Biosensors and capillary electrophoresis microchip devices for analytical applications

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BIOSENSORS AND CAPILLARY

ELECTROPHORESIS MICROCHIP DEVICES FOR ANALYTICAL APPLICATIONS

APPAN ROYCHOUDHURY

CENTRE FOR BIOMEDICAL ENGINEERING INDIAN INSTITUTE OF TECHNOLOGY DELHI

MARCH 2019

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© Indian Institute of Technology Delhi (IITD), New Delhi, 2019.

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BIOSENSORS AND CAPILLARY

ELECTROPHORESIS MICROCHIP DEVICES FOR ANALYTICAL APPLICATIONS

by

APPAN ROYCHOUDHURY

CENTRE FOR BIOMEDICAL ENGINEERING

Submitted

in fulfilment of the requirements of the degree of Doctor of Philosophy to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

MARCH 2019

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Dedicated to

All those people who have always supported and

encouraged me to pursue my dreams

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“Little by little, through patience and repeated effort, the mind will become stilled in the Self”

– Bhagavad Gita

“The secret of getting ahead is getting started”

– Mark Twain

“Excellence is a continuous process and not an accident”

– Dr. A.P.J. Abdul Kalam

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CERTIFICATE

This is to certify that the thesis entitled ‘Biosensors and capillary electrophoresis microchip devices for analytical applications’ being submitted by Mr. Appan Roychoudhury to the Indian Institute of Technology Delhi for the award of Doctor of Philosophy is a record of bonafide research work carried out by him. Mr. Appan Roychoudhury has worked under our guidance and supervision and has fulfilled the requirements for the submission of this thesis, which to our knowledge has reached the requisite standard.

The results presented in this thesis are original and have not been submitted, in part or full, to any other University or Institute for the award of any other degree or diploma.

Dr. Sandeep Kumar Jha Prof. Suddhasatwa Basu

Centre for Biomedical Engineering, Department of Chemical Engineering, Indian Institute of Technology Delhi, Indian Institute of Technology Delhi, Hauz Khas, New Delhi – 110016 Hauz Khas, New Delhi – 110016 India India

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ACKNOWLEDGEMENTS

My doctoral research at the Indian Institute of Technology Delhi has been enriched through the interaction with many wonderful people. The completion of my Ph.D. would not have been possible without their continuous support and guidance, for which I am truly grateful.

At the very outset, I would like to express my sincere gratitude to my doctoral supervisor Dr. Sandeep Kumar Jha for his valuable instructions, support and encouragement that worked as a motivating force to conduct meaningful research during my Ph.D. studies. His insightful guidance, scientific approach and scholarly advice have helped me significantly to work harder and accomplish the task in time. It was my privilege and matter of pride to be an IIT Delhi student and having Dr. Jha as my mentor.

I owe my sincere gratitude to my doctoral co-supervisor Prof. Suddhasatwa Basu for providing thoughtful guidance, continuous encouragement and enthusiasm throughout the entire research period. Working under his supervision was a great achievement and truly enriching experience for me.

I would like to thank my SRC members; Prof. Veena Koul, Dr. Dinesh Kalyanasundaram and Dr. Shalini Gupta for their critical comments, inputs and suggestions to my research work.

Besides, I also wish to thank all faculties and staff members of the Centre for Biomedical Engineering for their requisite guidance and cooperation.

I am thankful to Ministry of Human Resource Development (MHRD), Government of India and IIT Delhi for providing me research assistantship and facilitating my research work. Apart from that, the characterization facilities at Nanoscale Research Facility (NRF) and Central Research Facility (CRF) of IIT Delhi are gratefully acknowledged.

I would like to express my sincere thanks to Prof. D. Sakthi Kumar, Bio Nano Electronics Research Centre, Toyo University, Japan for providing necessary facilities to conduct the research work in his laboratory. I am also thankful to Prof. Toru Maekawa, Dr. Neha Chauhan and all the other research associates and staff members of the Bio Nano Electronics Research Centre for giving me generous support and cooperation during my short stay in Japan.

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I express my special thanks to Arneish Prateek, Jay Patel and Kevin Antony Francis for providing immense help, support and coordination at different parts of my research work.

Furthermore, I would like to thank my colleagues at CBME and my fellow LoCB labmates;

Anuradha Soni, Amit Kumar Singh, M Marieshwaran, Rishi Raj, Tanu Bharadwaj, Jayant Kalra, Smriti Bala and Rajeev Kumar for their assistance and cooperation. I am also thankful to my Fuel Cell labmates; Dr. Pankaj Kumar Tiwari, Dr. Ayan Mukherjee, Dr. Sankalpita Chakrabarty, Dr.

Debabrata Chandra, Sundar Singh, Harikrishnan Narayanan, Nimai Bhandary, Biswajit De and all the other lab members for their kind help, fruitful discussions and technical suggestions.

I owe special thanks to Sangeeta Kalita for offering valuable advice, moral support, courage and confidence to achieve and accomplish my goals.

Finally, I would like to express my deepest appreciation for my parents and family members. Much of what I have accomplished would not have been possible without their constant patience, support and encouragement. I am truly blessed to have my loving, supportive and wonderful parents, brother and sister-in-law to guide me in all the aspects of my life. Throughout my life they have inspired me to pursue my dreams.

