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My name is Yvonne Higgins and I work as a science teacher in
East Glendalough secondary school in Co. Wicklow. This summer, I am working in the
NCBES in NUI Galway for a period of eight weeks as part of the STARs project. It was at an Irish Science Teachers Association (
ISTA) meeting that I first heard about the STARs programme.
This programme, introduced this year by
SFI, provides secondary school science teachers with an opportunity to work with research groups in third level institutions. I decided to take part in this programme as I recognised it as a wonderful opportunity to enhance my skills as a science teacher. Above all, it would allow me to introduce my students to the world of cutting-edge research right here in Ireland!
Thus, I set about finding a project that would encompass my two main areas of interest; chemistry and biology. Firstly, I accessed the STARs projects on the SFI website. Here, I came across a list of projects from various scientific disciplines, but it was the following one that caught my attention; Fluorescence Lifetime Based Sensing: Methods and Instrumentation. This project is located in the Nanoscale Biophotonics laboratory, under the direction of Dr. Alan G. Ryder. The next step was to contact Dr. Ryder, and a joint application was made to SFI, which thankfully, was accepted!
What are the benefits of such a scheme? Personally, I have found that:
Finally, I have thoroughly enjoyed my work experience in the NCBES and would recommend any science teacher to take part in the STARs programme. In addition to the many advantages mentioned above, it has provided me with an opportunity to work with scientists and engineers of many different nationalities including Australian, French, Norwegian, and Polish. All I can say is that I look forward to the prospect of taking part in further projects!
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One of the researchers I am working with is an IAESTE (International Association for the Exchange of Students for Technical Experience) exchange student from Australia, Joanne Gashumba. Joanne is an undergraduate student studying science and engineering at Monash University, Melbourne Australia. She is currently completing a 12 week internship in the same lab. working on this project " Development of Fluorescence lifetime based sensors". Joanne is carrying out some of the more Physics orientated tasks of this interdisciplinary project. |
Aim: The overall goal of this research is to develop a real-time, minimally invasive pH sensor for monitoring foetal blood pH during labour. This involves the use of a fluorescent dye, acridine, which exhibits a large difference in fluorescent lifetime between the neutral and protonated states, making it an ideal candidate for pH sensing. The fluorescent lifetime of a molecule is the average time it spends in the excited state before returning to the ground state. Nafion, a perfluorinated ionomer polymer, is being used as a matrix for the dye.
Project work:
Before I commenced any practical work, I had to prepare all the necessary glassware. All glass-ware was washed with detergent and warm water, rinsed several times with water and then four times with deionised water. Finally, all glass-ware was rinsed with acetone and dried.
Solutions were prepared as follows:
10
-4 M acridine solution: dissolving 0.00448g of acridine in 250ml ethanol.
1M HNO
3: 64.286ml concentrated HNO
3 in 1L using deionised water.
1M NaOH: dissolving 40g NaOH in 1L deionised water.
200mM stock solution of Na
2HPO
4: dissolving 53.614g Na
2HPO
4.7H
2O in 1L deionised water.
200mM stock solution of NaH
2PO
4: dissolving 27,598g NaH
2PO
4.H
2O in 1L deionised water.
Buffered solutions:
For pH 5.8-8.0, buffered solutions were made up according to the following table:
|
pH |
200mM Na 2HPO 4(ml) |
200mM NaH 2PO 4 (ml) |
Deionised water (ml) |
|
5.8 |
4.0 |
46.0 |
40 |
|
6.0 |
6.15 |
43.85 |
40 |
|
6.2 |
9.25 |
40.75 |
40 |
|
6.4 |
13.25 |
36.75 |
40 |
|
6.6 |
18.75 |
31.25 |
40 |
|
6.8 |
24.5 |
25.5 |
40 |
|
7.0 |
30.5 |
19.5 |
40 |
|
7.2 |
36.0 |
14.0 |
40 |
|
7.4 |
40.5 |
9.5 |
40 |
|
7.6 |
43.5 |
6.5 |
40 |
|
7.8 |
45.75 |
4.25 |
40 |
|
8.0 |
47.35 |
2.65 |
40 |
At pH values above 8, a 100mM stock buffer solution was made up by dissolving 26.807g Na 2HPO 4.7H 2O in 800ml deionised water. This solution was divided into 80ml samples, these were then titrated to the following pH values; 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 and 12.0 using either 1M HNO 3 or 1M NaOH, and made up to 100ml with deionised water.
