VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY INTERNATIONAL UNIVERSITY DEVELOPMENT OF a VERSATILE AND COMPACT PAPER BASED MICROFLUIDIC BIOSENSOR FOR DETECTION OF COPPER IN FOOD PRODUCTS

VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY INTERNATIONAL UNIVERSITY DEVELOPMENT OF A VERSATILE AND COMPACT PAPER-BASED MICROFLUIDIC BIOSENSOR FOR DETECTION OF COPPER IN FOOD PRODUCTS A thesis submitted to The School of Biotechnology, International University in partial fulfillment of the requirements for the degree of B.S in Biotechnology Student’s name: Nguyễn Lan Thảo - ID:BTIU09046 Supervisor: Dr Nguyễn Thái Lộc June 2013 Acknowledgement First of all, it is of great importance to deliver my thankfulness to Dr Loc T Nguyen, who dedicated his generous time and sound knowledge to my research as a mentor Truthfully, it was my absolute honor to work with Dr Nguyen Thanks to his insightful instruction, valuable experiences and motivating encouragement, I became brave and confident enough to take on plenty of challenges Had it not been for his constant supports, I would not have successfully accomplished this study Secondly, my sincere gratitude is expressed to Dr Khoi T Nguyen for consulting me with suitable techniques and stimulating ideas to deal with difficulties that arose during the study of unfamiliar subjects That was his inspiration which kept me persistent until the end Moreover, I owe my gratefulness to all the laboratory managers and staffs, particularly MSc Long H Nguyen, MSc Dao Q T Tran, MSc Lieu B T Truong and BSc Anh L Dang, who devoted themselves to ensure a wellequipped and safe laboratory and did not even mind working overtime or at weekend I also would like to take this opportunity to say special thanks to Ms Han B Nguyen, Ms Yen K T Dang and Mr Trong Q Luu for standing by me throughout hardships, as well as cheering me up in various situations Besides, the supports from Mr Thien H Nguyen are much appreciated His interesting lessons enabled me to perform sophisticated image analysis as well as to turn ideas into vivid figures, all of which contributed to the best presentation I have ever made It would be incomplete not to thank you all – my colleagues in lab 101, including Ms Dung M T Nguyen, Mr Dang D Nguyen, Ms Hien T T Nguyen, Ms Khanh K T Nguyen, Ms My H T Nguyen, Ms Oanh K T Nguyen, Ms Vien L Ngo and Ms Tram N L Nguyen for supporting and sharing with me no matter in good times or in bad that made every moment priceless Together they created the most memorable semester Last but not least, it is my family who I want to convey the deepest appreciation to Thank my mom, Mrs Lan T Ta and my dad, Mr Hy D Nguyen for giving me such a favorable environment that I could concentrate and the best in the study Thank my brothers for their sweet treats and assistance in delivering necessary research equipment No matter what the situations are, my family is always the greatest back-up that I can count on For this moment, my heart is filled with endless love and sympathy from many other people that I cannot list here due to limited scope To all of those, I would love to personally say big thank you and promise to continuously improve myself DEVELOPMENT OF A VERSATILE AND COMPACT PAPER-BASED MICROFLUIDIC BIOSENSOR FOR DETECTION OF COPPER IN FOOD PRODUCTS a Thao L Nguyen , Loc T Nguyen a,* : School of Biotechnology, International University – Vietnam National University - HCMC a *: Corresponding author’s email address: ntloc@hcmiu.edu.vn Abstract In this contribution, a versatile, inexpensive and compact paper-based microfluidic biosensor was developed to detect heavy metals in food products The underlying principle was based on sensing of ammonia (NH3) released during ureasecatalyzed hydrolysis of urea At the presence of heavy metals, the amount of NH3 produced diminished due to inhibition of urease enzyme Therefore, the concentration of target heavy metals could be indirectly determined from NH 3, qualitatively, semi-quantitatively or quantitatively Hydrophobic barriers of functional areas the sensor were fabricated by paraffin-dipping method Urease enzyme was physically immobilized onto the sensor and phenol red was used for qualitative and semi-quantitative detection of heavy metal via image analysis The sensor was also integrated into an electrochemical system using simple screenprinted electrodes In the current study, Cu++ was used as a model heavy metal for testing performance of the sensor Qualitative results showed that a strong contrast between safe and unsafe sample, which was critical for the practical applicability of the sensor Relationship between color intensity and Cu++ concentration was characterized by a R2 of 0.98 and the linear range covered Cu++ concentrations from 0.01 – ppm Detection limit was estimated to be 0.018 ppm which was well below standard limit established by WHO and EC for Cu++ Quantitative tests were still at beginning