- Email: [email protected]
Nitrite detection using plastic optical fiber (POF); an early stage investigation towards the development of oral cancer sensor using POF
Accepted Manuscript Title: Nitrite Detection using Plastic Optical Fiber (POF); an early stage investigation towards the development of oral cancer sensor using POF Author: Sayidatul Nadia Elias Norhana Arsad Sabiran Abu Bakar PII: DOI: Reference:
S0030-4026(15)00590-2 http://dx.doi.org/doi:10.1016/j.ijleo.2015.07.038 IJLEO 55753
To appear in: Received date: Accepted date:
27-5-2014 4-7-2015
Please cite this article as: S.N. Elias, N. Arsad, Nitrite Detection using Plastic Optical Fiber (POF); an early stage investigation towards the development of oral cancer sensor using POF, Optik - International Journal for Light and Electron Optics (2015), http://dx.doi.org/10.1016/j.ijleo.2015.07.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TITLE: Nitrite Detection using Plastic Optical Fiber (POF); an early stage investigation
cr
AUTHORS: Sayidatul Nadia Elias1, Norhana Arsad2, Sabiran Abu Bakar2
ip t
towards the development of oral cancer sensor using POF
us
MAILING ADDRESS: Department of Electric, Electronic and System Engineering, Faculty of Engineering and Built and Environment,
te
FAX NUMBER: +603 8925 9080
d
PHONE NUMBER: +603 8921 6320
M
Selangor, Malaysia.
an
The National University of Malaysia,
Ac ce p
EMAIL ADDRESS: [email protected] [email protected] [email protected]
Page 1 of 19
Abstract: Oral Squamous Cell Carcinoma (OSCC) has a higher chance of survival rate if being detected at an early stage. Overconsumption of nitrite in human body may result in OSCC formation. Nitrite and nitrate is a part of reactive nitrogen species (RNS) in saliva and
ip t
is found to have higher percentage in OSCC patients’ saliva, thus chosen to be investigated as a potential OSCC biomarker using plastic optical fiber (POF) with evanescent wave sensing
cr
method. The experiment is done using UV/VIS light source and the peak absorbance is found to be approximately 540nm. The sensor displays moderate sensitivity with higher sensitivity
us
towards lower end of the concentration.
M
an
Keywords: Beer-Lambert law; evanescent wave sensing; nitrite; OSCC; plastic optical fiber
INTRODUCTION
d
Oral squamous cell carcinoma (OSCC) is generally referred as oral cancer [1]. Oral cancer is
te
the sixth most common cancers among the world population, and third in the developing countries including Malaysia [2]. Number of oral cancer incidence worldwide is 170,496 with
Ac ce p
83, 109 mortality for men, and 92,524 incidence with 44,545 mortality for women [3]. Estimated incidence rate per 100,000 of oral cancer cases in Malaysia is 17.3 for male and 8.6 for female, while the mortality rate per 100,000 is 9.7 for male and 4.6 for female [4]. Some of the risk factors identified are tobacco smoking, alcohol consumption, viruses, dieting habit and deficiency states [5]. Despite numerous advancement in treatment, the five-year survival rate for the oral cancer patients is still 50% for the past few decades, mainly due to lack early stage diagnosis, with 60% of the patients are examined to have oral cancer when the disease is at stage III and IV [1, 6, 7]. An early stage detection of oral cancer is vital to ensure a higher survival chance for the patients by 60%-80% as compared to only 30%-40 % percent when the cancer is detected at an advanced state [8].
Page 2 of 19
Nitrates (NO3) and nitrites (NO2) are inorganic ions that occur naturally in our environment [9]. The main concern of health hazard on human body caused by nitrates and nitrites is that when nitrates are consumed by human they change into nitrites [10]. Over-consumption of
ip t
nitrites can cause damage to the nervous system, spleen liver, reducing the oxygen transport capacity by blood, and most importantly the reaction between nitrites and secondary or
cr
tertiary amines. This reaction causes the formation of N-nitroso compounds, with some of these compounds are carcinogenic, tetratogenic, or mutagenic which may result in the
us
formation of OSCC [9, 11-14].
Reactive nitrogen species (RNS) in the form of nitrosamines (nitrites NO2 and nitrates NO3)
an
found in human saliva can cause DNA base alterations, strand breaks, damaged tumor
M
suppressor genes, and enhanced expression of protooncogenes which lead the development of cancer [15]. A study conducted found significant elevated concentration of RNS in the saliva
d
sample of OSCC patients, with the percentage of nitric oxide (NO) 60%, nitrites (NO2) 190%,
te
and nitrates (NO3) 93% higher as compared to their concentrations in the saliva of nonpatients [16]. Thus, for this work nitrite is chosen as the sample to be investigated.
