DEVELOPMENT AND APPLICATIONS OF ADVANCED MATERIALS BASED BIOSENSORS EMRIL MOHAMED ALI (B Eng (Hons)) NUS (MSc) Imperial College London A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES & ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2008 List of Publications E M Ali, E A B Kantchev, H H Yu and J Y Ying, “Carboxylic AcidFunctionalized Polyethylenedioxythiophenes (PEDOTs): Syntheses, Characterizations, and Electronic Performances,” Proceedings of the 233rd American Chemical Society National Meeting, Division of Polymeric Materials: Science & Engineering, PMSE Preprint, 96 (2007), March 25-29, 2007, Chicago, Illinois, USA, pp 304-305 E M Ali, E A B Kantchev, H H Yu and J Y Ying "Conductivity shift of polyethylenedioxythiophenes (PEDOTs) in aqueous solutions from side-chain charge perturbation" Macromolecules (2007) 40, 6025-6027 E M Ali, Y Zheng, H H Yu and J Y Ying " Ultrasensitive Pb2+ detection by nature-mimicking, glutathione-capped quantum dots" Analytical Chemistry (2007), 79, 9452-9458 S C Luo, E M Ali, N C, Tansil, H H Yu, E A B Kantchev and J Y Ying, “PEDOT nanobiointerfaces: thin, ultrasmooth, and functionalized poly(3,4-ethylenedioxythiophene) films with in vitro and in vivo biocompatibility" accepted in Langmuir (2008) E M Ali, E A B Kantchev, S C Luo, H H Yu and J Y Ying “Conductivity Behavior of Polyethylenedioxythiophenes from Side-Chain Perturbation and Polymer Dimensional Influence in Aqueous Solutions” manuscript in preparation Patents J Y Ying, H H Yu and E A Mohamed, “Robust and Photostable Luminescent ZnO Films: Applications as Fluorescence Resonance Energy Transfer (FRET) Donors,” US Provisional and PCT Patent filed December 2005 J Y Ying, H H Yu, E A Mohamed and J R Nikhil, “ ‘Turn-Off’ Luminescence Detection by Switching Photostability of Nanocrystals,” US Provisional and PCT Patent filed December 2005 i Acknowledgements First and foremost, I would like to thank my supervisors, Professor Jackie Y Ying and Dr Bruce Yu for their close guidance, encouragement and scientific directions This research would not have been possible without their help They were not only supervisors but also mentors who gave me great support during the difficult early phase of my PhD research Coming from a mechanical engineering background, the chemistry aspects of material synthesis were quite a challenge initially but close laboratory guidance from Dr Bruce made my transition relatively easy Professor Ying was not only my supervisor but also the executive director of Institute of Bioengineering and Nanotechnology Together with Noreena AbuBakar, they gave me the privilege to work in this world class research institute, which not only provided fantastic research equipment but also wonderful colleagues, too many to mention, who were fun to work with and readily shared their wealth of scientific knowledge Thank you all for making this journey so memorable and I will be looking forward to working with everyone again for my post-doctoral training Taking four years away from work would not have been financially possible without Philip Yeo and A*star Thank you for this truly privileged opportunity Special mention goes to Associate Professor Francis Tay from NUS who gave insightful and objective views on the research Besides the scientific and financial support from work, truly important pillars of strength came from my Mum and Dad Thank you for standing by me, through the difficult early phases, softening the blow whenever I faced difficulties at work and ii with my personal life Parent’s love has no boundaries and that was one of the many lifelong lessons I acquired in the past four years Both of you are not only my pillars of support but also great friends No words can ever describe my love for both of you I love you guys so much! Not forgetting my brother Norham who is not only a husband to his wife Norizan but also a father to my adorable nephew, Dani Thank you