Theory and Use of Fluorescent Ion Sensors

Ariana Watson, Biology/Pre-Med/Anthropology/German

In this experiment four samples with different calcium concentrations and the same amount of indo-1 dye are individually placed in a cuvette, a small, clear, tube-like container, then the spectrophotometer sends UV-light through the cuvette to display a graph of the concentration of fluorescence onto a computer. Using the data of the given graph, points can be plotted to show how accurately they align, proving how the intensity of concentration of calcium and indo-1 dye correlates with fluorescence. This process can be used on a variety of sample subjects, however, in this experiment calcium ions will be used. Then using the data of the given graph, points can be plotted to show how accurately they align, proving how the concentration of calcium is affected by indo-1 dye.

What is fluorescence? Fluorescence is the process of using light as a source of excitement, the electrons in the given sample will jump from their typical ground state to a higher, excited energy state. Then, once the excitement wears off and the electrons reach ground state again, they release a photon, thus giving off fluorescence. 

What is spectroscopy? Spectroscopy is the study of how certain substances interact with light to measure composition, concentration, structure, and other physical properties.

Fluorescent spectroscopy is the analysis of the measurement of light emitted by a sample being tested upon using either a spectrometer or spectrophotometer. In my lab, I will be using the latter, since it can clearly identify color after absorption. Using light as a source of excitement, the electrons in the given sample will jump from their typical ground state to a higher, excited energy state. Then, once the excitement wears off and the electrons reach ground state again, they release a photon, thus giving off what we call fluorescence. Calcium ions act as intracellular messengers, making them crucial for multiple processes throughout the body, and when using an ion sensitive fluorescent probe dye such as indo-1, a dual emission calcium indicator, the presence of calcium ions can be easily detected.

1. How do specific concentrations of calcium ions and indo-1 dye show fluourescence using a spectrophotometer?

2. Why is calcium important?

3. What is the importance of ion sensing dyes, such as indo-1?

To begin, four samples of various concentrations of the calcium samples must be collected. I will be using 4.63 pCa and 7.00 pCa as the two extreme polar ends with 5.68 pCa and 6.82 pCa acting as the in-between intervals. Although the same amount of indo-1 dye will be added to each sample cuvette, the concentrations of the calcium buffer solutions will differ per sample. For the lower most extreme, 4.63 pCa, 2.988mL of CaEGTA and .012mL K₂EGTA are added using a pipette. The two middle intervals, 5.68 pCa and 6.82 pCa are composed of 2.8mL CaEGTA and .2 K₂EGTA, and 1.5mL CaEGTA and K₂EGTA respectively. Finally, the high extreme, 7.00 pCa is made up of 1.2 mL CaEGTA and 1.8 mL K₂EGTA. Now they are ready to be analyzed in the spectrophotometer. However, before any measurements can be taken, the spectrophotometer must be properly calibrated. This includes; covering the light sensor with a black darkening cloth to prevent the infiltration of unneeded light, then the computer must be set up and the graphing program, such as SpectraWiz has to be opened first to prevent confusion and system overload on the program and computer. Once the program has been opened, the cord joining the spectrophotometer to the computer can be plugged in, as well as the spectrophotometer outlet cord. To examine each sample, place one cuvette into the cuvette/cell holder located at the top of the spectrophotometer. Then, whilst allowing the UV to shine through the fluid solution, a graph of the individual concentration will appear on the computer program. Each graph must be saved in-between samples so that they may be plotted together to identify their relationship. Once each sample has been examined, every data point will be graphed into Origin and that data will be used to find how the fluorescence of calcium is directly affected by the concentrations of calcium in proportion to indo-1 dye.

The results given by the experiment proved that as the concentration of calcium increased, the pCa did the opposite and decreased. This can be denoted by the following logarithmic function: pCa = -log10[Ca2+]

The lesser the concentration of calcium, the smaller its intensity, thus harboring a longer and higher its wavelength. Transversely, when using larger concentrations of calcium, its intensity will be posses more counts and its wavelengths will in turn be shorter and lower.

Using the data from this experiment, further research on intracellular signaling, better neural activity monitoring, and drug discovery in the human body can be achieved. Through the specialized and highly precise measurements, further studies on disease localization and dysfunction can be more accurately identified.

I want to thank the STEMM Scholars program for giving me the opportunity to join a research lab this early in my academic career. I’m truly grateful for the ability to present my research. 

I also want to thank Dr. Paul Urayama for accepting me into his lab and making me feel so welcome. 

In addition, I want to give many thanks to Anna Lagona, a grad student in the Urayama Physics Lab, who took the time to help me, guide me, and kindly mentor me for my research.

Ryan, Jordan, and Paul Urayama. "Characterizing the Dual-Wavelength Dye Indo-1 for Calcium-Ion Sensing Under Pressure." Analytical Methods, vol. 4, no. 1, 2012, pp. 80-84. Royal Society of Chemistry.

Urayama, Paul. "Fluorescent Probe Dyes for Metabolic-Ion Sensing Under High Hydrostatic Pressures." Annals of the New York Academy of Sciences, vol. 1175, no. 1, 2009, pp. 115-23. Wiley Online Library.

Urayama, Paul, et al. "A High-Pressure System for Pressure-Jump Integrated with Fluorescence Detection." Analytical Biochemistry, vol. 384, no. 2, 2009, pp. 327-32. ScienceDirect.

Salerno, Brooke A., et al. "Characterization of Dual-Wavelength Seminaphthofluorescein and Seminaphthorhodafluor Dyes for pH Sensing Under High Hydrostatic Pressure." Analytical Biochemistry, vol. 362, no. 1, 2007, pp. 117-24. ScienceDirect.

Over the course of conducting this experiment, I have gained the skills of career and self development, better communication, professionalism, and technology. Through the process of being open about my vulnerability with some aspects of my research, I was able to recognize my strengths and weaknesses to better my project, and ultimately my communication. My professionalism also improved through composing several emails daily, finding meetings times that suit everyone, and ensuring respect is shared between all. Finally, I learned how to operate certain technology and machinery that I had never had to understand before.

Over the course of conducting this experiment, I have gained the skills of career and self development, better communication, professionalism, and technology. Through the process of being open about my vulnerability with some aspects of my research, I was able to recognize my strengths and weaknesses to better my project, and ultimately my communication. My professionalism also improved through composing several emails daily, finding meetings times that suit everyone, and ensuring respect is shared between all. Finally, I learned how to operate certain technology and machinery that I had never had to understand before.