Ac ce p
Plastic optical fiber (POF) differs from the other types of optical fibers by having its core being made of polymethylmethacrylate (PMMA) with the core radius typically between 125409 µm [17, 18]. POF is easy to handle, flexible, lower density, higher elastic deformation limits, and cheaper because it allows the use of low precision connector due to its large core diameter, causing a cost decrement on the overall system [17]. Fiber optic is usually chosen as a chemical sensor for the determination of cations and anions because it has good sensitivity and selectivity, can be fabricated easily, and having low cost [19]. As a biosensor, optical fiber is very small, flexible and light, thus conviniet for insertion into catheters and needles hence allows particular measurements inside tissues and blood. The materials used in fiber
Page 3 of 19
structure (glass or plastic) are sturdy, non toxic, biocompatible with human body, diaelectric and uses low power for the light source, ensuring patient’s intrinsic safety [20]. Evanescent wave sensor, a type of intensity based fiber optic sensor is a popular choice for a
ip t
chemical and biological sensor because this method proves excellent sensitivity over chemical reagents, great control over interaction parameters, able to interact with molecules within the
cr
depth of light penetration and low cost [21-25]. Evanescent wave sensor makes use of the change in light energy that flows into the cladding from the core. The sensing probe for this
us
sensor is created by removing a portion of the cladding on fiber’s body, exposing the core. The unclad portion (sensing probe) is the being brought into direct contact with the chemical
an
sample being measured, or coating the unclad portion with chemically-sensitive materials
M
[26].
In this work, a simple POF evanescent wave sensor is constructed for the detection of nitrite
d
with different concentration. This experiment is done as an early stage towards the design and
Ac ce p
THEORY
te
development of OSCC sensor using POF.
When a portion of the fiber cladding is removed and the unclad fiber is immersed in the sample, there will be an interaction between the evanescent field in the fiber and the lightabsorbing elements in the sample. The sample acts as a new ‘cladding’ for the fiber and will absorbs a particular wavelength of light transmitted in the fiber depending on the concentration of the elements that absorbs light at that wavelength [21]. In this work, the fiber is uncladded and being brought directly in contact with the sample; this method is known as direct spectroscopic evanescent wave sensing [27]. Thus, Beer-Lambert law that relates the element’s absorbance with its concentration can be applied [28]: (1)
Page 4 of 19
Where A is absorbance, P0 and P is the intensity of incident and transmitted light energy respectively, ε is molar absorptivity (M-1 cm-1), b is optical path length (cm) and c is concentration of the solution (M).
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If the optical path length is being kept constant at 1cm, molar absorptivity is the slope of the concentration versus absorbance curve:
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(2)
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METHODOLOGY
Optical Fiber Preparation
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A.
4 single core POFs with each having 125μm core radius, 2mm diameter and 40cm in length
M
were prepared. 3 of the POFs were stripped in the middle from their protective jacket with an optical path length of 1cm each, using coaxial stripper and utility knife. The diameter of the
d
cladding for all 3 POFs were 1mm. 2 of the unjacketed POFs were the then mechanically
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etched using fine sand paper to give out 0.8mm and 0.6mm cladding diameter respectively. For all cladding diameter measurements, POFs cladding were viewed under Olympus
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Microscope Imaging System and using DigiAcquis 2.0 software, the images were captured and the claddings’ diameter was measured. All POFs were later washed with distilled (DI) water and leaved to dry. To measure the fiber loss in all 4 POFs, a simple setup was done using and an optical fiber communication trainer (OFT) having 850nm LED transmitter and PIN diode receiver. A function generator was connected to OFT input and the output was connected to an oscilloscope. The unjacketed section was encapsulated to prevent external disturbances. This step was done to find out the most sensitive POF to be used for nitrite detection. Fig. 1 shows the experimental setup to measure POFs loss. B.
Sample Preparation
Page 5 of 19
For this experiment, a nitrite solution was prepared by diluting 0.0138g sodium nitrite into 200ml DI water using volumetric method to give out 1mM nitrite concentration sample. Solution samples with varying concentration was produced by diluting the 1mM nitrite
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solution with DI water into another 5 samples, each having 50μM, 25μM, 12.5μM, 6.25μM, and 3.125μM nitrate concentration respectively. Greiss reagent consisting of two solutions
cr
was also prepared by adding 0.0581M of sulphanilamide into 5% acid for the first solution, and the second solution having 0.0038M of N-1-napthylamine. Colorimetric analysis of nitrite
us
was performed by diluting each sample with sulphanilamide acid, and then being added with 1ml N-1-napthylamine. The mixture gave out a reddish-violet colored solution. Each sample
an
had a total volume of 3ml.
Experimental Setup
M
C.