stage and were worth further investigations The findings from this study demonstrated that the proposed paper-based biosensor could be a promising platform to develop low-cost test kits for detection of heavy metals in foods Keywords: Paper-based biosensor, wax-dipping, screen-printing electrodes, SPEs, colorimetric test, amperometric test 1 Introduction Heavy metals are ubiquitous pollutants and their presence in the environment can be attributed to natural or human activities Many heavy metals are carcinogens and may be involved in several dangerous diseases (Hossain et al., 2011) In general, heavy metals are not easily degraded and tend to accumulate in soils and sediments (Dominguez-Renedo et al 2013) Major sources of soil contamination with heavy metals are wastewater irrigation, solid waste disposal, sludge applications, vehicular exhaust and industrial activities (Khan et al 2008) Plants grown on contaminated soils may build up excessive content of heavy metals and eventually have negative effects on food quality and safety Therefore, it is of critical importance to monitor the contamination of heavy metals in food products Currently, analytical methods for heavy metals usually rely on inductively coupled plasma/atomic, emission spectrometry (ICP/AES), inductively coupled plasma, mass spectrometry (ICPMS), atomic absorption spectroscopy (AAS), or wet chemical methods such as titrimetry, gravimetry, colorimetric assays, etc (Hossain et al., 2011) Despite their high sensitivity, selectivity, reliability, and accuracy, these methods are time-consuming, require sophisticated instrumentation, skilled personnel and complicated sample pretreatment (Turdean, 2011) Inherent drawbacks of these assays restrict their use mainly in centralized laboratory Thus, inexpensive, easy-to-use and portable test kits which can screen contamination of heavy metals for a large number of samples are highly desirable Recently, the emergence of paper-based microfluidic devices has become important resources for low-cost diagnosis The underlying principle of these devices is to pattern hydrophilic hydrophobic micron-size capillary channels on paper using various methods including wax-printing, ink-jet printing, flexography printing, screen printing, etc (Li et al 2012) Paper has advantages of being inexpensive, lightweight, available everywhere and compatible with biological samples (Martinez et al 2010) Paper-based devices can be easily disposed after use, require very small volume of sample and reagents, capable of analyzing multianalytes at the same time and easily mass produced (Nie et al 2010) Several paper-based assays with diverse formats developed for health care, environmental monitoring, food quality control and forensic science (Li et al 2012) Paper-based devices can be applied for either qualitative or quantitative analysis When coupled with appropriate detection methods, paper-based devices can produce quantitative results with reasonable accuracy Popular detection methods such as colorimetric, electrochemical, electrochemiluminescence or chemiluminescence were extensively reviewed by Li et al (2012) Paper-based microfluidic devices can also serve as an excellent platform for biosensor development Biosensors with exceptional performance such as high specificity and sensitivity, rapid response, low cost, compact size and easy-to-use were considered as important means in clinical, food and environmental monitoring (Amine et al 2006) One of the most widely used technique in monitoring pollutants and toxic compounds is enzyme inhibition-based biosensing in which concentrations of target compounds can be determined from the extent to which the enzyme is inhibited, signifying by the product concentration Popular enzymes used as bio-recognition elements for detection of heavy metals are horseradish peroxidase, urease, glucose oxidase, alcohol oxidase, glycerol 3-phosphate oxidase, invertase and acetylcholinesterase (Amine et al, 2006) For analysis of heavy metals, several paper-based test kits with significant contribution in term of sensing methods, sensitivity, selectivity were developed for mercury (Gu et al., 2011; Hossain et al 2011; Torabi et al 2011; Aragay et al 2012), cadimium (Abe et al 2011; Hossain et al 2011; Marzo et al 2013), copper (Fang et al 2010; Hossain et al 2011), iron (Apilux et al., 2010), lead (Mazumdar et al., 2010; Hossain et al 2011), chromium (Hossain et al 2011; Liu et al., 2012) and gold (Apilux et al., 2010) Considering the end-use of paper-based devices as low-cost diagnostic kits in developing countries, they should be able to perform analyses at different levels of complexity In certain situation, an inexpensive qualitative test requiring no advanced analytical skills is sufficient to make sure that heavy metals in foods, agricultural products or water sources are under a safe limit If more accurate results are expected, the same device can be