for stepping up to be a husband and father before me You are definitely two steps ahead of me in that aspect Thanks to one truly special person, its only one step for now… Sofia Joanne Chong Mei San, you are like the final revelation of my life For the last four years of my life, I went through many trials and tribulations It was not only a journey about scientific learning but one of self discovery; learning about me, overcoming challenges and coming out stronger each time However, I didn’t feel complete After each small accomplishment I made, there was still a sense of emptiness I realized I had no one to share my life with It was a void that my loving parents could not fill That was until 21st July 2007 The day I felt complete, the day I met you, the day you walked into my life and filled the emptiness with love, hope, joy and completeness This thesis is dedicated to you This work may have just taken the last four years but I have spent the entire 30 years of my life looking for you I am truly blessed to have found you a year ago I just can’t wait to build our life together You complete me iii Table of Contents List of Publications i Acknowledgements ii Table of Contents iv Summary viii List of Tables x List of Figures xi List of Figures xi Chapter : Introduction to Materials-based Biosensors and Literature Review Research Abstract Background Information Biosensors Based on Advanced Materials Literature Review Quantum dot based biosensors Conducting polymer based biosensors Development of DNA sensors 12 Research outline 15 References 15 Chapter : Application of GSH-Capped Quantum Dots to Pb2+ Detection 20 Introduction 20 Experimental Section 22 iv Materials and Reagents 22 Quantum Dot Synthesis 23 High-Throughput Fluorescence Measurements 23 Selectivity Measurements 24 Fluorescence Quenching Measurements 24 Physical Characterization of Fluorescence Quenching with Pb2+ 25 Interference Fluorescence Quenching Measurements 26 Results and Discussion 26 Selective Fluorescence Quenching of GSH-Capped QDs by Pb2+ 27 Mechanism of Pb2+ Detection by GSH-Capped QDs 28 Detection Limit for Pb2+ Detection 37 Pb2+ Detection in the Presence of Other Metal Ions 40 Conclusion 42 Reference 43 Chapter : Side Chain Charge Modulation Study of Polyethylenedioxythiophene (PEDOT) 46 Introduction 46 Experimental Section 48 Materials and Reagents 48 Synthesis of Functionalized Monomer 48 Film Electropolymerization 51 Film Surface Analysis 52 Electrical Characterization of Polymer 53 v Results and Discussion 55 Characterization of Functionalized EDOTs 55 Negative Charge Modulation via pH Variation 58 Charge Perturbation of Co-Poly(EDOT-OH)-Poly(C4-EDOT-COOH) 65 Post Film Functionalization Study 69 Conclusions 72 Reference 73 Chapter : Integration of PEDOT with microfabricated device towards the application of ‘label-free’ DNA detection 77 Introduction 77 Experimental Section 79 Materials and Reagents 79 Device Fabrication 80 EDOT integration with device 84 Electrical characterization setup 85 DNA probe immobilization 88 DNA hybridization and concentration-dependent study 88 DNA hybridization characterization 89 Device surface analysis 89 Results and Discussion 90 Electropolymerization on microjunction electrode devices 90 Study of electrode dimensions effect on EDOT electrical behavior 97 vi Further electrical characterization of TMJ/C2-EDOT-COOH system .104 PEDOT nanowire FET characteristics .106 DNA detection with EDOT nanowires 109 Conclusion 117 Reference 119 Chapter : Conclusion and Future Work 123 Conclusion 123 Future Work .126 Reference 127 vii Summary Quantum dots (QDs) and conducting polymers (CPs) are examples of novel advanced novel materials that possess intrinsic properties suitable for measurement Fluorescence of QDs and conductivity of CPs can be easily quantified by devices such as fluorescence microplate reader and electrical instrumentation, respectively Hence QDs and CPs are