Above all, I praise God, the almighty, merciful and passionate, for providing me this opportunity and granting me the capability to proceed successfully.

Appan Roychoudhury

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ABSTRACT

Over the past decade, analytical detection techniques have undergone a rapid development in variety of fields including medical diagnostics, food quality testing, environmental pollution monitoring and pharmaceutical research etc. The conventionally used analytical techniques for separation and detection of compounds are primarily based on high performance liquid chromatography (HPLC) and gas chromatography (GC) techniques, equipped with an optical or electrochemical detector. These methods are being used continuously for high throughput screening of the compounds and provide a promising platform for performing chemical or biological analysis. However, the precise and ultrasensitive detection of analytes using analytical devices is still a challenge due to unavailability of higher detection volume. Apart from that, the currently available analytical techniques are limited with the difficulties of being extremely sophisticated, time consuming, requiring high sample volume and complicated due to complex instrumentation. Above all, majority of the present techniques are not miniatuarizable to point-of- care devices or point-of-analysis and not suitable for on-site or in-line operations. Therefore, considerable efforts are being made to address these problems and in this regard, the development of sensitive and selective biosensors, particularly in microfluidic format can provide the appropriate solutions. Beside this, the possibilities of massive parallelization of multiple processes and multi-analyte detection at the same time make the microchip capillary electrophoresis technology highly favorable in sensing applications. Most importantly, these analytic tools have potential to exhibit simple, rapid, reliable, yet cost-effective determination of the analytes with on- site detection abilities. Hence, it’s worth to inspect the applicability of both biosensors and capillary electrophoresis microchips for analytical purposes by involving the key techniques of immobilization, miniaturization and multianalyte detection.

Against these background, the present dissertation work comprising of five chapters aims to explore the possibilities of biosensor and capillary electrophoresis microchip systems to detect multianalyte mixtures which have significant role in diagnosis and other essential applications.

The first chapter is contained with introduction and extensive literature review on biosensors, microfluidics and capillary electrophoresis microchip systems, while in the last chapter the overall work summary along with the future perspectives of the conducted research work are revealed.

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In the second chapter, analytic applicability of biosensors has been established after evaluating a clinically relevant neurochemical dopamine by developing asimple, easy to fabricate, highly reusable and sensitive biosensing system based onnickel oxide (NiO) nanoparticles (NPs) and tyrosinase enzyme conjugate. For further improvement of sensing parameters for dopamine, the synthesized NiO NPs were surface modified with Prussian blue (PB) to utilize as a matrix for covalent immobilization of tyrosinase enzyme. After several refinements of interfacial chemistry and sensor measurement technique, the sensitivity was enhanced significantly, while the detection limit was lowered many folds. As the main objective of the present research focuses on the assessment of a multianalyte mixture, hence in the subsequent work, a reduced graphene oxide (rGO)-NiO nanocomposite based sensor probe was developed for simultaneous detection of two neurochemicals; dopamine and epinephrine in a single run. With the developed sensor, a sensitive and selective detection of dopamine and epinephrine was attained in synthetic samples, even in presence of interferents and in real samples. However, detecting more than a few neurochemicals would even remain a challenge. This prompted us to miniaturize the technique onto a microchip format to automate and simplify the detection. Thus, capillary electrophoresis microchip detector has been selected to first segregate the analyte from a mixture followed by their analysis, because it is practically impossible to detect multiple analytes in a highly complex sample such as blood without first achieving separation of individual components.

Third chapter demonstrates the detection of more than two neurochemicals simultaneously using the aforementioned paper microfluidic capillary electrophoresis-amperometric detector (CE- AD) microchip which was utilized to separate and simultaneously detect three clinically relevant neurochemicals; dopamine, epinephrine and serotonin. The developed system was contained with promising features such as miniaturized size (75 mm × 38 mm), portability, low sample volume requirement (2 µL) and ease in integration to the point-of-care diagnostic devices for on-site detection etc. Using this system, we could achieve simultaneous detection of multiple neurochemicals in synthetic as well as real samples and concluded that such system shall be useful in developing more such assay protocols.

We wanted to further explore the use of developed CE-AD microfluidic chip and we tested another domain to monitor analytes in food and beverages. Thus, the fourth chapter explores use of developed CE-AD device for testing taste and aroma inducing polyphenolic compounds present

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in fermented beverages such as beer and wine. The microchip was fabricated using polydimethylsiloxane (PDMS) framework, keeping in mind the possible use of such chip for inline monitoring of fermentation products. The developed chip was successfully used for separation and rapid simultaneous detection of three test analytes; vanilic acid, caffeic acid and quercetin with requirement of extremely low sample volume. The applicability of the microchip was evaluated with commercially available wine and beer samples.Therefore, it was concluded thatthe fabricated microchip has the potential to be integrated to a fermentation broth for inline/in-situ monitoring owing to miniaturized size (75 mm×38 mm) and portability and it may also be used for automation and replacement of subjective manual testers in beer and wine quality analysis.