All instruments used for spectra measurements are from Ocean Optics. The UV-VIS-NIR
d
light source used is of model DH-2000-BAL with deuterium tungsten halogen light source.
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The spectrometer model HR4000 was connected to 3648-element CCD-array Toshiba detector. SMA connector was used to connect POF to the light source and spectrometer. The
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exposed cladding of the POF was inserted into a tube containing the sample and totally encapsulated with a black box. Data spectrum was displayed and recorded using Spectrasuite software. The experimental setup is as shown in Fig. 2.
RESULT AND DISCUSSION
Exposing the cladding of the fiber will result in loss due to light energy that leaks from the cladding to the environment. In evanescent wave sensing this leaked energy is important for its interaction with the sample, hence higher loss of fiber means more light is able to interact with the light-absorbing elements in the sample. Measurement of loss in different prepared POFs was carried out in order to verify that POF has the same loss property as silica fiber, and
Page 6 of 19
also to determine the best POF preparation to be used for nitrite detection. Fig. 3 shows the loss in the prepared POFs and it can be seen that POF with 0.6mm cladding exhibited the most loss. This is because the etched cladding is the nearest to the core, hence allowing more
ip t
leakage of light. Based on the graph, it can be assumed that diameter lower than 0.6mm will give out higher loss, with the most sensitive POF having a totally exposed core. However the
cr
sensor setup caused strain on the thin exposed cladding, thus any cladding diameter below 0.6mm would break.
us
Fig. 4 shows the absorption spectrum of nitrite samples with varying concentration in the visible wavelength of 400-700nm using POF with 0.6mm cladding. The peak absorbance
M
increasing concentration of nitrite samples.
an
wavelength is found to be at approximately 540nm. The amount of absorption increases with
According to Beer-Lambert law, absorbance is linearly related to concentration. Hence, in an
d
ideal case when the concentration of nitrite increases, absorbance will increase in direct
te
proportion with the concentration. Fig. 5 shows the calibration curve plot of nitrite concentration versus absorbance at the peak absorbance wavelength of approximately 540nm.
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The absorbance increases as the concentration of the sample increases, but not in the form of a straight curve.
Fig. 6 describing the same result as in Fig. 5 but in the form of a scattering plotted graph, taking into consideration the colleration coefficient (R2) and the linear regression line. The correlation coefficient for this graph is 0.9475, showing a strong positive linear correlation approaching +1 which proves that this graph possess an almost linear characteristic. The linear trend indicates that the sensor being used in this experiment does not deviate far from Beer-Lambert law, and thus the sensor is said to exhibit an adequate sensitivity. Evanescent field absorption is the key element in this experiment; hence even the slightest disturbances on the surface will causes error to the measurement. The sensing region was carefully cleaned
Page 7 of 19
in between sample changes, but minute particles in the environment would still affect the sensor performance. Fig. 7 shows the comparison between the calibration curve based on experimental results and
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theoretical calculation using (1). There is smaller difference between the two curves at the lower end of the concentration, indicating that the sensor is more sensitive towards smaller
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concentration of solution. The sensor deviates further away from the theoretical curve beyond 60µM.
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Based on a number of previous researches, the uses of optical sensors that apply evanescent wave field absorbance theory are prone to demonstrate higher sensitivity at a lower level of
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concentration [27, 29-31]. The effect of concentration on the sensor can induce a phenomenon
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in which at a concentration of more than 0.01 M, a deviation from Beer-Lambert law can be seen. Some of the causes that lead to such phenomenon are electrostatic interaction, light
d
scattering due to particulates in the sample, sample fluorescence or phosphorescence, changes
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in the refraction index at high analyte concentration, and non-monochromatic radiation [32]. Molar absorptivity can be found from the slope of Fig. 5 using (2) and slope formula for non-
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linear relationship. Molar absorptivity of this experiment is found to be 3.4 x 104M-1cm-1. Based on Savvin’s criteria of sensitivity [33], this value falls into moderate sensitivity. Comparing with the molar absorptivity of the theoretical curve’s slope of 3.5 x 104M-1cm-1, the percentage error of the experimental data is 4.33%.
CONCLUSION
As a conclusion, an experimental setup for the detection of nitrite in varying concentration using POF has been carried out. Loss in 4 different prepared POFs has being studied to find the most suitable POF structure for nitrite detection. 6 nitrite samples with different concentration are used in this work, developed using greiss reaction. The peak absorbance
Page 8 of 19
wavelength is found to be at approximately at 540nm. Calibration curve is plotted based on Beer-Lambert law and it shows that the sensor exhibits adequate sensitivity. Comparison between experimental data and theoretical calculation indicates that the sensor has higher
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sensitivity and the lower end of the concentration. Molar absorptivity of the nitrite samples is calculated from the slope of the calibration curve with the value of 3.4 x 10-4M-1cm-1 and
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based on Savvin’s criteria of sensitivity, the sensor has moderate sensitivity. Percentage error of the experimental molar absorptivity as compared to the theoretical value is 4.33%. For
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future work, we aim to increase the sensitivity of the sensor and improvise the setup in order
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to detect nitrite in human saliva.