attractive platforms for the development of biosensing transducers that can directly translate a biological binding event into fluorescence and electrical signals This research investigates the mechanism correlating the biological binding event with the change of materials’ intrinsic property The investigations were subsequently used to develop sensory systems and apply them for sensing important biological analytes QDs used were capped with glutathione (GSH) shells GSH and its polymeric form, phytochelatin, are employed by nature to detoxify heavy metal ions Detailed studies show that competitive GSH binding of Pb2+ with the QD core changed both the surface and photophysical properties of QDs Coupling the GSH-capped QDs with high-throughput detection system, a simple scheme for the quick and ultrasensitive Pb2+ detection without the need for additional electronic devices was developed Functionalized 3, 4-ethylenedioxythiophene (EDOT) monomers were synthesized and the conductivity profile of poly(C4-EDOT-COOH)-coated electrode junctions in aqueous buffers could be manipulated by modulating the negative charge density in the polymer matrix through side-chain functional groups Upon fixing the viii applying voltage of interdigitated electrodes at the transitional stages, the polymer coated device was utilized as pH resistive sensors Nanowire EDOT polymers were further developed Fabricated MEMS electrode junction devices, integrated with EDOT nanowires, immobilized with DNA probes, were utilized as a liquid gated field-effect transistor and the hybridization of the negatively charged complimentary DNA was found to increase the conductivity of the nanowire The development potential of a ‘Lab on Chip’ device in the application of ‘Label-free’ DNA detection was demonstrated by this integrated EDOT and MEMS system ix Figure 4-20 Isd-Vsd measurement of poly(C2-EDOT-COOH) nanowires with immobilized ssDNA probes on separate devices incubated with (a) nM and (b) 50 nM of complimentary ssDNA Measurements are obtained before ( _) and after ( -) h of ssDNA incubation FET measurements are conducted at pH with 0.1 M of LiClO4 as the supporting electrolyte Vg = V Figure 4-21 Isd-Vsd measurement of poly(C2-EDOT-COOH) nanowires with immobilized ssDNA probes on separate devices incubated with (a) 100 nM and (b) µM of complimentary ssDNA Measurements are obtained before ( _) and after ( -) h of ssDNA incubation FET measurements are conducted at pH with 0.1 M of LiClO4 as the supporting electrolyte Vg = V 114 Figure 4-22 Percentage change of Isd (rI/Io) with increasing concentration of target ssDNA oligonucleotide Vsd = 0.5 V, Vg = V In the previous DNA hybridization experiments, the DNA incubation was conducted in 0.1 M of NaCl solution, buffered at pH 7, as reported by other groups.49, 50 However, the FET measurements were conducted in pH to prevent the deprotonation of the carboxyl side groups, which would introduce negative charges into the polymer matrix This would counteract the conductance increase triggered by the DNA hybridization In practical DNA detection application, the hybridization and FET measurements would have to be conducted simultaneously and in the same pH conditions Due to the current limitations presented by the carboxylic side groups, the detection experiments would have to be conducted at pH DNA hybridization at pH To verify the hybridization of the DNA at pH 5, QCM-D experiment was conducted with a quartz crystal that was previously coated 115 with C2-EDOT-COOH and immobilized with DNA capture probes 10 µM of complimentary DNA targets were dissolved in pH and buffered solutions with 0.1 M of NaCl flowed into the quartz chambers of the Q-Sense microfluidic system The hybridization of DNA on the PEDOT reflected a quartz mass increase that corresponded to a resonance frequency and dissipation change (see Figure 4-23) The frequency and dissipation change was observed in