The obtained results of the present research work might foster the development of efficient biosensing and capillary electrophoresis microchip systems that would be particularly useful for point-of-analysis and point-of-care applications.

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सार

पिछले एक दशक में, पिश्लेषणात्मक िहचान तकनीकों ने चचककत्साननदान, खाद्य गुणित्ता िरीक्षण,

ियाािरण प्रदूषण ननगरानी और दिा अनुसंधान आदद सदहतपिभिन्न क्षेत्रों में तेजी से पिकास ककया है।

यौचगकों कोअलग करनेऔर िहचानने केभलए िारंिररकरूि से उियोग कीजाने िाली पिश्लेषणात्मक तकनीकें मुख्य रूि से उच्च प्रदशान तरल क्रोमैटोग्राफी (एचिीएलसी) और गैस क्रोमैटोग्राफी (जीसी) तकनीकोंिरआधाररतहोतीहैं, जोएकऑप्टटकलयापिद्युतरासायननकडिटेक्टरसेसुसप्जजतहोतीहैं।

इनपिचधयोंकाउियोगयौचगकोंकीउच्चथ्रूिुटस्क्क्रीननंगकेभलएलगातारककयाजारहाहैऔररासायननक याजैपिकपिश्लेषणकरनेकेभलएएकआशाजनकमंचप्रदानकरताहै।हालांकक, पिश्लेषणात्मकउिकरणों

का उियोगकरके पिश्लेषण करनेिाले सटीक और अल्ट्रासेंभसदटि कािता लगाना अिी िीएक चुनौती

है, जो उच्च मात्रा का िता लगाने की अनुिलब्धता के कारण है। इसके अलािा, ितामान में उिलब्ध पिश्लेषणात्मक तकनीक बेहद जटिल, समय लेने िाली, उच्च नमूना मात्रा की आिश्यकता और जदटल इंस्क्ूमेंटेशन के कारण जदटल होने की कदिनाइयों से सीभमत हैं। इन सबसे ऊिर, ितामान तकनीकों के

अचधकांशिॉइंट-ऑफ-केयरडििाइसयािॉइंट-ऑफ-एनाभलभससकेभलएलघुरूपणनहींहैंऔरऑन-साइट या इन-लाइन ऑिरेशन के भलए उियुक्त नहीं हैं। इसभलए, इन समस्क्याओं को दूर करने के भलए काफी

प्रयासककएजारहे हैंऔर इससंबंधमें, संिेदनशील औरचयनात्मकबायोसेंसरकेपिकास, पिशेषरूिसे

माइक्रोफ्लुइडिक प्रारूि में उचचत समाधान प्रदान कर सकते हैं। इसके अलािा, एक ही समय में कई प्रकक्रयाओंऔर बहु-पिश्लेषण काितालगानेकेबडेिैमाने िरसमानांतरकरणकीसंिािनाएं माइक्रोचचि

केभशका िैद्युतकणसंचलन प्रौद्योचगकी को संिेदन अनुप्रयोगों में अत्यचधक अनुकूल बनाती हैं। सबसे

महत्वपूणण बात यह है कि इन ववश्लेषणात्मि उपिरणों में ऑन-साइि डििेक्शन क्षमताओं िे साथ एनाललटिक्सिे सरल, तीव्र, ववश्वसनीय, अभीति लागत प्रभावीननर्ाणरण िोप्रदलशणतिरनेिीक्षमता

है। इसभलए, यहदोनों बायोसेंसरऔर केभशकािैद्युतकणसंचलनमाइक्रोचचटस पिश्लेषणात्मकप्रयोजनों

के भलए प्स्क्िरीकरण, लघुरूिण और बहुिक्षीय िहचान की प्रमुख तकनीकों को शाभमल करके ननरीक्षण करनेकेलायकहै।

इनपृष्ठभूलममें, पांचअध्यायोंवालेवतणमानअनुसंर्ानिायणप्रबंर्िाउद्देश्यबहुसंयोजिलमश्रणों

िा पता लगानेिे ललएबायोसेंसर और िेलशिा वैद्युतिणसंचलन माइक्रोचचप लसस्िम िी संभावनाओं

िा पता लगाना है जजनिी ननदान और अन्य आवश्यि अनुप्रयोगों में महत्वपूणण भूलमिा है।

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पहला अध्याय बायोसेंसर, माइक्रोफ्लुइडिक्स और िेलशिा वैद्युतिणसंचलन माइक्रोचचप लसस्िम पर पररचयऔर व्यापिसाटहत्य समीक्षािे साथननटहतहै, जबकि वपछलेअध्याय में आयोजजत अनुसंर्ान

िायणिेभववष्यिेदृजष्ििोणिे साथ-साथसमग्रिायण सारांशिापताचलाहै।

दूसरे अध्याय में, ननिल ऑक्साइि (NiO) नैनोपाटिणिल्स (NPs) और िाइरोलसनेज एंजाइम संयुग्म पर आर्ाररत एि सरल, आसान, पुन: प्रयोज्य और संवेदनशील बायोसेंलसंग प्रणाली वविलसत