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ACKNOWLEDGMENT
I would like to express my sincere gratitude towards Dr. Norhana Bt. Arsad, Department of
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Electrical, Electronic and System Engineering, Faculty of Engineering and Built
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Environment, National University of Malaysia for her valuable advices, helpful suggestions, study materials, and thorough support during this research. I would also like to thank Mr.
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Sabiran bin Abu Bakar, a Master’s student from the Department of Electrical, Electronic and System Engineering, Faculty of Engineering and Built Environment, National University of Malaysia for his guidance in preparing the experimental setup and sample solutions.
REFERENCE
[1] M.W. Lingen, J.R. Kalmar, T. Karrison, P.M. Speight, Critical Evaluation of Diagnostic Aids for the Detection of Oral Cancer, Oral oncology 44 (2008) 10-22. [2] N. Johnson, Tobacco Use and Oral Cancer: A Global Perspective, Journal of dental education 65 (2001) 328-339.
Page 9 of 19
[3] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of Worldwide Burden of Cancer in 2008: Globocan 2008, International Journal of Cancer 127 (2010) 2893-2917.
ip t
[4] M. Kimman, R. Norman, S. Jan, D. Kingston, M. Woodward, The Burden of Cancer in
Cancer Prev 13 (2012) 411-420.
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Member Countries of the Association of Southeast Asian Nations (Asean), Asian Pac J
[5] R. Radhakrishnan, B. Shrestha, D. Bajracharya, Oral Cancer–an Overview.
us
[6] K.U. Ogbureke, C. Bingham, Overview of Oral Cancer, 2012.
[7] M.a.T. Canto, S.S. Devesa, Oral Cavity and Pharynx Cancer Incidence Rates in the United
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States, 1975–1998, Oral oncology 38 (2002) 610-617.
journal for clinicians 55 (2005) 74-108.
M
[8] D.M. Parkin, F. Bray, J. Ferlay, P. Pisani, Global Cancer Statistics, 2002, CA: a cancer
d
[9] M.J. Moorcroft, J. Davis, R.G. Compton, Detection and Determination of Nitrate and
te
Nitrite: A Review, Talanta 54 (2001) 785-803.
[10] D. Xia, D. Deng, S. Wang, Destruction of Parotid Glands Affects Nitrate and Nitrite
Ac ce p
Metabolism, Journal of dental research 82 (2003) 101-105. [11] A.A. Ensafi, A. Kazemzadeh, Simultaneous Determination of Nitrite and Nitrate in Various Samples Using Flow Injection with Spectrophotometric Detection, Analytica chimica acta 382 (1999) 15-21.
[12] A. Kazemzadeh, A.A. Ensafi, Sequential Flow Injection Spectrophotometric Determination of Nitrite and Nitrate in Various Samples, Analytica chimica acta 442 (2001) 319-326. [13] A.A Ensafi, B. Rezaei, S. Nouroozi, Simultaneous Spectrophotometric Determination of Nitrite and Nitrate by Flow Injection Analysis, Analytical sciences 20 (2004) 1749-1753.
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[14] H. Kroupova, J. Machova, V. Piackova, J. Blahova, R. Dobsikova, L. Novotny, Z. Svobodova, Effects of Subchronic Nitrite Exposure on Rainbow Trout (Oncorhynchus Mykiss), Ecotoxicology and environmental safety 71 (2008) 813-820.
ip t
[15] N. Ma, T. Tagawa, Y. Hiraku, M. Murata, X. Ding, S. Kawanishi, 8-Nitroguanine Formation in Oral Leukoplakia, a Premalignant Lesion, Nitric Oxide 14 (2006) 137-143.
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[16] G. Bahar, R. Feinmesser, T. Shpitzer, A. Popovtzer, R.M. Nagler, Salivary Analysis in Oral Cancer Patients, Cancer 109 (2006) 54-59.
us
[17] L. Bilro, N. Alberto, J.L. Pinto, R. Nogueira, Optical Sensors Based on Plastic Fibers, Sensors 12 (2012) 12184-12207.
an
[18] J. Zubia, J. Arrue, Plastic Optical Fibers: An Introduction to Their Technological
M
Processes and Applications, Optical Fiber Technology 7 (2001) 101-140. [19] A.A. Ensafi, M. Amini, A Highly Selective Optical Sensor for Catalytic Determination
d
of Ultra-Trace Amounts of Nitrite in Water and Foods Based on Brilliant Cresyl Blue as a
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Sensing Reagent, Sensors and Actuators B: Chemical 147 (2010) 61-66. [20] M. Anna Grazia, B. Francesco, Biomedical Sensors Using Optical Fibres, Reports on
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Progress in Physics 59 (1996) 1.
