Use of Fluorescence to Detect Urobilin in Water as a Potential...
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METHOD INTRODUCTION DISCUSSION Use of Fluorescence to Detect Urobilin in Water as a Potential Indicator of Fecal Contamination Jhoana M. Duran; Mayra Sarria; Daniel Gerrity, Ph.D., Assistant Professor Department of Civil and Environmental Engineering and Construction, University of Nevada, Las Vegas A major cause of death worldwide (~2,300 people per day) is the lack of access to clean drinking water (water.org/water-crisis/ water-facts/water). In addition to inadequate sanitation and treatment, another limitation is the lack of an affordable and reliable test to determine whether water is safe for drinking. As a result, many people may unknowingly ingest or come into contact with water contaminated with fecal material and human pathogens. Today, non-specific indicators of human waste contamination can be detected and quantified, but the methods used for these tests are not entirely reliable (i.e., positive/negative results do not necessarily indicate the presence/absence of human pathogens). Existing methods include quantification of ammonia, coliform bacteria, and coprostanol (Miyaba et al., 1992). However, these methods are time consuming, complex, not human specific, and expensive (Miyaba et al., 1992). Urobilin is a final byproduct of hemoglobin metabolism in mammals that is excreted in both fecal material and urine (Jones-Lepp, 2002), thereby making it a good candidate to serve as an indicator for the presence of fecal material in water. Even though other mammals produce urobilin, humans are considered a primary source because they generate larger quantities and higher concentrations (Jones-Lepp, 2002). Finding a consistent and accurate method of detection for aqueous urobilin would be beneficial to public health because it would provide a rapid and economic alternative to existing indicator methods. Urobilin could serve as a valuable indicator of fecal contamination in developing countries, in places recovering from natural disasters, and even in urban runoff. A 1 μM urobilin hydrochloride stock solution was serially diluted in dechlorinated tap water; adjusted to pH 5.0-8.5; and analyzed for fluorescence in the presence of varying zinc concentrations. Fluorescence was quantified at an excitation wavelength of 480 nm and emission wavelengths ranging from 490 nm to 620 nm. Complexation with zinc results in an augmented, ‘red shifted’ urobilin peak, which allows for detection at lower concentrations (Figure 3). The area of the fluorescence peak was found to be directly proportional to the concentration of urobilin (method detection limit near 20 nM) (Figures 5 and 7). Urobilin was found to be relatively stable at pH ≤ 7.5 but showed signs of instability at pH ≥ 8.5. In addition, fluorescence peaks were lower at elevated pH levels. This indicates that adjustment to a standard, more acidic pH may be necessary to quantify urobilin in environmental samples. The spectrofluorometer was able to reliably detect the presence of urobilin at concentrations between 25 nm and 525 nm with 0.03% zinc. Lower detection limits may be possible with higher zinc concentrations, and samples with higher urobilin concentrations will require dilution to prevent saturation of the detector (i.e., plateaus). A concern throughout the course of the experiments was the amount of dissolved zinc present in the samples. Particularly at higher pH, adding more zinc did not always result in increased fluorescence. This may be attributable to increased precipitation rates caused by zinc’s lower solubility at higher pH. For example, zinc ions can react with hydroxide to form Zn(OH) 2 (s), which has a solubility product (K sp ) of 1.2x10 -17 . Zinc also reacts with carbonate to form ZnCO 3 (s) (K sp = 1.4x10 -11 ) and phosphate to form Zn 3 (PO 4 ) 2 (K sp = 9.0x10 -33 ). Standardizing this method is crucial if it is to be used for analysis of environmental samples. In addition, the effects of background fluorescence from bulk organic matter must also be considered. There are a variety of chemicals that are present in surface water that might mimic the fluorescence of urobilin, which in turn would make it more difficult to detect accurately. Once these issues are addressed, urobilin could be used as a valuable indicator of fecal contamination. OBJECTIVES The primary objectives were to (1) verify the fluorescence properties of urobilin-zinc complexes, (2) evaluate the impacts of pH on compound stability and zinc efficacy, and (3) develop standard curves to quantify urobilin in unknown water samples with the ultimate goal of using urobilin as a potential indicator of fecal contamination. REFERENCES 1. Jones-Lepp, T. (2002). Chemical markers of human waste contamination: Analysis of urobilin and pharmaceuticals in source waters. Journal of Enviornmental Monitoring 8, 472-478. 