िरिे नैदाननि रूप से प्रासंचगि न्यूरोिेलमिल िोपामाइन िा मूल्यांिन िरने िे बाद बायोसेंसर िी

ववश्लेषणात्मिप्रयोज्यतास्थावपतिीगईहै।िोपामाइनिेललएसंवेदनमापदंिोंिेआगेसुर्ारिेललए, संश्लेवषत ननिल ऑक्साइि (NiO) नैनोपाटिणिल्स (NPs) सतह थे, जो प्रलशया नीले (PB) िे साथ संशोचर्तथे, जोिाइरोलसनेजएंजाइमिेसहसंयोजिजस्थरीिरणिेललएएिमैटिक्सिेरूपमें उपयोग कियाजाताथा। इंिरफैलसअलिेलमस्िी औरसेंसरमापतिनीििेिईशोर्निे बाद, संवेदनशीलतामें

िाफी वृविहुई थी, जबकि पता लगाने िीसीमा िई गुना िम हो गई थी। चूंकि वतणमान अनुसंर्ान िा

मुख्यउद्देश्यएिबहुपक्षीयलमश्रणिेमूल्यांिनपरिेंटितहै, इसललएबादिेिाममें, एिरेिूसेिग्राफीन ऑक्साइि (rGO)-ननिल ऑक्साइि (NiO) नैनोिोम्पोसाइि आर्ाररत सेंसर जांच एि साथ दो

न्यूरोिेलमिल्सिापतालगानेिेललएवविलसतिीगईथी; एिहीसमयमें िोपामाइनऔरएवपनेफ्रीन।

वविलसतसेंसरिेसाथ, िृत्रिमनमूनोंमें िोपामाइनऔरएवपनेफ्रीनिाएिसंवेदनशीलऔरचयनात्मि

पतालगाना हस्तक्षेप िीउपजस्थनत में और वास्तववि नमूनों में भीप्राप्त किया गयाथा।हालााँकि, िुछ से अचर्ि न्यूरोिेलमिल्स िा पतालगाना भी एि चुनौती बनारहेगा। इसने हमें एि माइक्रोचचप प्रारूप पर तिनीि िो छोिा िरने और पता लगाने िो सरल बनाने िे ललए प्रेररत किया। इस प्रिार, िेलशिा

वैद्युतिणसंचलन माइक्रोचचप डििेक्िरिोउनिेववश्लेषण िे बादलमश्रणसे ववश्लेषण िोअलगिरने

िे ललए चुना गया है, क्योंकि यह अत्यचर्ि जटिल नमूने में िई ववश्लेषणों िा पता लगाने िे ललए व्यावहाररिरूपसेअसंभवहै जैसेकिरक्तमें व्यजक्तगतघििोंिोअलगकिए त्रबना।

तीसरा अध्याय एि साथ पूवोक्त िागज माइक्रोफ्लुइडिि िेलशिा वैद्युतिणसंचलन-

एम्परोमेटिि डििेक्िर (CE-AD) माइक्रोचचप िा उपयोग िरिेएि साथ दो से अचर्ि न्यूरोिेलमिल्स

िा पतालगानेिो प्रदलशणतिरता है जोतीन नैदाननि रूपसे प्रासंचगिन्यूरोिेलमिल्स िो अलगिरने

और एि साथ उपयोग िरने िे ललए उपयोग किया गया था; िोपामाइन, एवपनेफ्रीन और सेरोिोननन।

वविलसत लसस्िम लघु-आिार (75 mm × 38 mm), पोिेत्रबललिी, िम नमूना मािा िी आवश्यिता (2 µL) और ऑन-साइि डििेक्शन िे ललए पॉइंि-ऑफ-िेयर िायग्नोजस्िि डिवाइसेस िे एिीिरण में

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आसानी िे साथ आशाजनि ववशेषताओं िे साथ समाटहत थी। इस लसस्िम िा उपयोग िरिे, हम लसंथेटिि िे साथ-साथ वास्तववि नमूनों में िई न्यूरोिेलमिल्स िा एि साथ पता लगा सिते हैं और ननष्िषणननिालाहै किइसतरहिेलसस्िम अचर्िपरखप्रोिोिॉल वविलसतिरनेमें उपयोगीहोंगे।

हमपिकभसत CE-AD माइक्रोफ्लुइडिि्चचि केउियोगकाऔरितालगाना चाहतेिेऔरहमने

खाद्य और िेयिदािों में पिश्लेषणकी ननगरानी केभलएएक औरिोमेन कािरीक्षण ककया। इसप्रिार, चौथाअध्यायबीयर औरवाइनजैसे फमेन्िेिपेयपदाथों में मौजूदपॉलीफेनोललि यौचगिोंिेस्वाद और सुगंर्िेपरीक्षणिेललएवविलसत CE-AD डिवाइसिेउपयोगिीपड़तालिरताहै।फेरमेंिशन उत्पादों

िी इनलाइन ननगरानी िे ललए इस तरह िे चचप िे संभाववत उपयोग िो ध्यान में रखते हुए, पॉलीडिमेचथललसलोक्सेन (PDMS) ढांचे िाउपयोगिरिे माइक्रोचचप तैयार िीगई थी।वविलसत चचप