[21] N.A. George, P. Sureshkumar, P. Radhakrishnan, C. Vallabhan, V. Nampoori, Chemical Sensing with Microbent Optical Fiber, Optics Letters 26 (2001) 1541-1543. [22] B.G.S. Khijwania, Experimental Studies on the Response of the Fiber Optic Evanescent Field Absorption Sensor, Fiber & Integrated Optics 17 (1998) 63-73. [23] S. Khijwania, B. Gupta, Fiber Optic Evanescent Field Absorption Sensor: Effect of Fiber Parameters and Geometry of the Probe, Optical and Quantum Electronics 31 (1999) 625-636. [24] B.D. Maccraith, Optical Fiber Chemical Sensor Systems and Devices, Optical Fiber Sensor Technology: Volume 4: Chemical and Environmental Sensing 4 (1999) 15.
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[25] M. Marazuela, M. Moreno-Bondi, Fiber-Optic Biosensors-an Overview, Analytical and Bioanalytical Chemistry 372 (2002) 664-682. [26] M. Connelly, Fiber Sensors, Elsevier Ltd., Limerick, 2005.
ip t
[27] S.M. John, Evanescent Wave Fibre Optic Sensors: Design, Fabrication and Characterization, 2011.
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[28] D. Swinehart, The Beer-Lambert Law, Journal of Chemical Education 39 (1962) 333.
[29] J. Heo, M. Rodrigues, S.J. Saggese, G.H. Sigel Jr, Remote Fiber-Optic Chemical Sensing
us
Using Evanescent–Wave Interactions in Chalcogenide Glass Fibers, Applied Optics 30 (1991) 3944-3951.
an
[30] J.B. Jensen, L.H. Pedersen, P.E. Hoiby, L.B. Nielsen, T.P. Hansen, J.R. Folkenberg, J.
M
Riishede, D. Noordegraaf, K. Nielsen, A. Carlsen, A. Bjarklev, Photonic Crystal Fiber Based Evanescent-Wave Sensor for Detection Of Biomolecules in Aqueous Solutions, Opt. Lett. 29
d
(2004) 1974-1976.
te
[31] P. Suresh Kumar, S. Thomas Lee, C. Vallabhan, V. Nampoori, P. Radhakrishnan, Design and Development of an Led Based Fiber Optic Evanescent Wave Sensor for Simultaneous
30.
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Detection of Chromium and Nitrite Traces in Water, Optics Communications 214 (2002) 25-
[32] B.-L. Law, L.S. Regression, C.L. Squares, I.L. Squares, The Beer Lambert Law, Chemistry 311 (2003) 1.
[33] S.B. Savvin, K. Hiiro, Organic Reagents in Photometric Analysis, C R C Critical Reviews in Analytical Chemistry 8 (1979) 55-109.
Page 12 of 19
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Fig. 1. Experimental setup to measure POF loss.
Page 13 of 19
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Fig. 2. Experimental setup for nitrite detection.
Page 14 of 19
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Fig. 3. Fiber loss in 4 different prepared POFs.
Page 15 of 19
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Fig. 4. Absorption spectrum of nitrite sample soultion with varying concentration.
Page 16 of 19
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Fig. 5. Calibrated curve plot of nitrite absorbance at peak wavelength based on Beer-Lambert
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law.
Page 17 of 19
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line.
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Fig. 6. Scattering plot of nitrite absorbance with correlation coefficient and linear regression
Page 18 of 19
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Fig. 7. Comparison between experimental data and theorotical calculation of varying nitrite
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concentration at peak wavelength absorbance.
Page 19 of 19
S0030-4026(15)00590-2 http://dx.doi.org/doi:10.1016/j.ijleo.2015.07.038 IJLEO 55753
To appear in: Received date: Accepted date:
27-5-2014 4-7-2015
Please cite this article as: S.N. Elias, N. Arsad, Nitrite Detection using Plastic Optical Fiber (POF); an early stage investigation towards the development of oral cancer sensor using POF, Optik - International Journal for Light and Electron Optics (2015), http://dx.doi.org/10.1016/j.ijleo.2015.07.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TITLE: Nitrite Detection using Plastic Optical Fiber (POF); an early stage investigation
cr
AUTHORS: Sayidatul Nadia Elias1, Norhana Arsad2, Sabiran Abu Bakar2
ip t
towards the development of oral cancer sensor using POF
us
MAILING ADDRESS: Department of Electric, Electronic and System Engineering, Faculty of Engineering and Built and Environment,
te
FAX NUMBER: +603 8925 9080
d
PHONE NUMBER: +603 8921 6320
M
Selangor, Malaysia.