both pH conditions, confirming that pH conditions were suitable for DNA hybridization to take place Figure 4-23 Frequency shift of EQCM quartz crystal coated with DNA probe immobilized poly(C2-EDOT-COOH) after the introduction of 10 µM of complimentary target ssDNA at (a) pH and (c) pH buffer Target ssDNA was prepared with 10 mM of Tris buffer and 0.1 M of NaCl The corresponding quartz crystal dissipation change after the introduction of ssDNA at (b) pH and (d) pH DNA detection limit To investigate the DNA detection limit of the PEDOT DNA probe immobilized nanowires, experiments were conducted on a PEDOT nanowire device at pH The concentration of target DNA incubated on the device was steadily increased at 1-h intervals Isd-Vsd measurements were conducted at the 116 end of each interval to progressively monitor the nanowire FET characteristics upon DNA hybridization As shown in Figure 4-24, a 22% increase in Isd current was observed at 10 nM of DNA With increasing DNA concentration, the Isd current increased steadily before saturating at 60% beyond 50 nM The surface negative field potential on the nanowire surface increased as more DNA hybridized on the nanowire surface Hence, the Isd current increased quantitatively with the DNA target concentrations before reaching a plateau when the capture probes were fully saturated with DNA targets Figure 4-24 (a) FET characterization curve of DNA probe immobilized poly(C2EDOT-COOH) with increasing target DNA The dash line represents the initial FET measurement with µM of target DNA (b) (rI/Io) % with increasing amount of target ssDNA oligonucleotide Vsd = 0.5 V, Vg = V Conclusion We have fabricated microjunction electrode devices that allow 5-µl droplet of test solution to be isolated and form an electrolyte chamber that connects the working, counter and reference electrodes to create a liquid gated FET system This 117 configuration enables electrical experiments to be conducted with microliter electrolyte solutions, paving the way for the development of a PEDOT ‘lab-on-achip’ system capable of droplet-based detection Functionalized PEDOTs were integrated onto the electrode junctions of the devices through various electropolymerization methods The application of alternating potential directly combines nanowire synthesis and device integration, eliminating the ohmic contact problems when integrating pre-synthesized conducting polymer nanowires with electrode junction devices The comparative study between the TMJ and SMJ devices shows that reduced ohmic contact between the device electrode and the PEDOT polymer results in reduction in the polymer’s intrinsic conductance instead of an onset potential shift, when subjected to negative charges modulated by its side chains This electrical behavior extends to poly(C2-EDOT-COOH) nanowires that have significantly reduced dimensions Further studies highlight the important distinction between the intrinsic and surface negative charge effects on the FET characteristics of PEDOT nanowires The modulation of negative charges within the polymer’s intrinsic matrix causes a reduction in the polymer conductance However, the negative charges on the PEDOT nanowire surface create a negative potential field that increases its conductance The effect of negative surface charges was evident from the immobilization and hybridization of DNA on the PEDOT nanowire surface This phenomenon was used in the application of DNA detection, achieving a detection limit of 10 nM of 118 DNA The coupling of the PEDOT nanowires and a suitable microjunction device, connected to a potentiostat via simple chip fixtures, demonstrates the application of a DNA sensing transducer based on the PEDOT conducting polymer The hybridization between the target DNA and its capture probe is directly translated to a conductance change within the PEDOT nanowires, which are electrically monitored by the potentiostat This direct detection