AIP Conference Proceedings 2193, 040009 (2019); https://doi.org/10.1063/1.5139371 2193, 040009
© 2019 Author(s).
Photoelectrochemical measurement of
Epstein-Barr virus DNA in peripheral blood serum of nasopharyngeal carcinoma patients
Cite as: AIP Conference Proceedings 2193, 040009 (2019); https://doi.org/10.1063/1.5139371 Published Online: 10 December 2019
Kusmardi Kusmardi, Aryo Tedjo, Yurnadi Hanafi Midoen, et al.
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Photoelectrochemical Measurement of Epstein-Barr Virus DNA in Peripheral Blood Serum of Nasopharyngeal
Carcinoma Patients
Kusmardi Kusmardi
1, Aryo Tedjo
2,a), Yurnadi Hanafi Midoen
3, Michael Adrian
41Departement of Anatomic Pathology, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
2Departement of Medical Chemistry, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
3Departement of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
4Student at Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
a)Corresponding author: [email protected]
Abstract. Epstein-Barr Virus (EBV) DNA presents in the serum of nasopharyngeal carcinoma (NPC) patients indicates the existence of the viral DNA in the circulation and may be used as an early marker in NPC diagnosis. EBV DNA isolated from the serum and then amplified by using polymerase chain reaction (PCR) could be used to assess the response of the DNA to various NPC therapies. This study compares the results of EBV DNA measurement in the serum of NPC patients using photoelectrochemical technique and PCR. The presence of a specific oligonucleotide sequence of a standard DNA (EBNA-1) may be detected by photoelectrochemical hybridization of the standard with a DNA probe immobilized on TiO2 electrode, in which the DNA is intercalated by an electrochromic material.The purpose of this study is to obtain a photoelectrochemical system with a relatively good response and sensitivity to the changes in the concentration of EBV DNA in the peripheral blood serum of NPC patients. This research attempts to provide a device and methods that are simple, relatively inexpensive and easy to use, yet sensitive enough to see the prognosis of NPC patients. This study also attempts to develop the measurement instrumentation technology in the field of biomedical research. The photoelectrochemical system of this research used anthocyanin as DNA intercalator. The anthocyanin was isolated from purple cabbage leaves (also known as red cabbage), strawberries, and grapes and the extractant of these anthocyanins were ethanol and water. The best extraction with the highest level of total anthocyanin was achieved by alcohol as extractant.
Alcohol gave higher yield of anthocyanin compared to water and ethyl acetate. Purple cabbage leaves have the highest level of anthocyanin compared to strawberry fruits and grapes. Anthocyanins contained in the extract of purple cabbage, strawberries, and grapes may function as DNA intercalators. Electrochromic substance used as DNA intercalator produces difference between the current strength generated by photoelectrochemical system with light and without light. There is a very strong correlation between diluted concentration of DNA and current strength shown by the electrochemical system of this study. There is also correlation between EBV EBNA-1 DNA concentration readings by using photoelectrochemical system and PCR. The sensitivity and specificity to EBV DNA in the serum of NPC patients of photoelectrochemical techniques are significantly higher compared to PCR.
Keywords: EBV, photoelectrochemical, PCR, anthocyanin, intercalation
INTRODUCTION
Nasopharyngeal Carcinoma (NPC) is a malignancy in the head and neck with multifactor etiology [1]. Generally, NPC is an alteration in the malignancy of the squamous cells. Common NPC etiology is Epstein-Barr Virus (EBV)
infection. However, some etiologies related to lifestyle are also cited. In researches conducted in 48 countries, it is reported that the NPC etiologies related to lifestyle are, among others, salted fish consumption and smoking cigarettes.
Socioeconomic status is also reported to increase the risk of NPC incidence. Milk consumption, on the contrary, is reported to reduce the possibility of having NPC [2]. Generally, NPC is a rare malignancy and endemic, especially in North America [3]. However, the incidence is higher in Asia [4]. NPC incidence in Asian countries is reported to achieve 5-9 times higher than in the North American countries [5].
