry. Computer interfacing through digital-to-analog conversion (DAC) and analog-to-digital conversion (ADC) provides the necessary intermediary between digital computers and analog instrument components. For example, LabVIEW is used in time-correlated, single-photon counting systems (1), clinical monitoring (2), laser scanning microscopes (3), and surface science techniques such as X-ray photoelectron spectroscopy (4). LabVIEW is based on graphical code (G-code) rather than conventional, syntax-based line code used in languages such as C or BASIC. Programs written in LabVIEW are built on a front panel using virtual controls and indicators. The underlying code is created on a block diagram that largely resembles an electronic circuit, and determines the flow of data through virtual wires and modular functions.
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Wavelength / nm Figure 2. Normalized spectra illustrating that fluorescein is ideal for use with the home-built fluorimeter. Note the overlap between the LED emission (λmax = 460 nm) and fluorescein absorption. Also note how the filter absorption (λcutoff = 495 nm) is well-separated from the fluorescein emission yet overlaps with the LED emission.
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
with the LED and filter combination used. The absorption and fluorescence spectra of fluorescein are also shown in Figure 2. All components are interfaced through LabVIEW and a 12-bit USB data acquisition (DAQ) module. In LabVIEW, students are asked to write a program that can modulate the light source, read, display, and average data from the photodiode, automatically subtract the blank or background signal, and save the collected data. A screenshot of the front panel and block diagram for a basic LabVIEW program that satisfies these requirements is shown in Figure 3.
Hazards This lab presents no special hazards. Standard lab safety practices should be used, including safety goggles, lab coat, and gloves. Use care to avoid getting any electronic equipment wet. Experiments Once the fluorimeter is built and the software developed, students test the instrument by measuring a calibration curve for fluorescein concentration and are then asked to perform a sec-
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Figure 3. (A) Block diagram and (B) front panel for a LabVIEW program written to control the operation of the modular fluorimeter.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 1 January 2009 • Journal of Chemical Education
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In the Laboratory
Discussion The LabVIEW interfacing laboratory activity replaced an activity in which students were asked to analyze an infrared spectrum by programming a simulation of the rotational spectrum of hydrogen chloride using BASIC. Feedback from students suggested that they did not embrace programming. Students found that this previous module was tedious and the programming language was awkward. In contrast, feedback regarding the LabVIEW interfacing activity consistently suggests that it is one of the more challenging, but also more rewarding activities in the course. Although students with little programming experience initially feel some trepidation towards the assigned task, they quickly find that the learning curve is gentle and the task manageable. Aside from the very practical skills learned by the students, a major advantage of this activity is that it tends to demystify the “black box” aspect of most instruments and encourages students to think about how their data are collected rather than just their value. If, in future employment or graduate studies, students are required to develop or modify instrumentation to meet the needs of an experiment, the experience of this activity will be very valuable. The main drawback of the activity is the potentially high cost of the initial implementation and the amount of laboratory time required. The main expenses include a desktop computer, USB acquisition modules, and software required for each station. Although other components are individually inexpensive, their combined total is approximately $1000–2000 per station, depending on the resources already available. Time constraints can be dealt with by providing more or less resources to the students. The costs and possible remedies to time constraints are discussed in the online supplement. A large variety of learning opportunities are found within this laboratory activity. In addition to its function as an introduction to interfacing and LabVIEW, we have found that the activity can be used to introduce students to basic electronics. Although we provide a photodiode amplifier for the students to use, we also provide them with a circuit diagram and an “open box” amplifier for inspection. This is often the first exposure many chemistry students have to the practical application of resistors, capacitors, and operational amplifiers in chemistry laboratories. Instructors tend to briefly review how these electronic components work and how they can be combined to create circuits such as current-to-voltage converters. Furthermore, students are also expected to learn how photodiodes, LEDs, and other components of the fluorimeter work. The most important result of this experiment is a working fluorimeter, which is demonstrated by the calibration curve. 70
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Fluorescence Signal / V
ond experiment. Suggested second experiments include an investigation of the Stern–Volmer quenching of fluorescein by iodide, or the pH dependence of the fluorescence of fluorescein. A typical calibration curve is shown in Figure 4: this was prepared by measuring serial dilutions of fluorescein in basic buffer solution. Students fit the linear portion of the calibration curve and determine the limit of detection, limit of linearity, and dynamic range. For data in Figure 4, the LOD is 0.09 ± 0.03 μM, the limit of linearity is estimated to be 1.25 μM, and thus the dynamic range is from 0.09–1.25 μM. Details concerning experimental methods and results for the Stern–Volmer quenching of fluorescein by iodide and the pH-dependent fluorescence of fluorescein can be found in the online supplement.