िोसफलतापूवणितीनपरीक्षणववश्लेषणोंिेपृथक्िरणऔरतेजीसेएिसाथपतालगानेिेललएउपयोग किया गया था; अत्यंत िम नमूना मािा िी आवश्यिता िे साथ वैननललि एलसि, िैकफि एलसि और क्वेरसेटिन। व्यावसानयि रूपसे उपलब्र् वाइनऔर बीयर िे नमूनों िे साथमाइक्रोचचप िीप्रयोज्यता

िा मूल्यांिनकियागया था।इसललए, यह ननष्िषणननिाला गयाकि गढे हुएमाइक्रोचचप में इनलाइन / इन-सीिू मॉननिररंग िेललएफेरमेंिशन ब्रोथिो एिीिृतिरनेिीक्षमताहै जोलघुआिार (75 mm ×

38 mm) और पोिेत्रबललिी िे िारण होता है और इसिा उपयोग स्वचालन और बीयर और वाइन िी

गुणवत्ताववश्लेषण में व्यजक्तपरिमैनुअलपरीक्षिोंिेप्रनतस्थापनिेललएभीकियाजासिताहै।

वतणमान अनुसंर्ान िायण िे प्राप्त पररणाम एजफ्फलसएंि बायोसेंलसंग और िेलशिा

वैद्युतिणसंचलन माइक्रोचचप लसस्िम िे वविास िो बढावा दे सिते हैंजो पॉइंि-ऑफ-एनालललसस या

पॉइंि-ऑफ-िेयरडिवाइसअनुप्रयोगोंिे ललएववशेषरूपसेउपयोगी होंगे।

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List of Figures and Illustrations

Figure

Number Figure Title Page

Number Fig. 1.1 Schematic representation of a biosensor with different components 3 Fig. 1.2 Schematic illustration of a capillary electrophoresis microchip with

electrochemical detector

13

Fig. 2.1 Schematic representation of the fabrication of NiO NPs-tyrosinase enzyme conjugate based dopamine biosensing platform

40

Fig. 2.2 Nanoparticle characterization: (A) XRD; and (B) SAED pattern of synthesized NiO NPs; (C & D) TEM micrographs of NiO NPs

42

Fig. 2.3 Nanoparticle characterization: (A) Particle size distribution plot for NiO NPs acquired from DLS studies; (B) EDX spectra of NiO NPs; (C) FTIR spectra of NiO NPs; (D) FTIR spectra of Tyrosinase-NiO NPs conjugate

43

Fig. 2.4 Nanoparticle characterization: 2-dimensional AFM micrographs of (A) NiO/ITO; and (B) Tyrosinase/NiO/ITO; and 3-dimensional AFM micrographs of (C) NiO/ITO; and (D) Tyrosinase/NiO/ITO electrodes

45

Fig. 2.5 (A) Cyclic voltammograms of (a) bare ITO (b) NiO/ITO (c) Tyrosinase/NiO/ITO in absence of dopamine, and (d) Tyrosinase/NiO/ITO (e) NiO/ITO (f) ITO electrodes in presence of 100 µM dopamine; (B) Cyclic voltammograms of the Tyrosinase/NiO/ITO electrode with varying scan rate between 10 to 500 mV/s;

(C) Calibration curve of the biosensor based on cathodic peak current of o- dopaquinone reduction as a function of square root of scan rate; (D) Calibration curve of biosensor based on o-dopaquinone reduction peak potential with logarithmic function of scan rate

49

Fig. 2.6 (A) Electrochemical response of Tyrosinase/NiO/ITO electrode at different concentration of dopamine (0-500 µM); (B) Calibration curve of biosensor using cathodic peak currents for o-dopaquinone reduction at linear concentration (2 - 100 µM) range for dopamine, (inset) shows calibration curve of the biosensor at whole concentration range (0.1-500 µM)

51

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Fig. 2.7 (A) Selectivity studies of the developed dopamine biosensor in presence of ascorbic acid (AA) and uric acid (UA); (B) Determination of response time of dopamine biosensor with Tyrosinase/NiO/ITO electrode; (C) Reusability studies on dopamine biosensor; (D) Shelf-life studies of the fabricated biosensor based on Tyrosinase/NiO/ITO electrode

53

Fig. 2.8 Detection of dopamine in (a) standard samples, (b) real samples, (c) real samples with the presence of interferents ascorbic acid (0.5 mM) and uric acid (3 mM)

55

Fig. 2.9 Schematic of step-wise fabrication of the PB-NiO NPs-tyrosinase enzyme based dopamine bio-sensing platform

59

Fig. 2.10 (A) XRD spectra of (a) NiO NPs and (b) PB-NiO NPs; (B) Raman spectra of (a) NiO NPs and (b) PB-NiO NPs