integrated into colorimetric, electrochemical or other detection systems to produce semiquantitative or quantitative results Some authors (Fang et al 2010; Abe et al 2011; Gu et al., 2011; Torabi et al 2011; Aragay et al., 2012; Liu et al 2012; Marzo et al 2013) propose approaches using fluorescence or strip reader to obtain quantitative results besides qualitative test However, these methods still require large sample volume, bulky and sometime complicated fabrication process Recently, methods such as wax printing, wax dipping appeared as alternative approaches for producing inexpensive micron size devices The fundamental principle of creating a microfluidic device was to pattern hydrophilic channels bounded by hydrophobic barriers One of the simplest method was to use a printer to deposit patterns of solid wax on the paper which was then heated to enable the wax to penetrate into the entire thickness of paper, thus generating complete hydrophobic barrier An alternative approach was wax dipping which was first proposed by Songjaroen et al (2011) which are capable of both qualitative tests by visually observing color change or quantitative analysis using digital camera In this method, a mould was used to produce the hydrophobic areas The whole assembly of the mould and paper was quickly dipped and withdrawn from the melting wax Wax deposited on the uncovered parts, resulting in desired hydrophobic areas Paraffin was used in this study for its easy availability and low prices while having similar characteristics as wax The fact is that digital cameras and scanners are not as selective and sensitive as conventional analytical instrumentation, nevertheless, highly selective and sensitive detectors are still required for low analyte concentrations (Dungchai et al 2009) Electrochemistry-based method is attractive detection soucheme for paper-based biosensors due to its compact size, low-cost, high sensitivity and selectivity Using this method, Apilux et al (2010) successfully detected gold in waste stream Nonetheless, few studies have yet to develop paper-based biosensors capable of multiple testing schemes for detection of heavy metals in foods and food products In this contribution, our objectives were a) to design and fabricate a paperbased biosensor using wax-printing technology; b) to apply the produced biosensors for qualitative and semi-quantitative detection of heavy metals and c) to integrate the biosensors with electrochemical analyzer Materials and methods 2.1 Chemicals and solution preparation Urease (type III, EC 3.5.1.5, 33U/mg) from Canavalia ensiformis (Jack bean) was purchased from Sigma Aldrich (USA) Carbon ink (C-200) and Ag/AgCl inks (AGCL-375) were obtained from Hudson (USA).Tris-HCl (min 99.0%) was from HiMedia Labs (Mumbai, India) Urea, phenol red, CuSO4, KCl, NaOH and HCl were of analytical grade and used as provided Filter papers (60x60cm) and white pellet paraffin wax were sourced from local chemical stores Stock solutions of enzyme (1000U/mL), Cu ++ (680 ppm) were prepared in Tris-HCl buffer solution (50mM, pH 7.0) The enzyme stock solution was made on a weekly basis and kept in refrigerator after use Urea (0.1M) and KCl (0.1M) mixture was prepared in distilled water 2.2 Fabrication of paper-based microfluidic biosensor The design and dimensions of the sensor used in this study was illustrated in Figure 1A The sensor was developed to accommodate both colorimetric and electrochemical tests simultaneously The design was made using CorelDraw x3 (Corel Inc., Mountain View, USA) Initially, reference, working and counter electrodes were patterned on reaction zone (Figure 1A) using screen printing method Carbon ink (C-200) was used for working and counter electrodes whereas silver/silver chloride ink (AGCL-375) was used for reference electrode The printed electrodes sensor was dried at 65oC in oven for about 30 The printing patterns of the electrodes were tested for continuity by a multimeter Figure (A)Schematic design of the paper-based biosensor illustrating sensing zones, loading area and conductive pads to interface electrochemical system (B)Steel mould used to pattern hydrophilic-hydrophobic channels on the sensor In the next stage, the microfluidic channels were created A mould (Figure 1B) was cut from a 0.3 mm steel plate using computer numerical control (CNC) machine by a local workshop The mould can be used many times without deformation and decrease in resolution of the hydrophilic channels About 200g of paraffin was melted in a 500 ml beaker using hot plate (IKA RH Basic 2) Experiment were conducted at different dipping temperature (55-80oC) and time (1-5s) to determine the optimal conditions for patterning hydrophylic barrier Prior to wax dipping, the filter paper containing printed electrodes were cut into rectangles (50 mm width x 80 mm length) And sensor was sandwiched between the mould and a glass slide The mould was positioned so