Purification of Nafion film:
Ten strips of nafion film were cut to fit 1mm quartz cuvettes. These were placed in concentrated HNO
3 and stirred at 60°C for 24hr. The film was then placed in 60, 40, and 20% nitric acid respectively and stirred for 1hr in each. The film was then washed thoroughly with deionised water. The nafion strips were converted to the sodium form by placing them in 0.1M NaOH solution with stirring for 24hr. Once again, the film was washed thoroughly with deionised water.
Incorporating acridine into nafion film:
The films were then washed three times with ethanol and left to stand for 5 minutes in each rinse. Acridine was then incorporated into the films by placing them in 20ml 1 x 10
-4
M acridine solution for 5 minutes. Following this, the films were rinsed three times with ethanol before finally being rinsed thoroughly with deionised water. The films were stored in deionised water until use.
Adjusting film to the desired pH:
Before recording absorption and emission spectra and fluorescence lifetimes, the film was placed in buffer solution of the required pH for 30 minutes, replacing the solution after 10, 20, and 25 minutes. The film was then placed in a 1mm quartz cuvette containing the same pH buffer. Following the recording of each spectrum, the film was removed and washed thoroughly with deionised water.
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UV-Visible Absorption and Fluorescence Emission spectroscopy:
Picture: At work in the laboratory inserting the Nafion strips into cuvettes for spectroscopic measurements. |
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Fluorescence Lifetime Measurements The average fluorescent lifetime of a molecule is the average time the molecule spends in the excited state before returning to the ground state. These lifetimes are very short, from the picosecond (10 -12) to microsecond (10 -6) range. The fluorescence lifetimes of acridine in buffer and acridine in Nafion were found as a function of pH for different emission wavelengths. The FluoTime 200 Fluorescence Lifetime Spectrometer (right) was used to obtain lifetime measurements. Initially a 380nm LED excitation source was used for exciting the samples of acridine in buffer for the physiologically important pH range of 5.7 to 8. |
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For obtaining the average lifetimes of acridine in Nafion, a 375nm laser source was used at an intensity of 50% - measurements were taken for the pH range of 8 - 12 rather than for the pH range of 6 - 8 as the greatest change in average lifetime for acridine in Nafion occurs during the former pH range. The instrument response function was measured using a diluted solution of silica and the decay curves for a number of different wavelengths were also recorded using this same software. The picture (left) shows Joanne using the FluoTime 200. |
| Acridine is a very pH sensitive dye. The figure to the right shows the fluorescence decay curve (in Violet excitation 375nm) of a solution of acridine in buffer and the Instrument Response Function of the FluoTime 200 in Red. The pH value of the solution was 6.54 and the emission wavelength was 440nm. The low ÷ 2 value of 1.095 represents a good exponential fit, furthermore it can be seen that the decay curve comprises of two emitting species - thus the decay curve was fitted using two exponentials. Using this same software (Fluofit) the average lifetime of the acridine at this pH was found to be 16.09ns. |
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Lifetime Measurements - Acridine in Buffer
The plots to the left show the change in the average lifetime for the pH range of 5.7 to 8 which is of physiological importance. The plots are for average lifetime at varying pH for different emission wavelengths and from these plots it can be seen that the average lifetime is longer for longer emission wavelengths. Furthermore it can be seen that the average lifetime reduces as the pH increases. |
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Lifetime Measurements - Acridine in Nafion
Acridine fluorescence can be quenched by certain species such as halide ions making it an unsuitable pH sensor. Therefore to prevent fluorescence quenching Nafion was used as it is permselective, meaning that it passes only cations and not anions. Thus Nafion acts as a protective support material, however this shifts the pKa to a higher value. |
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At the moment we are trying to modify the chemical structure of Nafion to improve the pH response of acridine in Nafion by moving the pKa back towards 7. (Results to be added soon)
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