61

Fig. 2.11 UV-vis absorption spectra of (A) NiO NPs; (B) PB-NiO NPs 62 Fig. 2.12 TEM and HR-TEM images of (a-c) NiO NPs and (e-g) PB-NiO NPs; size

distribution histogram of (d) NiO NPs and (h) PB-NiO NPs

63

Fig. 2.13 EDX spectra of (A) NiO NPs; (B) PB-NiO NPs 64

Fig. 2.14 Wide scan XPS spectra of the synthesized (a) NiO NPs and (b) PB-NiO NPs;

individual XPS spectra of (c) Ni2p, (d) O1s, (e) Fe2p, (f) N1s and (g) C1s

65

Fig. 2.15 FTIR spectra of (a) SPCE, (b) PB-NiO/SPCE and (c) Tyrosinase/PB-NiO/SPCE electrodes

67

Fig. 2.16 SEM images of (a & b) SPCE, (c & d) PB-NiO/SPCE and (e & f) Tyrosinase/PB- NiO/SPCE electrodes

68

Fig. 2.17 (A) Cyclic voltammograms of (a) SPCE, (b) PB-NiO/SPCE, (c) Tyrosinase/PB- NiO/SPCE and (d) Tyrosinase/PB-NiO/SPCE in presence of 100 µM dopamine;

(B) Cyclic voltammograms of the Tyrosinase/PB-NiO/SPCE with varying scan rate between 10 to 250 mV/s; (C) Calibration curve of the biosensor based on anodic peak current of dopamine oxidation as a function of square root of scan rate; (D) Calibration curve of the biosensor based on dopamine oxidation peak potential with logarithmic function of scan rate

72

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Fig. 2.18 (A) Determination of sensor response time; (B) Amperometric response studies of the fabricated Tyrosinase/PB-NiO/SPCE at different concentration of dopamine (0-250 µM); (C) Calibration curve of the biosensor at linear concentration (0.5-100 µM) range for dopamine, (inset) shows calibration curve of the biosensor at entire concentration range (0.1-250 µM)

76

Fig. 2.19 (A) Interference studies of the fabricated dopamine (DA) biosensor in presence of ascorbic acid (AA) and uric acid (UA); (B) Reproducibility studies of five different Tyrosinase/PB-NiO/SPCE electrodes prepared in same batch; (C) Reusability studies of the dopamine biosensor; (D) Shelf-life studies of the fabricated biosensor based on Tyrosinase/PB-NiO/SPCE

78

Fig. 2.20 Detection of dopamine in (a) standard samples, (b) real samples, (c) real samples with interferents ascorbic acid (0.5 mM) and uric acid (3 mM)

79

Fig. 2.21 Schematic illustration of the fabrication of rGO-NiO based sensing platform for simultaneous determination of dopamine and epinephrine

85

Fig. 2.22 XRD spectra of (A) NiO NPs; (B) GO sheets; (C) rGO-NiO nanocomposite; and UV-Vis absorption spectra of (D) NiO NPs; (E) GO sheets; (F) rGO-NiO nanocomposite; and Raman spectra of (G) NiO NPs; (H) GO sheets; (I) rGO- NiO nanocomposite

89

Fig. 2.23 FTIR spectra of (A) NiO NPs; (B) GO sheets; (C) rGO-NiO nanocomposite; and EDX spectra of (D) NiO NPs; (E) GO sheets; (F) rGO-NiO nanocomposite

90

Fig. 2.24 HR-TEM images of (a & b) NiO NPs; and TEM images of (c & d) GO sheets; (e

& f) rGO-NiO nanocomposite. Inset of images (a, c, e) display SAED pattern of the respective samples

92

Fig. 2.25 (A) SEM image of rGO-NiO nanocomposite; and elemental mapping of (B) carbon; (C) oxygen; (D) nickel in rGO-NiO nanocomposite

93

Fig. 2.26 2-dimensional AFM micrographs of (a) ITO, (e) GO/ITO, (i) rGO-NiO/ITO electrodes and 3-dimensional AFM micrographs of (b) ITO, (f) GO/ITO, (j) rGO- NiO/ITO electrodes; and SEM images of (c & d) ITO, (g & h) GO/ITO, (k & i) rGO-NiO/ITO electrodes

95

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Fig. 2.27 (A) Cyclic voltammograms of (a) bare ITO, (b) GO/ITO, (c) rGO-NiO/ITO electrodes in absence of dopamine and (d) bare ITO, (e) GO/ITO, (f) rGO- NiO/ITO electrodes in presence of 100 µM dopamine solution, (inset) CV spectra of (a), (b) and (c); (B) Cyclic voltammograms of rGO-NiO/ITO electrode in presence of 100 µM dopamine solution as a function of pH (varying from 5.7 to 8); (C) Cyclic voltammograms of the rGO-NiO/ITO electrode with varying scan rate between 10 to 250 mV/s with 100 µM dopamine solution, (inset) anodic peak current of dopamine oxidation as a function of square root of scan rate; (D) Cyclic voltammograms of the rGO-NiO/ITO electrode with varying scan rate between 10 to 250 mV/s with 100 µM epinephrine solution, (inset) anodic peak current of epinephrine oxidation as a function of square root of scan rate

98

Fig. 2.28 (A) Square wave voltammograms of developed rGO-NiO/ITO electrode for simultaneous detection of dopamine and epinephrine in a concentration range of 0 to 250 µM for both the analytes; (B) calibration curves for dopamine, linear range (0.5-50 µM, with red line) and entire range (0.5-250 µM, with blue line);