In Southeast Asia, the incidence of NPC is almost evenly-spread. One research conducted in Brunei Darussalam comparing several ethnicity, such as Malay, Chinese, and indigenous people, showing a similar incidence of NPC in the three.3 Furthermore, NPC patients were also reported to be evenly-spread in every region in Indonesia. There are about 100 NPC cases per year at Dr. Cipto Mangunkusumo General Hospital (RSCM), Jakarta, while Hasan Sadikin Hospital in Bandung has 60 cases in average, Makassar 25 cases, Palembang 25 cases, Denpasar 15 cases, Padang and Bukittinggi 11 cases in average per year, as well as Medan, Semarang, Surabaya, and other cities showing that NPC is found everywhere in Indonesia [6,7,8].
As the main etiology in NPC, EBV infection becomes the focus of many researches [9]. Some modes of diagnosis need to be done to investigate the presence of EBV infection [10]. The virus genome found in the tissue and blood (including serum and plasma) is a sign of the presence of this viral infection [11]. In addition, various protein products of the virus may also be used as markers [12]. Antibody against the virus products found in the blood may be used as a marker in the diagnosis of this infection as well [13].
EBV DNA used as a parameter of diagnosis shows higher sensitivity and specificity than other markers [14]. EBV DNA examination in the biopsy tissues is correlated to the EBV DNA in the plasma or serum [11]. As a method of examination that is relatively interventive, the use of plasma or serum simplifies the process of examination compared to biopsy technique of the nasopharyngeal tissue [15]. So far, PCR is commonly chosen to confirm the presence of EBV infection [16].
EBV DNA, just like other viral DNAs, may be intercalated by an intercalating agent. This ability of intercalation could open the possibility of an intercalating agent to insert into viral DNA [17,18,19]. Furthermore, by photoelectrochemical measurement following the intercalation event, it may be predicted the quantity of the EBV DNA [20,21,22]. This will open the opportunity of complimentary use of photoelectrochemistry with PCR in DNA examination [19].
Anthocyanins are chemical compounds contained in plants that may be used as intercalating agents [20,21,23].
Anthocyanins have substantial potential as DNA intercalator for they are easily isolated and the levels are sufficient in some natural materials [17,18,22]. This research has an objective to compare the results of EBV DNA measurement in the serum of NCP patients in Jakarta by using PCR and by using photoelectrochemistry with anthocyanin as the intercalating agent.
METHOD
Research Subject.
Research subjects are NCP patients checking up at the Department of Radiotherapy, Faculty of Medicine, Universitas Indonesia (FKUI). All research subjects were previously provided with informed concerned before participated in this research. NCP diagnosis was made based on the examination of biopsy tissue histopathology performed by otorhinolaryngologists of the Department of Nose, Ear, and Throat (ENT) and anatomic pathology specialists of FKUI/RSCM, Jakarta.
EBV DNA Isolation.
Peripheral blood sample (3 mL) of an NCP patient in an EDTA (an anticoagulant) containing vacutainer was centrifuged to separate the blood into 2 parts, i.e., serum and blood cells. EBV DNA isolation was conducted according to the following steps. To a 1.5 mL Eppendorf tube, it was added 100 L serum, 300 L TE, 50 L 10% SDS, and 1
L proteinase-K. The mixture was then vortexed for 1 minute and incubated at 650C for 1 hour. After the incubation, 200 L phenol and 200 L chloroform iso-amyl-alcohol (C-IAA) were added. The mixture was vortexed for 3 minutes and centrifuged at 10,000 rpm for 10 minutes at 200C until a clear aqueous layer formed. The aqueous layer was then transferred into a new Eppendorf tube. Afterward, 100 L phenol and 300 L C-IAA were added to the tube and the mixture was vortexed for 3 minutes, centrifuged at 10,000 rpm for 10 minutes at 200C [24].
The above method was repeated several times (2-3 times) until it was obtained a clear aqueous layer and ready to be transferred into a new Eppendorf tube. Further, 30-40 mL natrium acetate (1/10 volume) pH 4.2 and 1 mL cold absolute ethanol were added to precipitate the DNA by turning the tube up and down several times and then stored at -200C overnight.