that electrochemical reaction zone was aligned with the printed electrodes A permanent magnet was used to hold the mould against the glass slide The whole assembly was dipped into the melted paraffin and quickly withdrawn in predetermined time After the paraffin was cooled to ambient temperature, the mould was removed and the sensor was visually examined for any defections The hydrophilic channels should remain clear and sharp The paraffin needed to penetrate evenly into the filter paper Those sensors which did not meet the requirements were discarded The process was described in Figure Figure Fabricating procedures of paper-based biosensor includes: (1) Screen-printing working electrode (WE) and counter electrode (CE) using carbon inks, and (2) reference electrode (RE) and conductive pads with Ag/AgCl inks (3-4) The sensor was wax-dipped to create hydrophobic/hydrophilic pattern (5) Tape was attached to the back side 2.3 Qualitative and semi-quantitative analysis The fundamental principle of the qualitative and semi-quantitative analysis was based on hydrolysis reaction of urea ((NH2)2CO) (1) (NH2 ) CO  H O Urease  CO  2NH3 (1) The production of ammonia (NH3) led to an increase in pH of the solution, hence change in color of phenol red As heavy metals were added to the solution, their binding to thiol group in the active center of urease diminished catalytic activity of the enzyme Since the amount of NH3 produced or pH was affected by the heavy metals, concentrations of heavy metals can be determined from the magnitude of color changes For the proof of concept, the proposed biosensor was Sensing areas shift from a dark violet to yellow color with increasing Cu++ concentration As evidenced from the data, there is a strong distinction between samples containing (blank) and 10 ppm of Cu++ Considering standard limits of Cu++ in drinking water (1 ppm, Food and Drug Administration (FDA)) and vegetables (5 ppm, CODEX STAN 179-1991), the sensor fabricated in this study could easily detect the presence of Cu++ in water or food products Experimental data were used to develop a color chart which served as standard reference for qualitative test (Figure 10) Samples classified as positive or negative if the corresponding colors on the sensor match the right or the left end of the chart, respectively Figure 10 Color chart developed for qualitative test of Cu++ in food sample In this research, due to time constraint, the interferences of other heavy metals were not investigated The toxicity of heavy metals toward urease immobilized on chitosan membrane was ranked following the sequence: Hg++> Ag++> Cu++> Ni++> Cd++> Zn++> Co++> Fe++> Pb++> Mn++ (Krajewska 1991) However, effects of heavy metals on urease immobilized on paper have not been reported yet It would be helpful to determine the levels of heavy metals which begin to interfere with the final results In the presence of other heavy metals above a critical limit, the results can be only interpreted as the overall effects of the mixtures, rather than Cu++ alone Nevertheless, the sensor is useful as it can provide an early warning about the contamination of heavy metals in foods 3.3 Semi-quantitation The colorimetric response of the sensor to different levels of Cu++ (0-1ppm) was shown in Figure 11A At the presence of Cu++, urease enzyme was inhibited, leading to diminished production of NH3 The amount of Cu++, therefore, had a direct impact on color intensity inside the sensing areas The images were converted into digital values by by splitting the original color into Red, Green, Blue constitute channels (Figure 11B) using Adobe Photophop CS2 Correlation between color intensity of individual channels and concentrations of target compound was analyzed 14 It was found that the coefficients were higher for Red and Green channels and lower for Blue channels with 0.83, 0.98 and 0.01 respectively (Table 1) The results suggested a tight relationship between color intensity and measured value in Green channels Table Correlation of color intensity in signal channel Regression coefficient (R ) Red Green Blue 0.83 0.98 0.01 Figure 11 Deductive analysis of RGB picture (A)Original sensor in full color (B) The image was separated into gray scale with using red, blue and green filter for further analysis The relationship between Red, Blue values and concentrations of Cu++ was not significant, indicated by low values of R2, 0.83 and 0.01, respectively (Table 1) In addition, a closed examination of Red and Blue channels showed that they did not represent color in the sensing areas Instead, the background or periphery color due to diffusion of reagents through damaged paraffin borders was captured 15 Color in Green channels was most visually closed to original developed color in the sensing zones and a strong correlation between Green values and content of Cu++ in the samples was obtained (R2 = 0.98; Figures 12) As a result, values of Green