2. Miyabara, Y., Sakata, Y., Suzuki, J., & Suzuki, S. (1992). Estimation of faecal pollution based on the amounts of urobilins in urban rivers. Environmental Pollution 84, 117-122. 3. Millions lack safe water. (n.d.). Retrieved December 1, 2014, from http://water.org/water-crisis/water-facts/water/ . Figure 1: Horiba Aqualog – Fluorometer for the characterization and quantification of dissolved organic matter (DOM) y = 0.1418x 7.2899 R² = 0.97975 y = 0.1401x 7.3588 R² = 0.9775 y = 0.1379x 6.9989 R² = 0.9779 0 30 60 90 120 150 0 200 400 600 800 1000 1200 Peak Area Urobilin Concentra1on (nM) pH = 6.5, Zinc = 0.03%, and Integra1on Time = 3 sec 1 2 3 y = 0.0803x + 0.9342 R² = 0.98493 y = 0.0819x + 0.625 R² = 0.99526 y = 0.0797x + 0.3578 R² = 0.99275 0 30 60 90 120 150 0 200 400 600 800 1000 1200 Peak Area Urobilin Concentra1on (nM) pH = 8.5, Zinc = 0.03%, and Integra1on Time = 3 sec 1 2 3 Figure 5: Peak Area of Fluorescence vs. Concentration at pH 6.5 Figure 7: Peak Area of Fluorescence vs. Concentration at pH 8.5 Figure 6: Peak Area of Fluorescence vs. Concentration at pH 8.5 Figure 4: Fluorescence vs. Emission Wavelength at pH 6.5 Peak Area = (0.140±0.002)*C urobilin – (7.22±0.19) Peak Area = (0.081±0.001)*C urobilin + (0.64±0.29) Figure 2: Potential Application of Urobilin in the Las Vegas Valley Figure 3: Fluorescence v Emission Wavelength for Control Samples BACKGROUND AND RESULTS 0 0.5 1 1.5 2 2.5 3 490 510 530 550 570 590 610 Fluorescence (AFU) Emission Wavelength (nm) pH = 5.0, Urobilin = 750 nM, and Integra1on Time = 3 sec Urobilin Zinc (0.25%) Urobilin+Zinc (0.25%) Urobilin+Zinc (0.1%) Urobilin+Zinc (0.075%) Urobilin+Zinc (0.05%) Urobilin+Zinc (0.03%) Urobilin+Zinc (0.01%) Urobilin+Zinc (0.005%) 0 0.5 1 1.5 2 2.5 3 490 510 530 550 570 590 610 Fluorescence (AFU) Emission Wavelength (nm) pH = 6.5, Zinc = 0.03%, and Integra1on Time = 3 sec 25 nM 90 nM 263 nM 313 nM 375 nM 482 nM 525 nM 750 nM 963 nM 0 0.5 1 1.5 2 2.5 3 490 510 530 550 570 590 610 Fluorescence (AFU) Emission Wavelength (nm) pH = 8.5, Zinc = 0.03%, and Integra1on Time = 3 sec 25 nM 90 nM 263 nM 313 nM 375 nM 482 nM 500 nM 525 nM 750 nM 963 nM
Transcript of Use of Fluorescence to Detect Urobilin in Water as a Potential...
METHOD INTRODUCTION
DISCUSSION
Use of Fluorescence to Detect Urobilin in Water as a Potential Indicator of Fecal Contamination
Jhoana M. Duran; Mayra Sarria; Daniel Gerrity, Ph.D., Assistant Professor Department of Civil and Environmental Engineering and Construction, University of Nevada, Las Vegas
A major cause of death worldwide (~2,300 people per day) is the lack of access to clean drinking water (water.org/water-crisis/water-facts/water). In addition to inadequate sanitation and treatment, another limitation is the lack of an affordable and reliable test to determine whether water is safe for drinking. As a result, many people may unknowingly ingest or come into contact with water contaminated with fecal material and human pathogens. Today, non-specific indicators of human waste contamination can be detected and quantified, but the methods used for these tests are not entirely reliable (i.e., positive/negative results do not necessarily indicate the presence/absence of human pathogens). Existing methods include quantification of ammonia, coliform bacteria, and coprostanol (Miyaba et al., 1992). However, these methods are time consuming, complex, not human specific, and expensive (Miyaba et al., 1992). Urobilin is a final byproduct of hemoglobin metabolism in mammals that is excreted in both fecal material and urine (Jones-Lepp, 2002), thereby making it a good candidate to serve as an indicator for the presence of fecal material in water. Even though other mammals produce urobilin, humans are considered a primary source because they generate larger quantities and higher concentrations (Jones-Lepp, 2002). Finding a consistent and accurate method of detection for aqueous urobilin would be beneficial to public health because it would provide a rapid and economic alternative to existing indicator methods. Urobilin could serve as a valuable indicator of fecal contamination in developing countries, in places recovering from natural disasters, and even in urban runoff.
A 1 µM urobilin hydrochloride stock solution was serially diluted in dechlorinated tap water; adjusted to pH 5.0-8.5; and analyzed for fluorescence in the presence of varying zinc concentrations. Fluorescence was quantified at an excitation wavelength of 480 nm and emission wavelengths ranging from 490 nm to 620 nm. Complexation with zinc results in an augmented, ‘red shifted’ urobilin peak, which allows for detection at lower concentrations (Figure 3).