The Eppendorf tube was centrifuged at 12,000 rpm for 30 minutes at 200C until the white pellet formed at the bottom of the tube. The supernatant was then removed, and the precipitate was added with cold, sterile 70% ethanol to wash the DNA and then centrifuged again at 10,000 rpm for 10 minutes at 200C. The supernatant was then carefully removed, and the pellet was wind-dried for 30 minutes by tilting the Eppendorf tube toward a tissue paper. The dried DNA pellet was rehydrated with 20 L TE and then stored at -200C. The DNA was ready for the next steps.
EBV DNA presence analysis.
DNA amplicons from the serum of NPC patients were assayed by separating the DNA fragments with electrophoresis on 1.5% agarose gel added with 1 g/L ethidium bromide using a horizontal electrophoresis apparatus in 1x TAE buffer. To each well of the agarose gel, it was added a mixture of 10 L DNA fragments and 3
L loading buffer (0,25% bromophenol blue, xylene cyanol, sucrose 4% w/v) and then electrophoretically separated at 90 Volt for 60 minutes. DNA ladder 100 bp was used as the marker. The DNA fragment bands were then visualized by using UV illuminator, and the results were reported as positive if one band of 120 bp PIGR DNA was found. The specific DNA band was then documented by using polaroid camera for EBV DNA presence analysis [25].
Electrode Preparation.
TiO2 electrode was made from a mixture of 0.2 g TiO2 powder (Degussa, P25), 0,4 mL nitrate solution (0.1 M), 0.08 g polyethylene glycol and a few drops of Triton X-pa 100. The mixture was made into a paste and used to coat SnO2 conductive glass, which was then heated at 4500C for 2 hours [26].
Electrochromic dye preparation.
One gram materials, each from grapes, strawberries and purple cabbage leaves, were macerated with 100 mL aquadest and with 100 mL ethanol for 30 minutes. The extracts were then dried and weighed to the concentration to be used [26].
Electrochromic material test as DNA intercalator.
The test was performed by adding the extracts of the tested materials into solutions of double-stranded DNA from EBV DNA isolation in several variations of concentration. Observation of the capability of the material to intercalate DNA was performed by UV-Vis spectrophotometer. The data collected was the change in intensity of the absorbance peaks and shifts of the absorbance peaks from the absorption spectrum after the addition of the organic material [20].
Photoelectrochemical system test.
The photoelectrochemical system in this research used ‘sandwich’ electrodes consisting of TiO2 as working electrode and SnO2 as counter electrode. The electrolyte used was iodide-iodin (I2-KI) solution. The sample was double stranded DNA. The test conducted was sensitivity of the photoelectrochemical system to the change in DNA concentration. The data analyzed was the change in current generated by the system due to DNA concentration change [19].
Detection by using DNA probe.
DNA probe solution was immobilized onto TiO2 electrode layer by incubating 25 L DNA probe solution 200 M in HEPES buffer 50 mM pH 7 at 60°C for 6 hours. After the incubation, the electrode was washed with aquadest.
Hybridization was performed by adding 25 L EBV DNA in several variations of concentration to HEPES buffer 50 mM pH 7 at 60 °C for 6 hours. After the hybridization, the TiO2 electrode was washed with HEPES buffer 50 mM and aquadest. Electrochromic material (anthocyanin extract) and electrolyte were then added to the system, and the
change in current was monitored. The data on the current change of the system to concentration change in the target DNA was recorded. 22
Data analysis.
A linear regression was made from the data of current change in the photoelectrochemical system to the change in EBV DNA. T-test was used to calculate the significance of alcohol and aquadest as solvents in the isolation of anthocyanins of purple cabbage, strawberries, and grapes. Kendall correlation test was performed to the results of DNA level determination according to photoelectrochemistry and PCR. Chi-square was used to find out the sensitivity and specificity of the photoelectrochemical method compared to PCR. The correlation between photoelectrochemical readings and RGB image intensity in PCR was determined.