(C) calibration curves for epinephrine, linear range (0.5-50 µM, with red line) and entire range (0.5-250 µM, with blue line)

100

Fig. 2.29 Selectivity studies of developed rGO-NiO/ITO electrode in presence of ascorbic acid (AA) and uric acid (UA) during simultaneous detection of (A) dopamine and (B) epinephrine; Simultaneous detection of (C) dopamine and (D) epinephrine in (a) standard samples, (b) real samples, (c) real samples with interferents ascorbic acid (0.05 mM) and uric acid (0.3 mM)

104

Fig. 3.1 Developed microchip (A) front view and (B) rear view after alignment and fixing; (1) sample reservoir, (2) sample waste reservoir, (3) separation microchannel, (4) microelectrodes

119

Fig. 3.2 Schematic illustration of the set-up and procedure for simultaneous detection of dopamine, epinephrine and serotonin

120

Fig. 3.3 Cyclic voltammograms of (A) dopamine; (B) epinephrine; and (C) serotonin in PBS (50 mM, pH 7.0, 0.9% NaCl) with 50 mV/s scan rate

121

Fig. 3.4 Square wave voltammograms of (A) dopamine; (B) epinephrine; and (C) serotonin in PBS (50 mM, pH 7.0, 0.9% NaCl)

122

Fig. 3.5 Differential pulse voltammograms of (A) dopamine; (B) epinephrine; and (C) serotonin in PBS (50 mM, pH 7.0, 0.9% NaCl)

123

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Fig. 3.6 Electropherograms of (A) dopamine; (B) epinephrine; (C) serotonin; and (D) mixture solution comprising of dopamine, epinephrine and serotonin of concentration 50 µM each

125

Fig. 3.7 (A) Bar graph showing migration time profile for dopamine, epinephrine and serotonin; calibration curves of variation in peak current amplitude (baseline corrected) as a function of (B) dopamine; (C) epinephrine; and (D) serotonin concentration in the range of 0.1 to 50 µM

127

Fig. 3.8 Simultaneous detection of (A) dopamine; (B) epinephrine; and (C) serotonin in (a) standard samples and (b) real samples

135

Fig. 4.1 Surface profiler scan of the fabricated SU-8 mold 146 Fig. 4.2 Schematic illustration of the detection strategy and top view of the developed

microchip showing its different components

147

Fig. 4.3 Off-chip cyclic voltammograms of 50 mM (A) vanillic acid; (B) caffeic acid; and (C) quercetin in PBS (50 mM, pH 7.0, 0.9% NaCl) with 50 mV/s scan rate

149

Fig. 4.4 Off-chip square wave voltammograms of 50 mM (A) vanillic acid; (B) caffeic acid; and (C) quercetin in PBS (50 mM, pH 7.0, 0.9% NaCl)

150

Fig. 4.5 Off-chip differential pulse voltammograms of 50 mM (A) vanillic acid; (B) caffeic acid; and (C) quercetin in PBS (50 mM, pH 7.0, 0.9% NaCl)

151

Fig. 4.6 Electropherograms for mixture comprising (A) 100 µM vanillic acid, 10 µM caffeic acid and 10 µM quercetin; (B) 10 µM vanillic acid, 100 µM caffeic acid and 10 µM quercetin; (C) 10 µM vanillic acid, 10 µM caffeic acid and 100 µM quercetin; and (D) bar graph showing migration time profile for vanillic acid, caffeic acid and quercetin

153

Fig. 4.7 Calibration curves of variation in peak current amplitude (baseline corrected) as a function of (A) vanillic acid; (B) caffeic acid; (C) quercetin concentration in the range of 0 to 100 µM

156

Fig. 4.8 Electropherograms for analysis of real wine and beer samples (A) Folonari red wine sample; (B) Kressmann white wine sample; (C) Argento red wine sample;

(D) Kingfisher lager beer sample; (E) London Pride ale beer sample; (F) Flensburger Weizen wheat beer sample; (curve a) before and (curve b) after spiking of vanillic acid, caffeic acid and quercetin (10 µM each)

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List of Tables

Table

Number Table Title Page

Number Table 2.1 Percentage of the elemental distribution in the prepared NiO NPs 44 Table. 2.2 Interfacial kinetics studies of fabricated electrode surface 48

Table 2.3 Determination of the interfering effect of ascorbic acid (AA) and uric acid (UA) during dopamine (DA) detection

52

Table 2.4 Detection of dopamine (DA) in fetal bovine serum samples 55 Table 2.5 Percentage of the elemental distribution in the prepared NiO NPs 64 Table 2.6 Percentage of the elemental distribution in the prepared PB modified NiO

NPs

64

Table 2.7 Values of interfacial kinetics parameters for the PB-NiO/SPCE and Tyrosinase/PB-NiO/SPCE electrodes

71

Table 2.8 Determination of the interfering effect of ascorbic acid (AA) and uric acid (UA) during dopamine (DA) detection

77

Table 2.9 Detection of dopamine (DA) in fetal bovine serum samples 80 Table. 2.10 Sensing performance of the fabricated Tyrosinase/NiO/ITO and