an
The National University of Malaysia,
Ac ce p
EMAIL ADDRESS: [email protected] [email protected] [email protected]
Page 1 of 19
Abstract: Oral Squamous Cell Carcinoma (OSCC) has a higher chance of survival rate if being detected at an early stage. Overconsumption of nitrite in human body may result in OSCC formation. Nitrite and nitrate is a part of reactive nitrogen species (RNS) in saliva and
ip t
is found to have higher percentage in OSCC patients’ saliva, thus chosen to be investigated as a potential OSCC biomarker using plastic optical fiber (POF) with evanescent wave sensing
cr
method. The experiment is done using UV/VIS light source and the peak absorbance is found to be approximately 540nm. The sensor displays moderate sensitivity with higher sensitivity
us
towards lower end of the concentration.
M
an
Keywords: Beer-Lambert law; evanescent wave sensing; nitrite; OSCC; plastic optical fiber
INTRODUCTION
d
Oral squamous cell carcinoma (OSCC) is generally referred as oral cancer [1]. Oral cancer is
te
the sixth most common cancers among the world population, and third in the developing countries including Malaysia [2]. Number of oral cancer incidence worldwide is 170,496 with
Ac ce p
83, 109 mortality for men, and 92,524 incidence with 44,545 mortality for women [3]. Estimated incidence rate per 100,000 of oral cancer cases in Malaysia is 17.3 for male and 8.6 for female, while the mortality rate per 100,000 is 9.7 for male and 4.6 for female [4]. Some of the risk factors identified are tobacco smoking, alcohol consumption, viruses, dieting habit and deficiency states [5]. Despite numerous advancement in treatment, the five-year survival rate for the oral cancer patients is still 50% for the past few decades, mainly due to lack early stage diagnosis, with 60% of the patients are examined to have oral cancer when the disease is at stage III and IV [1, 6, 7]. An early stage detection of oral cancer is vital to ensure a higher survival chance for the patients by 60%-80% as compared to only 30%-40 % percent when the cancer is detected at an advanced state [8].
Page 2 of 19
Nitrates (NO3) and nitrites (NO2) are inorganic ions that occur naturally in our environment [9]. The main concern of health hazard on human body caused by nitrates and nitrites is that when nitrates are consumed by human they change into nitrites [10]. Over-consumption of
ip t
nitrites can cause damage to the nervous system, spleen liver, reducing the oxygen transport capacity by blood, and most importantly the reaction between nitrites and secondary or
cr
tertiary amines. This reaction causes the formation of N-nitroso compounds, with some of these compounds are carcinogenic, tetratogenic, or mutagenic which may result in the
us
formation of OSCC [9, 11-14].
Reactive nitrogen species (RNS) in the form of nitrosamines (nitrites NO2 and nitrates NO3)
an
found in human saliva can cause DNA base alterations, strand breaks, damaged tumor
M
suppressor genes, and enhanced expression of protooncogenes which lead the development of cancer [15]. A study conducted found significant elevated concentration of RNS in the saliva
d
sample of OSCC patients, with the percentage of nitric oxide (NO) 60%, nitrites (NO2) 190%,
te
and nitrates (NO3) 93% higher as compared to their concentrations in the saliva of nonpatients [16]. Thus, for this work nitrite is chosen as the sample to be investigated.
Ac ce p
Plastic optical fiber (POF) differs from the other types of optical fibers by having its core being made of polymethylmethacrylate (PMMA) with the core radius typically between 125409 µm [17, 18]. POF is easy to handle, flexible, lower density, higher elastic deformation limits, and cheaper because it allows the use of low precision connector due to its large core diameter, causing a cost decrement on the overall system [17]. Fiber optic is usually chosen as a chemical sensor for the determination of cations and anions because it has good sensitivity and selectivity, can be fabricated easily, and having low cost [19]. As a biosensor, optical fiber is very small, flexible and light, thus conviniet for insertion into catheters and needles hence allows particular measurements inside tissues and blood. The materials used in fiber
Page 3 of 19
structure (glass or plastic) are sturdy, non toxic, biocompatible with human body, diaelectric and uses low power for the light source, ensuring patient’s intrinsic safety [20]. Evanescent wave sensor, a type of intensity based fiber optic sensor is a popular choice for a
ip t
chemical and biological sensor because this method proves excellent sensitivity over chemical reagents, great control over interaction parameters, able to interact with molecules within the
cr
depth of light penetration and low cost [21-25]. Evanescent wave sensor makes use of the change in light energy that flows into the cladding from the core. The sensing probe for this
us
sensor is created by removing a portion of the cladding on fiber’s body, exposing the core. The unclad portion (sensing probe) is the being brought into direct contact with the chemical
an
sample being measured, or coating the unclad portion with chemically-sensitive materials
M
[26].
