Learning about Fluorescence in Undergraduate Biochemistry: Enzyme Kinetics Using a Low-Cost, Student-Built Fluorescence Spectrometer

Abstract

Techniques and instrumentation that involve fluorescence are used in many careers related to biology and biochemistry. However, due to the expense of instrumentation, the theory and practice of fluorescence spectroscopy is commonly concentrated in instrumental analysis in most curricula, a class rarely required for biology and biochemistry majors. Biochemistry represents a course taken by these majors, providing an opportunity to expose them to the fundamentals of fluorescence. A three session laboratory series has been developed where the kinetic analysis of lactate dehydrogenase is carried out through the use of inexpensive, student-built fluorometers. These instruments are optimized for the detection of NADH fluorescence and constructed with interlocking building blocks (such as LEGO blocks), an LED source, a phototransistor detector, and other commercially available materials for a total cost of $52. Biochemistry students have built these spectrometers and used them to determine the K m of Gallus gallus lactate dehydrogenase for L-lactate and the k cat of this enzyme in accordance with literature values. Learning outcomes were assessed and indicate proficient understanding of instrument use and design.

A Low-Cost Dual-Beam Smartphone Visible Spectrometer

“…Nevertheless, a digital camera attached to a tripod is essential for this design, resulting in a bulky device. For this reason, more compact and affordable designs were proposed by replacing the costly high-photosensitivity detector with a typical webcam. − Unfortunately, these webcam-based spectrometers still require an additional power supply and a computer for signal processing and display. Thus, on-site and rapid investigation using a spectrometer is limited.…”

An Instrument Assembly and Data Science Lab for Early Undergraduate Education

“…The modern workforce and academic research have become more intensively data-science-driven, requiring skills ranging from fluency in spreadsheets at the most basic level to Python programming or high-throughput data science tools. , Upper division chemistry curricula have responded with coursework in instrumentation and advanced data analysis, , often associated with an analytical, physical, or biochemistry lab . With advanced analysis and computational skills now critical in a variety of fields, there is a growing need to find affordable ways to introduce these skills earlier than the junior and senior years in college. , …”

“…2,3 Upper division chemistry curricula have responded with coursework in instrumentation and advanced data analysis, 4,5 often associated with an analytical, 6 physical, 7 or biochemistry lab. 8 With advanced analysis and computational skills now critical in a variety of fields, there is a growing need to find affordable ways to introduce these skills earlier than the junior and senior years in college. 9,10 By the time most students start to see a more in-depth connection between data science and instrumentation in areas like spectroscopy, they may have already fulfilled their chemistry requirements.…”

Taking School Instrumentation One Step Forward: A Do-It-Yourself Type Spectrophotometer and a Jupyter Notebook That Enable Real Time Spectroscopy during School Lessons

“…The introduction of spectroscopy in schools worldwide faces a significant challenge: the commercially available analytical instruments are relatively expensive. Moreover, even when available, their educational impact is up to a point questionable, since there is always the problem of students viewing them as black boxes. − In order to diminish instrument cost and properly discuss instrumentation in the classroom, the construction of several do-it-yourself (DIY)-type devices has been reported in the past decade, e.g., spectrophotometers, ,− single wavelength spectrophotometers, − fluorometers, ,,,,,,− atomic emission spectrophotometers, polarimeters, nephelometers, and Raman spectrometers, , many of which contain “smart” phones as optical detectors. − Despite the fact that most of them are well built, their main drawback, when the discussion comes to full wavelength scanning, is that they require time-consuming postprocessing of the acquired spectral images, e.g., with ImageJ, Spectral Workbench, or other relative software.…”