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Fluorescein Concentration / (Nmol/L) Figure 4. Calibration curve for the measurement of fluorescein using the modular fluorimeter. The linear region of the calibration curve is indicated in the figure. Beyond the limit of linearity, the photodiode detector is saturated. The LOD is 0.09 ± 0.03 μM of fluorescein.
The secondary pH and quenching experiments allow students to use their validated fluorimeter to study a chemical system. Students are encouraged to elaborate on the chemistry of these experiments in the discussion section of their lab reports. For example, students can discuss the Stern–Volmer quenching of fluorescein-labeled amino acid residues by iodide to probe solvent accessibility in proteins (8–11). Similarly, fluorescein has an ionization equilibrium and pH-dependent spectroscopy that can be discussed (12, 13). From the results of these experiments, students can extract values for the bimolecular quenching constant and pKa of fluorescein that often show good agreement with published values. In addition to the experiments we have suggested, students in our course have also studied the temperature dependence of fluorescence, constructed an absorption photometer, and constructed a dual-beam fluorimeter. Summary We have described a laboratory activity for an upper-level undergraduate course in instrumental analysis that teaches the fundamentals of LabVIEW and interfacing through construction of a fluorimeter from modular components. Students use a DAQ module and LabVIEW to interface a desktop computer with a blue LED light source and photodiode detector. In addition to measuring a calibration curve for fluorescein, students have investigated the Stern–Volmer quenching of fluorescein by iodide and the pH dependence of fluorescein fluorescence. Literature Cited 1. Stryjewski, W. J. Rev. Sci. Instrum. 1991, 62, 1921–1925. 2. Kalkman, C. J. J. Clin. Monit. 1995, 11, 51–58. 3. Sridhar, M.; Basu, S.; Scranton, V.; Campagnola, P. J. Rev. Sci. Instrum. 2003, 74, 3474–3477. 4. Cautero, G.; Paolucci, G.; Brena, B.; Agostino, R. G.; Tommasini, R.; Comelli, G.; Rosei, R. Meas. Sci. Technol. 1995, 5, 1002–1011. 5. Gostowski, R. J. Chem. Educ. 1996, 73, 1103–1107. 6. Drew, S. M. J. Chem. Educ. 1996, 73, 1107–1111. 7. Muyskens, M. A.; Glass, S. V.; Wietsma, T. W.; Gray, T. M. J. Chem. Educ. 1996, 73, 1112–1114. 8. Nalefski, E. A.; Falke, J. J. Biochem. 1998, 37, 17642–17650. 9. Albani, J. R. Biochim. Biophys. Acta 1998, 1425, 405–410. 10. Swindlehurst, C. A.; Voss, E. W., Jr. Biophys. J. 1991, 59, 619–628.
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory 11. Watt, R. M.; Voss, E. W. J. Biol. Chem. 1979, 254, 1684–1690. 12. Sjoback, R.; Nygren, J.; Kubista, M. Spectrochim Acta A 1995, 51, L7–L21. 13. Klonis, N.; Sawyer, W. H. J. Fluoresc. 1996, 6, 147–157.