system does not require the DNA targets to be prelabeled, and represents a significant step to the development of a more efficient but yet simplified DNA detection system Reference Krishnamoorthy, K.; Gokhale, R S.; Contractor, A Q.; Kumar, A., Novel label-free DNA sensors based on poly(3,4-ethylenedioxythiophene) Chem Commun 2004, 820-821 Wang, J., Towards Genoelectronics: Electrochemical biosensing of DNA hybridization Chem Eur J 1999, 5, 1681-1685 Palecek, E.; Fojta, M.; Jelen, F., New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces Bioelectrochemistry 2002, 56, 85-90 Drummond, T G.; Hill, M G.; Barton, J K., Electrochemical DNA sensors Nat Biotechnol 2003, 21, 1192-1199 Star, A.; Tu, E.; Gabriel, J.-C P.; Joiner, C S.; Valcke, C., Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors PNAS 2006, 103, 921-926 Groenendaal, L.; Jonas, F.; Freitag, D.; Pielartzik, H.; Reynolds, J R., Poly(3,4-ethylenedioxythiophene) and Its Derivatives: Past, Present, and Future Adv Mater 2000, 12, 481-494 Groenendaal, L.; Zotti, G.; Aubert, P H.; Waybright, S M.; Reynolds, J R., Electrochemistry of poly(3,4-alkylenedioxythiophene) derivatives Adv Mater 2003, 15, 855-879 Roncali, J.; Blanchard, P.; Frère, P., 3,4-Ethylenedioxythiophene (EDOT) as a versatile building block for advanced functional Pi-conjugated systems J Mater Chem 2005, 15, 1589-1610 119 10 11 12 13 14 15 16 17 18 19 20 21 Kirchmeyer, S.; Reuter, K., Scientific importance, properties and growing applications of poly(3,4-ethylenedioxythiophene) J Mater Chem 2005, 15, 2077-2088 Kros, A.; Sommerdijk, N A J M.; Nolte, R J M., Poly(pyrrole) versus poly(3,4-ethylenedioxythiophene): Implications for biosensor applications Sens Actuators B 2005, 106, 289 Sapp, S A.; Sotzing, G A.; Reynolds, J R., High contrast ratio and fastswitching dual polymer electrochromic devices Chem Mater 1998, 10, 2101-2108 Mouffouk, F.; Higgins, S J., Oligonucleotide-functionalised poly(3,4ethylenedioxythiophene)-coated microelectrodes which show selective electrochemical response to hybridisation Electrochem Commun 2006, 8, 317-322 Wang, J.; Chan, S.; Carlson, R R.; Luo, Y.; Ge, G.; Ries, R S.; Heath, J R.; Tseng, H R., Electrochemically fabricated polyaniline nanoframework electrode junctions that function as resistive sensors Nano Lett 2004, 4, 1693-1697 James, F.; Hulvat, S I S., Liquid-crystal templating of conducting polymers Angew Chem Int Ed 2003, 42, 778-781 Hatano, T.; Bae, A.-H.; Takeuchi, M.; Fujita, N.; Kaneko, K.; Ihara, H.; Takafuji, M.; Shinkai, S., Helical superstructure of conductive polymers as created by electrochemical polymerization by using synthetic lipid assemblies as a template Angew Chem Int Ed 2004, 43, 465-469 Choi, J W.; Han, M G.; Kim, S Y.; Oh, S G.; Im, S S., Poly(3,4ethylenedioxythiophene) nanoparticles prepared in aqueous DBSA solutions Synth Met 2004, 141, 293-299 Jang, J.; Chang, M.; Yoon, H., Chemical sensors based on highly conductive poly(3,4-ethylenedioxythiophene) nanorods Adv Mater 2005, 17, 1616-1620 Yoon, H.; Chang, M.; Jang, J., Formation of 1D poly(3,4ethylenedioxythiophene) nanomaterials in reverse microemulsions and their application to chemical sensors Adv Funct Mater 2007, 17, 431-436 Caras-Quintero, D.; Bauerle, P., Synthesis of the first enantiomerically pure and chiral, disubstituted 3,4-ethylenedioxythiophenes (EDOTs) and corresponding stereo- and regioregular PEDOTs Chem Commun 2004, 926927 Ha, Y.-H.; Nikolov, N.; Dulcey, C.; Wang, S.-C.; Mastrangelo, J.; Shashidhar, R., Conductivity tuning of poly(3,4-ethylenedioxythiophene) through sidegroup cleavage Synth Met 2004, 144, 101 Trippé, G.; Le Derf, F.; Lyskawa, J.; Mazari, M.; Roncali, J.; Gorgues, A.; Levillain, E.; Sallé, M., Crown-tetrathiafulvalenes attached to a pyrrole or an EDOT unit: Synthesis, electropolymerization and recognition properties Chem Eur J 2004, 10, 6497-6509 120 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Ansorge, W.; Sproat, B.; Stegemann, J.; Schwager, C.; Zenke, M., Automated DNA sequencing: Ultrasensitive detection of fluorescent bands during electrophoresis Nucleic Acids Res 1987, 15, 4593-4602 Pease, A C.; Solas, D.; Sullivan, E J.; Cronin, M T.; Holmes, C P.; Fodor, S P., Light-generated oligonucleotide arrays for rapid DNA sequence analysis Proc Natl Acad Sci USA 1994, 91, 5022-5026 Wei, F.; Wang, J.; Liao, W.; Zimmermann, B G.; Wong, D T.; Ho, C.