In this work, a simple POF evanescent wave sensor is constructed for the detection of nitrite
d
with different concentration. This experiment is done as an early stage towards the design and
Ac ce p
THEORY
te
development of OSCC sensor using POF.
When a portion of the fiber cladding is removed and the unclad fiber is immersed in the sample, there will be an interaction between the evanescent field in the fiber and the lightabsorbing elements in the sample. The sample acts as a new ‘cladding’ for the fiber and will absorbs a particular wavelength of light transmitted in the fiber depending on the concentration of the elements that absorbs light at that wavelength [21]. In this work, the fiber is uncladded and being brought directly in contact with the sample; this method is known as direct spectroscopic evanescent wave sensing [27]. Thus, Beer-Lambert law that relates the element’s absorbance with its concentration can be applied [28]: (1)
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Where A is absorbance, P0 and P is the intensity of incident and transmitted light energy respectively, ε is molar absorptivity (M-1 cm-1), b is optical path length (cm) and c is concentration of the solution (M).
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If the optical path length is being kept constant at 1cm, molar absorptivity is the slope of the concentration versus absorbance curve:
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(2)
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METHODOLOGY
Optical Fiber Preparation
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A.
4 single core POFs with each having 125μm core radius, 2mm diameter and 40cm in length
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were prepared. 3 of the POFs were stripped in the middle from their protective jacket with an optical path length of 1cm each, using coaxial stripper and utility knife. The diameter of the
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cladding for all 3 POFs were 1mm. 2 of the unjacketed POFs were the then mechanically
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etched using fine sand paper to give out 0.8mm and 0.6mm cladding diameter respectively. For all cladding diameter measurements, POFs cladding were viewed under Olympus
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Microscope Imaging System and using DigiAcquis 2.0 software, the images were captured and the claddings’ diameter was measured. All POFs were later washed with distilled (DI) water and leaved to dry. To measure the fiber loss in all 4 POFs, a simple setup was done using and an optical fiber communication trainer (OFT) having 850nm LED transmitter and PIN diode receiver. A function generator was connected to OFT input and the output was connected to an oscilloscope. The unjacketed section was encapsulated to prevent external disturbances. This step was done to find out the most sensitive POF to be used for nitrite detection. Fig. 1 shows the experimental setup to measure POFs loss. B.
Sample Preparation
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For this experiment, a nitrite solution was prepared by diluting 0.0138g sodium nitrite into 200ml DI water using volumetric method to give out 1mM nitrite concentration sample. Solution samples with varying concentration was produced by diluting the 1mM nitrite
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solution with DI water into another 5 samples, each having 50μM, 25μM, 12.5μM, 6.25μM, and 3.125μM nitrate concentration respectively. Greiss reagent consisting of two solutions
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was also prepared by adding 0.0581M of sulphanilamide into 5% acid for the first solution, and the second solution having 0.0038M of N-1-napthylamine. Colorimetric analysis of nitrite
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was performed by diluting each sample with sulphanilamide acid, and then being added with 1ml N-1-napthylamine. The mixture gave out a reddish-violet colored solution. Each sample
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had a total volume of 3ml.
Experimental Setup
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C.
All instruments used for spectra measurements are from Ocean Optics. The UV-VIS-NIR
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light source used is of model DH-2000-BAL with deuterium tungsten halogen light source.
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The spectrometer model HR4000 was connected to 3648-element CCD-array Toshiba detector. SMA connector was used to connect POF to the light source and spectrometer. The
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exposed cladding of the POF was inserted into a tube containing the sample and totally encapsulated with a black box. Data spectrum was displayed and recorded using Spectrasuite software. The experimental setup is as shown in Fig. 2.
RESULT AND DISCUSSION
Exposing the cladding of the fiber will result in loss due to light energy that leaks from the cladding to the environment. In evanescent wave sensing this leaked energy is important for its interaction with the sample, hence higher loss of fiber means more light is able to interact with the light-absorbing elements in the sample. Measurement of loss in different prepared POFs was carried out in order to verify that POF has the same loss property as silica fiber, and
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also to determine the best POF preparation to be used for nitrite detection. Fig. 3 shows the loss in the prepared POFs and it can be seen that POF with 0.6mm cladding exhibited the most loss. This is because the etched cladding is the nearest to the core, hence allowing more
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leakage of light. Based on the graph, it can be assumed that diameter lower than 0.6mm will give out higher loss, with the most sensitive POF having a totally exposed core. However the
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sensor setup caused strain on the thin exposed cladding, thus any cladding diameter below 0.6mm would break.