RESULTS
Anthocyanin extraction and level determination.
The results of measurement of anthocyanins isolated from purple cabbage, strawberries and grapes showed that the best extractant with the highest total anthocyanin level was alcohol (an average of 20.12 mg/100 g for purple cabbage, 5.57 mg/100 g for strawberries, and 4.48 mg/100 g for grapes). Water only gave anthocyanins in an average of 7.45 mg/100 g for purple cabbage, 1.47 mg/100 g for strawberries, and 0.74 mg/100 g for grapes. Therefore, anthocyanin isolated from purple cabbage was used in the next steps of this research.
Significance test by using t-test to ethanol and water as solvents in the extraction of purple cabbage anthocyanin resulting p-value of 0.00, which means that higher level of purple cabbage anthocyanin was achieved by ethanol (Table 1).
TABLE 1. Differences in purple cabbage anthocyanin level isolated by using ethanol and aquadest Solvent N Mean Std. Deviation Std. Error
Mean P
anthocyanin
(mg/100 g) ethanol 21 20.1238 9.88982 2.15814 0.000
aquadest 21 7.4529 3.90786 0.85277
Electrochromic material test as DNA intercalator.
The test results of electrochromic materials (anthocyanin) from purple cabbage, strawberries, and grapes in intercalating double-stranded DNA are shown in Figure 1. The concentrations of DNA used as intercalation samples were 2 ppm and 4 ppm. All anthocyanins extracted from purple cabbage, strawberries, and grapes mixed with DNA solution showed decrease in absorbances compared to the absorbance of anthocyanin without DNA. The decrease in absorbance increases with the increase in DNA concentration (Figure 1).
FIGURE 1. Electrochromic material test as DNA intercalator. The absorbance curve of grapes extract (A), purple cabbage leaves extract (B), strawberries extract (C), and EtBr solution (D), as control with two DNA concentrations show decrease in absorbance
after being added to DNA solution (--extract, --extract + 0.2 ppm DNA, --extract + 0.4 ppm)
Photoelectrochemical system test
This test was performed by using anthocyanin extract from purple cabbage. The change in the current generated based on the given external voltage in the system can be seen in Figure 2
FIGURE 2.The Change of Current Produced by Photoelectrochemical System to Detect Double-Stranded DNA for with Light (), without Light (♦); (a) System without dsDNA addition, (b) System with dsDNA addition and 10 mg/ml anthocyanin extract,
and (c) System with dsDNA addition and Ru(bpy)22+ solution. The dsDNA Concentration was 10 ng/mL and The Light Source was Bulb Lamp (25 watt, 535 Luxes at 25 cm)
Photoelectrochemical measurement of standard DNA.
The results of photoelectrochemical measurement of EBNA-1 DNA (standard) with concentrations of 0.5 nM, 1.0 nM, 1.5 nM and 2.0 nM are presented in Figure 3.
FIGURE 3. Regression line connecting standard (EBNA-1 DNA) concentration and current strength measured by using photoelectrochemistry
Measurement of EBNA-1 DNA using electrochemistry and PCR.
The amplification of EBNA-1 DNA found in nasopharyngeal carcinoma patients using PCR is shown in Figure 4.
The results of RGB image intensity reading using Adobe Photoshop for every band resulted from agarose gel electrophoresis are not provided. The results of electrochemical and PCR measurements of EBNA-1 DNA from each of 16 samples are shown in Figure 5. From Figure 5, it is shown that the current strength of all samples could be read by the created photoelectrochemical system. The concentration of EBNA-1 DNA may be calculated from the standard curve.
FIGURE 4. The results of PCR of EBNA-1 DNA (no serum, serum normal, 1-7 from the serum of NPC patients
Sensitivity and specificity of photoelectrochemical measurement.
Sensitivity and specificity tests of EBV DNA measurements from the samples collected were performed by photoelectrochemistry and then compared to PCR. From the ROC curve, it was obtained detection limit for electrochemistry (0,65 mA) and PCR (16,5 nM). Chi-square test resulted a 100% sensitivity and 87.5% specificity for photoelectrochemical measurement.