Tyrosinase/PB-NiO/SPCE for dopamine along with those reported in literature

81

Table 2.11 Atomic and mass percentage of Ni, O and C in the prepared NiO NPs, GO sheets and rGO-NiO nanocomposite

91

Table 2.12 Linear range and detection limit of the developed rGO-NiO/ITO electrode for simultaneous detection of dopamine (DA) and epinephrine (EP) along with those reported in literature

101

Table 2.13 Determination of interfering effects of ascorbic acid (AA) and uric acid (UA) during simultaneous detection of dopamine (DA) and epinephrine (EP)

103

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Table 2.14 Simultaneous detection of dopamine (DA) and epinephrine (EP) in fetal bovine serum samples

104

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List of Abbreviations

5-HIAA 5-Hydroxyindoleacetic Acid

AA Ascorbic Acid

Ab-AFB1 Antibodies of Aflatoxin B1

ADHD Attention Deficit Hyperactivity Disorder

AFM Atomic Force Microscopy

BSA Bovine Serum Albumin

CE Capillary Electrophoresis

CE-AD Capillary Electrophoresis Amperometric Detection

CE-pAD Capillary Electrophoresis-pulsed Amperometric Detection

CNTs Carbon Nanotubes

CSF Cerebrospinal Fluid

CV Cyclic Voltammetry

DA Dopamine

DLS Dynamic Light Scattering

DNA Deoxyribonucleic Acid

DOPAC 3,4-Dihydroxyphenylacetic Acid

DPV Differential Pulse Voltammetry

DW Deionised Water

EDC N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide Hydrochloride

EDX Energy Dispersive X-ray Spectroscopy

EP Epinephrine

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FTIR Fourier-transform Infrared

FWHM Full Width at Half Maxima

GC Gas Chromatography

GO Graphene Oxide

HIV-1 Human Immunodeficiency Virus Type 1

HPLC High Performance Liquid Chromatography

HRCA Hyperbranched Rolling Circle Amplification

HR-TEM High Resolution Transmission Electron Microscopy

ICU Intensive Care Unit

IEP Isoelectric Point

IPA Isopropanol Alcohol

ISFET Ion Selective Field Effect Transistors

ITO Indium Tin Oxide

JCPDS Joint Committee on Powder Diffraction Standards L-CDOPA L-Hydrazinomethyldihydroxyphenalyalanine

L-DOPA 3,4-Dihydroxy-L-phenylalanine

LIF Laser Induced Fluorescence

LOC Lab-on-a-chip

LOD Limit of Detection

LSPR Localized Surface Plasmon Resonance

μPADs Microfluidic Paper-based Analytical Devices

μTAS Micro Total Analysis System

MEMS Micro-electro-mechanical System

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NHS N-Hydroxysuccinimide

NiO Nickel Oxide

NPs Nanoparticles

PAT Process Analytical Technique

PB Prussian Blue

PBS Phosphate Buffer Saline

PCR Polymerase Chain Reaction

PDI Polydispersity Index

PDMS Polydimethylsiloxane

PEI Polyethylenimine

PET Polyethylene Terephthalate

POC Point-of-care

PSA Prostate Specific Antigen

PVA Polyvinyl Alcohol

rGO Reduced Graphene Oxide

RSD Relative Standard Deviation

SAED Selected Area Electron Diffraction

SDS Sodium Dodecyl Sulphate

SEM Scanning Electron Microscopy

SERS Surface-enhanced Raman Scattering

SPCE Screen Printed Carbon Electrode

SPR Surface Plasmon Resonance

SWV Square Wave Voltammetry

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TEM Transmission Electron Microscopy

TLC Thin Layer Chromatography

UA Uric Acid

XPS X-ray Photoelectron Spectroscopy

XRD X-ray Diffraction

Biosensors and capillary electrophoresis microchip devices for analytical applications

BIOSENSORS AND CAPILLARY

ELECTROPHORESIS MICROCHIP DEVICES FOR ANALYTICAL APPLICATIONS

APPAN ROYCHOUDHURY

CENTRE FOR BIOMEDICAL ENGINEERING INDIAN INSTITUTE OF TECHNOLOGY DELHI

MARCH 2019

© Indian Institute of Technology Delhi (IITD), New Delhi, 2019.

BIOSENSORS AND CAPILLARY

ELECTROPHORESIS MICROCHIP DEVICES FOR ANALYTICAL APPLICATIONS

by

APPAN ROYCHOUDHURY

CENTRE FOR BIOMEDICAL ENGINEERING

Submitted

in fulfilment of the requirements of the degree of Doctor of Philosophy to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

MARCH 2019

Dedicated to

All those people who have always supported and

encouraged me to pursue my dreams

“Little by little, through patience and repeated effort, the mind will become stilled in the Self”

– Bhagavad Gita

“The secret of getting ahead is getting started”

– Mark Twain

“Excellence is a continuous process and not an accident”

– Dr. A.P.J. Abdul Kalam

CERTIFICATE

ACKNOWLEDGEMENTS

ABSTRACT

सार

CONTENTS

List of Figures and Illustrations

List of Tables

List of Abbreviations