The area of the fluorescence peak was found to be directly proportional to the concentration of urobilin (method detection limit near 20 nM) (Figures 5 and 7). Urobilin was found to be relatively stable at pH ≤ 7.5 but showed signs of instability at pH ≥ 8.5. In addition, fluorescence peaks were lower at elevated pH levels. This indicates that adjustment to a standard, more acidic pH may be necessary to quantify urobilin in environmental samples. The spectrofluorometer was able to reliably detect the presence of urobilin at concentrations between 25 nm and 525 nm with 0.03% zinc. Lower detection limits may be possible with higher zinc concentrations, and samples with higher urobilin concentrations will require dilution to prevent saturation of the detector (i.e., plateaus). A concern throughout the course of the experiments was the amount of dissolved zinc present in the samples. Particularly at higher pH, adding more zinc did not always result in increased fluorescence. This may be attributable to increased precipitation rates caused by zinc’s lower solubility at higher pH. For example, zinc ions can react with hydroxide to form Zn(OH)2(s), which has a solubility product (Ksp) of 1.2x10-17. Zinc also reacts with carbonate to form ZnCO3(s) (Ksp = 1.4x10-11) and phosphate to form Zn3(PO4)2 (Ksp = 9.0x10-33). Standardizing this method is crucial if it is to be used for analysis of environmental samples. In addition, the effects of background fluorescence from bulk organic matter must also be considered. There are a variety of chemicals that are present in surface water that might mimic the fluorescence of urobilin, which in turn would make it more difficult to detect accurately. Once these issues are addressed, urobilin could be used as a valuable indicator of fecal contamination.
OBJECTIVES The primary objectives were to (1) verify the fluorescence properties of urobilin-zinc complexes, (2) evaluate the impacts of pH on compound stability and zinc efficacy, and (3) develop standard curves to quantify urobilin in unknown water samples with the ultimate goal of using urobilin as a potential indicator of fecal contamination.
REFERENCES 1. Jones-Lepp, T. (2002). Chemical markers of human waste
contamination: Analysis of urobilin and pharmaceuticals in source waters. Journal of Enviornmental Monitoring 8, 472-478.
2. Miyabara, Y., Sakata, Y., Suzuki, J., & Suzuki, S. (1992). Estimation of faecal pollution based on the amounts of urobilins in urban rivers. Environmental Pollution 84, 117-122.
3. Millions lack safe water. (n.d.). Retrieved December 1, 2014, from http://water.org/water-crisis/water-facts/water/.
Figure 1: Horiba Aqualog – Fluorometer for the characterization and quantification of dissolved organic matter (DOM)
y = 0.1418x -‐ 7.2899 R² = 0.97975
y = 0.1401x -‐ 7.3588 R² = 0.9775
y = 0.1379x -‐ 6.9989 R² = 0.9779
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30
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90
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150
0 200 400 600 800 1000 1200
Peak Area
Urobilin Concentra1on (nM)
pH = 6.5, Zinc = 0.03%, and Integra1on Time = 3 sec
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3
y = 0.0803x + 0.9342 R² = 0.98493
y = 0.0819x + 0.625 R² = 0.99526
y = 0.0797x + 0.3578 R² = 0.99275
0
30
60
90
120
150
0 200 400 600 800 1000 1200
Peak Area
Urobilin Concentra1on (nM)
pH = 8.5, Zinc = 0.03%, and Integra1on Time = 3 sec
1
2
3
Figure 5: Peak Area of Fluorescence vs. Concentration at pH 6.5
Figure 7: Peak Area of Fluorescence vs. Concentration at pH 8.5 Figure 6: Peak Area of Fluorescence vs. Concentration at pH 8.5
Figure 4: Fluorescence vs. Emission Wavelength at pH 6.5
Peak Area = (0.140±0.002)*Curobilin – (7.22±0.19)
Peak Area = (0.081±0.001)*Curobilin + (0.64±0.29)
Figure 2: Potential Application of Urobilin in the Las Vegas Valley Figure 3: Fluorescence v Emission Wavelength for Control Samples
BACKGROUND AND RESULTS
0
0.5
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490 510 530 550 570 590 610
Fluo
rescen
ce (A
FU)
Emission Wavelength (nm)
pH = 5.0, Urobilin = 750 nM, and Integra1on Time = 3 sec
Urobilin
Zinc (0.25%)
Urobilin+Zinc (0.25%)
Urobilin+Zinc (0.1%)
Urobilin+Zinc (0.075%)
Urobilin+Zinc (0.05%)
Urobilin+Zinc (0.03%)
Urobilin+Zinc (0.01%)
Urobilin+Zinc (0.005%)
0
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490 510 530 550 570 590 610
Fluo
rescen
ce (A
FU)
Emission Wavelength (nm)
pH = 6.5, Zinc = 0.03%, and Integra1on Time = 3 sec
25 nM
90 nM
263 nM
313 nM
375 nM
482 nM
525 nM
750 nM
963 nM
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490 510 530 550 570 590 610
Fluo
rescen
ce (A
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Emission Wavelength (nm)
pH = 8.5, Zinc = 0.03%, and Integra1on Time = 3 sec
25 nM
90 nM
263 nM
313 nM
375 nM
482 nM
500 nM
525 nM
750 nM
963 nM