-M., Electrochemical detection of low-copy number salivary RNA based on specific signal amplification with a hairpin probe Nucleic Acids Res 2008, 36, e65(1-7) Wanekaya, A K.; Chen, W.; Myung, N V.; Mulchandani, A., Nanowirebased electrochemical biosensors Electroanalysis 2006, 18, 533-550 Janata, J.; Josowicz, M., Conducting polymers in electronic chemical sensors Nature Mater 2003, 2, 19-24 Cheng, C D.; Gonela, R K.; Gu, Q.; Haynie, D T., Self-assembly of metallic nanowires from aqueous solution Nano Lett 2005, 5, 175-178 Thompson, L A.; Kowalik, J.; Josowicz, M.; Janata, J., Label-free DNA hybridization probe based on a conducting polymer J Am Chem Soc 2003, 125, 324-325 Minehan, D S.; Marx, K A.; Tripathy, S K., Kinetics of DNA binding to electrically conducting polypyrrole films Macromolecules 1994, 27, 777-783 Wang, J.; Jiang, M.; Fortes, A.; Mukherjee, B., New label-free DNA recognition based on doping nucleic-acid probes within conducting polymer films Analytica Chimica Acta 1999, 402, Ong, P L.; Wei, J.; Tay, F E H.; Iliescu, C., A new fabrication method for low stress PECVD-SiNx layers J Phys.: Conf Series 2006, 34, 764-769 Iliescu, C.; Wei, J.; Chen, B.; Ong, P L.; Tay, F E H., Low stress silicon nitride layers for MEMS applications Proc of SPIE 2006, 6415, 64150L-1 Minot, E D.; Janssens, A M.; Heller, I.; Heering, H A.; Dekker, C.; Lemay, S G., Carbon nanotube biosensors: The critical role of the reference electrode Appl Phys Lett 2007, 91, 093507 Wanekaya, A K.; Bangar, M A.; Yun, M.; Chen, W.; Myung, N V.; Mulchandani, A., Field-effect transistors based on single nanowires of conducting polymers J Phys Chem C 2007, 111, 5218-5221 Das, A.; Lei, C H.; Elliott, M.; MacDonald, J E.; Turner, M L., Nonlithographic fabrication of PEDOT nano-wires between fixed Au electrodes Org Electron 2006, 7, 181-187 Bredas, J L.; Street, G B., Polarons, bipolarons, and solitons in conducting polymers Acc Chem Res 1985, 18, 309-315 Alam, M M.; Wang, J.; Guo, Y Y.; Lee, S P.; Tseng, H R., Electrolytegated transistors based on conducting polymer nanowire junction arrays J Phys Chem B 2005, 109, 12777-12784 121 38 39 40 41 42 43 44 45 46 47 48 49 50 Nilsson, D.; Chen, M.; Kugler, T.; Remonen, T.; Armgarth, M.; Berggren, M., Bi-stable and dynamic current modulation in electrochemical organic transistors Adv Mater 2002, 14, 51-54 Zhu, Z.-T.; Mabeck, J T.; Zhu, C.; Cady, N C.; Batt, C A.; Malliaras, G G., A simple poly(3,4-ethylene dioxythiophene)/poly(styrene sulfonic acid) transistor for glucose sensing at neutral pH Chem Commun 2004, 1556-1557 Epstein, A J.; Hsu, F.-C.; Chiou, N.-R.; Prigodin, V N., Electric-field induced ion-leveraged metal-insulator transition in conducting polymer-based field effect devices Curr Appl Phys 2002, 2, 339 Lu, J.; Pinto, N J.; MacDiarmid, A G., Apparent dependence of conductivity of a conducting polymer on an electric field in a field effect transistor configuration J Appl Phys 2002, 92, 6033-6038 Nilsson, D.; Kugler, T.; Svensson, P.