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Fig. 4 shows the absorption spectrum of nitrite samples with varying concentration in the visible wavelength of 400-700nm using POF with 0.6mm cladding. The peak absorbance
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increasing concentration of nitrite samples.
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wavelength is found to be at approximately 540nm. The amount of absorption increases with
According to Beer-Lambert law, absorbance is linearly related to concentration. Hence, in an
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ideal case when the concentration of nitrite increases, absorbance will increase in direct
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proportion with the concentration. Fig. 5 shows the calibration curve plot of nitrite concentration versus absorbance at the peak absorbance wavelength of approximately 540nm.
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The absorbance increases as the concentration of the sample increases, but not in the form of a straight curve.
Fig. 6 describing the same result as in Fig. 5 but in the form of a scattering plotted graph, taking into consideration the colleration coefficient (R2) and the linear regression line. The correlation coefficient for this graph is 0.9475, showing a strong positive linear correlation approaching +1 which proves that this graph possess an almost linear characteristic. The linear trend indicates that the sensor being used in this experiment does not deviate far from Beer-Lambert law, and thus the sensor is said to exhibit an adequate sensitivity. Evanescent field absorption is the key element in this experiment; hence even the slightest disturbances on the surface will causes error to the measurement. The sensing region was carefully cleaned
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in between sample changes, but minute particles in the environment would still affect the sensor performance. Fig. 7 shows the comparison between the calibration curve based on experimental results and
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theoretical calculation using (1). There is smaller difference between the two curves at the lower end of the concentration, indicating that the sensor is more sensitive towards smaller
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concentration of solution. The sensor deviates further away from the theoretical curve beyond 60µM.
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Based on a number of previous researches, the uses of optical sensors that apply evanescent wave field absorbance theory are prone to demonstrate higher sensitivity at a lower level of
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concentration [27, 29-31]. The effect of concentration on the sensor can induce a phenomenon
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in which at a concentration of more than 0.01 M, a deviation from Beer-Lambert law can be seen. Some of the causes that lead to such phenomenon are electrostatic interaction, light
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scattering due to particulates in the sample, sample fluorescence or phosphorescence, changes
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in the refraction index at high analyte concentration, and non-monochromatic radiation [32]. Molar absorptivity can be found from the slope of Fig. 5 using (2) and slope formula for non-
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linear relationship. Molar absorptivity of this experiment is found to be 3.4 x 104M-1cm-1. Based on Savvin’s criteria of sensitivity [33], this value falls into moderate sensitivity. Comparing with the molar absorptivity of the theoretical curve’s slope of 3.5 x 104M-1cm-1, the percentage error of the experimental data is 4.33%.
CONCLUSION
As a conclusion, an experimental setup for the detection of nitrite in varying concentration using POF has been carried out. Loss in 4 different prepared POFs has being studied to find the most suitable POF structure for nitrite detection. 6 nitrite samples with different concentration are used in this work, developed using greiss reaction. The peak absorbance
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wavelength is found to be at approximately at 540nm. Calibration curve is plotted based on Beer-Lambert law and it shows that the sensor exhibits adequate sensitivity. Comparison between experimental data and theoretical calculation indicates that the sensor has higher
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sensitivity and the lower end of the concentration. Molar absorptivity of the nitrite samples is calculated from the slope of the calibration curve with the value of 3.4 x 10-4M-1cm-1 and
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based on Savvin’s criteria of sensitivity, the sensor has moderate sensitivity. Percentage error of the experimental molar absorptivity as compared to the theoretical value is 4.33%. For
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future work, we aim to increase the sensitivity of the sensor and improvise the setup in order
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to detect nitrite in human saliva.
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ACKNOWLEDGMENT
I would like to express my sincere gratitude towards Dr. Norhana Bt. Arsad, Department of
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Electrical, Electronic and System Engineering, Faculty of Engineering and Built
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Environment, National University of Malaysia for her valuable advices, helpful suggestions, study materials, and thorough support during this research. I would also like to thank Mr.
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Sabiran bin Abu Bakar, a Master’s student from the Department of Electrical, Electronic and System Engineering, Faculty of Engineering and Built Environment, National University of Malaysia for his guidance in preparing the experimental setup and sample solutions.
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Fig. 1. Experimental setup to measure POF loss.
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Fig. 2. Experimental setup for nitrite detection.
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Fig. 3. Fiber loss in 4 different prepared POFs.
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Fig. 4. Absorption spectrum of nitrite sample soultion with varying concentration.
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Fig. 5. Calibrated curve plot of nitrite absorbance at peak wavelength based on Beer-Lambert
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law.
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line.
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Fig. 6. Scattering plot of nitrite absorbance with correlation coefficient and linear regression
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Fig. 7. Comparison between experimental data and theorotical calculation of varying nitrite
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concentration at peak wavelength absorbance.
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