Supplement
Detailed experimental procedures; Quenching and pH experiments; Results from the experiments; Instrument photographs; Circuit diagrams; Detailed description of the LabVIEW software program
http://www.jce.divched.org/Journal/Issues/2009/Jan/abs68.html
Student handouts
Abstract and keywords
Instructor notes from the authors’ experiences
Full text (PDF) with links to cited JCE articles
Prelaboratory questions and answers
Supporting JCE Online Material
JCE Concept Connections: Science Underlying 2008 Nobel Prizes JCE offers a wealth of materials for teaching and learning chemistry that you can explore at our Web site, JCE Online (http://www.jce.divched.org). In the list below, Bernadette Caldwell of the Editorial Staff suggests additional resources that are available through JCE for teaching the science behind some of the 2008 Nobel Prizes. Discovering and Applying the Chemistry of GFP The Royal Swedish Academy of Sciences awarded the 2008 Nobel Prize in Chemistry “for the discovery and development of the green fluorescent protein, GFP” to three scientists: Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien. These scientists led the field in discovering and introducing a fluorescing protein from jellyfish into cells and genes under study, which allows researchers to witness biochemistry in action. Now tags are available that emit light in different colors, revealing myriad biological processes and their interactions simultaneously. Identifying HPV and HIV, HIV’s Replication Cycle, and HIV Virus−Host Interactions The Nobel Assembly at Karolinska Institutet awarded the 2008 Nobel Prize in Medicine or Physiology “for their discovery of human immunodeficiency virus” (HIV) to two scientists: Françoise Barré-Sinoussi and Luc Montagnier; and “for his discovery of human papilloma viruses [HPV] causing cervical cancer” to one scientist, Harald zur Hausen. Diseases caused by these infectious agents significantly affect global health. While isolating and studying the virus, researchers discovered HIV is an uncommon retrovirus that infects humans and relies on the host to make its viral DNA, infecting and killing the host’s white blood cells, ultimately destroying the immune systems of infected humans. Related Resources at JCE Online The Journal has published articles relating to GFP specifically, and more generally to fluorescing compounds applied to biochemistry. The Journal has also published an article and a video on protease inhibition—a strategy to suppress HIV’s biological processes. With the video clips, an accompanying guide for teachers includes instructions for three student activities that use enzymes. The resources below may help introduce students to the science behind some of these Nobel Prizes. Turning on the Light: Lessons from Luminescence. O’Hara, P. B.; Engelson, C.; St. Peter, W. J. Chem. Educ. 2005, 82, 49. (See especially the bioluminescence section on page 51 that concisely explains GFP.) JCE Classroom Activity #68: Turning on the Light. O’Hara, P. B.; Engelson, C.; St. Peter, W. J. Chem. Educ. 2005, 82, 48A. JCE Classroom Activity #81: pHantastic Fluorescence. Muyskens, M. J. Chem. Educ. 2006, 83, 768A. Recombinant Green Fluorescent Protein Isoforms: Exercises To Integrate Molecular Biology, Biochemistry, and Biophysical Chemistry. Hicks, B. W. J. Chem. Educ. 1999, 76, 409. C-SNARF-1 as a Fluorescent Probe for pH Measurements in Living Cells: Two-Wavelength-Ratio Method versus Whole-Spectral-Resolution Method. Ribou, A-C.; Vigo, J.; Salmon, J-M. J. Chem. Educ. 2002, 79, 1471. An Attack on the AIDS Virus: Inhibition of the HIV-1 Protease: New Drug Development Based on the Structure and Activity of the Protease and Its Role in the Replication and Maturation of the Virus. Volker, E. J. J. Chem. Educ. 1993, 70, 3. From Chemistry Comes Alive!, five video clips demonstrate properties and mechanisms involved in the chemistry of HIV. HIV-1 Protease: An Enzyme at Work. http://www.jce.divched.org/JCESoft/jcesoftSubscriber/CCA/CCA5/MAIN/2BIOCHEM/BIOCHEM2/HIV/ MENU.HTM
All articles from Volume 1 to the current issue are available in full-text PDF at JCE Online. For browsing by year, month, and page, go to http://www.jce.divched.org/Journal/Issues/index.html. For title and author searching of all issues of JCE, go to http://www.jce.divched.org/Journal/Search/index.html.
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