µA
(a) (b)
FIGURE 5. Diagram showing the relation between the results of EBV DNA measurement by electrochemistry (a) and PCR (b)
DISCUSSION
Purple cabbage extracted with ethanol gives anthocyanin in an average value of 20 mg/100 g. This level is relatively lower than the level obtained by other researchers using Indian local-market purple cabbages, which gave anthocyanin level achieving 50 mg/100 g [27]. This relatively significant difference is probably due to the difference in the purple cabbage variety used and the difference in the soil condition and the climate between Indonesia and India [28]. Another possibility also comes from the purple cabbage used in this research that was cultivated in plant pots in the Laboratory of Biopharmaca Research Center, IPB University, Bogor.
It is demonstrated that anthocyanins in purple cabbage, strawberries, and grapes extracts may function as DNA intercalators. This could be observed from the hypochromic effects, i.e. decreasing absorbance values of respective spectra curve of extract after being added to double-stranded DNA. The increase in the level of DNA resulting in smaller absorbance value. This is due the increase in DNA level will increase the capability of anthocyanins to intercalate DNA since a larger intercalation space is available. The more the anthocyanins intercalating the double- stranded DNA, the lower the absorbance [17,23].
From Figure 2(a), it is seen that there is almost no difference in the current strength generated in control (no double- stranded DNA added) both measured in light and no light in photoelectrochemical system. On the contrary, the use of electrochromic substances as DNA intercalator (Figure 2(b) and 2(c)) resulting in difference in current strength between photoelectrochemical systems provided with light and without light.
From Figure 3, it is seen that serial dilution of standard EBNA-1 DNA gives a regression value of 0.919 with the regression line equation of y = 33,25x – 3,660, where y is the current strength generated by photoelectrochemistry and x is DNA concentration in nM. R2 value of 0.919 shows that there is a very strong correlation between serially diluted DNA concentration and the current strength generated by the created and standardized electrochemical system.
Figure 3 indicates that there is a linear correlation between serum EBV DNA measurement with photoelectrochemistry and PCR quantified with Adobe Photoshop. This linearity shows the potential of photoelectrochemistry to replace or substitute PCR, which requires more sophisticated equipment and skillful technician. Like in other common electrochrom, this linearity shows the capability of anthocyanin to intercalate double-stranded EBV DNA[23].
The sensitivity of an apparatus compared to the gold standard is the capability of the apparatus to reveal a sample as purely positive, while specificity of an apparatus is the capability of the apparatus to test a sample as pure negative.
From the test conducted to photoelectrochemical system of present research, anthocyanins intercalation has 100%
sensitivity compared to the gold standard of PCR. This shows that photoelectrochemistry with DNA intercalation is capable of revealing that a sample is positive containing EBV DNA with 100% matching to the results of PCR. This kind of sensitivity is the highest of a test to equalize the gold standard measurement. In addition, the specificity showed by the photoelectrochemical method is achieving 87.5%, which is considered very high [29]. Therefore, photoelectrochemistry using anthocyanins as intercalating agent is potential to be developed as a method and technique in EBV DNA measurement.
CONCLUSION
Alcohol is the best solvent to extract anthocyanins compared to water. Purple cabbage leaves have the highest anthocyanin level compared to strawberry fruits and grapes. Anthocyanins in purple cabbage, strawberries, and grapes may function as DNA intercalators. The use of electrochromic substances as DNA intercalator resulting in difference in current strength generated in photoelectrochemical system with light and without light. There is a very strong correlation between diluted DNA concentrations and current strength showed by the photoelectrochemical system of this research. There is also correlation between the readings of EBNA-1 DNA concentrations by photoelectrochemistry and PCR. The sensitivity and specificity of photoelectrochemical technique in analyzing serum EBV DNA of NPC patients is significantly higher than PCR.
ACKNOWLEDGMENTS
The publication of the article was supported by Directorate of Research and Public Services Universitas Indonesia
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