channel were used to develop the standard curve for semi-quantitative analysis Color Intensity 220 200 R² = 0.9837 180 160 140 120 -7 -6 -5 -4 -3 -2 Log(Cu,M) Figure 12 Calibration curve for Cu++ concentration from concentration of 0.001-1 ppm When Cu++ concentration was less than 0.018 ppm, color intensity was negligibly different from the blank sample (Figure 11A-B.Green) Above ppm, no color development was observed, indicating a total inhibition of the urease enzyme on the sensor As colorimetric response of the sensor to Cu++ content was linear from 0.01 -1 ppm, a standard curve was constructed for this range of data with R2 of 0.98 (Figure 12) Detection limit, defined as the analyte concentration that produce the signal equal to or higher than the average signal produced by the blank plus standard deviations, was estimated to be 0.018 Detection limit for Cu++ on paper-based sensor was reported to be 0.02 ppm when reagents were entrapped in a sol-gel matrix (Hossain et al 2011) Lateral flow nucleic acid biosensor based on paper substrate seemed to yield a lower detection limit of about 10 nM (Fang et al 2010) However, the detection limit in this study was still 10 times lower than safe limits of (CODEX STAN 179-1991) and ppm (No 1881/2006) set by World Health Organization (WHO) and European Commission (EC), respectively Moreover, as compared to previous studies (Hossain et al 2011; Fang et al 2010), the proposed method had the advantages of being simple, compact and did not require extra expensive instruments beside an office scanner or a digital camera This would be important for the application of the sensor in developing countries where advanced analytical facilities were not easily accessible In this 16 study, factors affecting detection limit of the developed sensor could be attributed to immobilization of enzyme and hydrophobic barriers Urease enzyme was manually deposited on the paper and the immobilization was solely dependent on physical absorption Therefore, the distribution of enzyme on sensing zone owing to spreading of the liquid droplets was not uniform If reagents were dispensed using a printer and the mobility of liquid phase was confined by a sol-gel matrix as described by Hossain et al (2011), the enzyme would be more uniformly distributed and there would be enhancement in sensitivity and reproducibility of the sensor Moreover, effects from flows of fluid into the channels further had a negative effect on the development of color in sensing zones Regarding the hydrophobic barriers, extended exposure of paraffin to testing reagents made the borders more permeable to fluids Consequently, hydrophobic barriers could not confine the solutions inside sensing zone as expected This could partly influence the reproducibility of the test as enzymes and other reagents could have been washed off the sensing zones Within the limited time frame of the current study, it was hardly possible to verify and address all the problems simultaneously Future studies should consider the use of a gel matrix for enzyme immobilization and a different method for patterning hydrophobic barriers Table The recovery (%) of Cu++ from validation test log(Cu,M) Founded Value Set Set Actual Value Recoveries(%) -3.46 -3 86.74 -4.48 -4 89.29 -5.88 -5 84.98 -3.31 -3 90.76 -4.79 -4 83.58 -5.60 -5 89.24 Average recovery (%) 87.43 To validate the performance of the sensor, samples with known concentrations were tested (Table 2) Recoveries varied from 83 – 91 % were obtained and t-test showed that there was no significant difference (P=0.01, tstat=0.34Ag+>Cu++>Pb++>Cd++>Zn++>Ni++>Mn++ with sensitivity decreasing accordingly 21 f Replacing the key enzyme and establishing new signaling pathway, the use of paper-based sensor could possibly be widen to other areas to detect also pesticide, fungal toxins, bacteria or even DNA (Hossain et al 2009; Luckham and Brannan 2010; Crew 2011; Ge et al 2012) Conclusion In summary, this study had evidenced the applicability of a versatile paper- based microfluidic biosensor for sensing heavy metals Information on fabrication protocols such as control of fluid flow, patterning hydrophobic barriers, electrochemical analysis could be helpful for future studies on paper-based biosensors As compared to safe levels of 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OF A VERSATILE AND COMPACT PAPER- BASED MICROFLUIDIC BIOSENSOR FOR DETECTION OF COPPER IN FOOD PRODUCTS a Thao L Nguyen , Loc T Nguyen a, * : School of Biotechnology, International University –. .. important framework for continued research on paper- based biosensor for detection of heavy metal 22 Reference Abe K., Nakamura K., Arao T., Sakurai Y., Nakano A. , Suginuma C., Tawarada K., Sasaki... hydrophobic areas Paraffin was used in this study for its easy availability and low prices while having similar characteristics as wax The fact is that digital cameras and scanners are not as selective