-O.; Berggren, M., An all-organic sensortransistor based on a novel electrochemical transducer concept printed electrochemical sensors on paper Sens Actuators B 2002, 86, 193 Pinto, N J.; Johnson, J A T.; MacDiarmid, A G.; Mueller, C H.; Theofylaktos, N.; Robinson, D C.; Miranda, F A., Electrospun polyaniline/polyethylene oxide nanofiber field-effect transistor Appl Phys Lett 2003, 83, 4244 Chakrabarti, R.; Klibanov, A M., Nanocrystals modified with peptide nucleic acids (PNAs) for selective self-assembly and DNA detection J Am Chem Soc 2003, 125, 12531-12540 He, L.; Musick, M D.; Nicewarner, S R.; Salinas, F G.; Benkovic, S J.; Natan, M J.; Keating, C D., Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection of DNA hybridization J Am Chem Soc 2000, 122, 9071-9077 Gaylord, B S.; Heeger, A J.; Bazan, G C., DNA hybridization detection with water-soluble conjugated polymers and chromophore-labeled single-stranded DNA J Am Chem Soc 2003, 125, 896-900 Storhoff, J J.; Elghanian, R.; Mucic, R C.; Mirkin, C A.; Letsinger, R L., One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes J Am Chem Soc 1998, 120, 1959-1964 Cao, Y W.; Jin, R.; Mirkin, C A., DNA-modified core-shell Ag/Au nanoparticles J Am Chem Soc 2001, 123, 7961-7962 Kwon, D.; Kim, K.; Kwak, J., Electrochemical DNA hybridization detection using DNA cleavage Electroanal 2008, 20, 1204-1208 Li, D.; Zou, X.; Shen, Q.; Dong, S., Kinetic study of DNA/DNA hybridization with electrochemical impedance spectroscopy Electrochem Commun 2007, 9, 191 122 Chapter : Conclusion and Future Work Conclusion In Chapter 2, GSH-capped QDs demonstrated selective fluorescence quenching in the presence of Pb2+ ions 50% of the fluorescence intensity of GSHZnCdSe QDs was quenched in the presence of as low as 20 nM of Pb2+ This naturemimicking system was capable of Pb2+ detection even in the presence of an ion mixture, and only became less sensitive when the ion mixture was present at a very high concentration The low detection limit of Pb2+ was primarily due to the superior fluorescence properties of QDs The fluorescence quenching was hypothesized to be attributed to the stronger binding between heavy metal ions and the surface GSH capping layer, as confirmed by detailed mechanistic studies of spectroscopy, microscopy, and dynamic light scattering By coupling the highly selective and sensitive GSH-capped QDs with a highthroughput detection system, we have developed a simple detection system for quick and ultrasensitive Pb2+ detection without the need for additional electronic devices The detection was made possible by the GSH-capped QDs that functioned as a Pb2+ sensing transducer The Pb2+ GSH binding event was directly translated to a fluorescence intensity change, reported by the fluorescence microplate reader In Chapter 3, C4-EDOT-COOH and C2-EDOT-COOH monomers were successfully synthesized and polymerized on interdigitated microelectrode devices The functionalized polymers showed great stability in aqueous medium, and more 123 importantly, the carboxylic acid side groups would allow for the conjugation of biological probes via conventional EDC/NHS coupling reactions The first example of conductimetric response from shifting the conductivity curve was demonstrated via the modulation of negative charges in the polymer matrix through side-chain carboxylic acid groups of poly(C4-EDOT-COOH) and poly(C2-EDOT-COOH)coated interdigitated microelectrodes The devices were utilized as resistive sensors, producing an immediate 0.5-mA current response from pH manipulation The device’s large, rapid amperometric response demonstrated its potential as a sensing device with low power requirements The study concluded that to utilize this methodology for biosensing applications, the targeted biological analytes would have to be negatively charged so as to induce the necessary charge density increase upon binding with the immobilized bioprobes Charge density above 80% was identified as the critical threshold point needed by the biological targets to successfully produce the desired amperometric response In Chapter 4, microjunction electrode devices were specially fabricated to form a liquid gated FET device that enabled experiments to be conducted with microliter electrolyte solutions, paving the way for the development of a PEDOT ‘lab-on-a-chip’ system capable of droplet-based detection Functionalized PEDOTs were integrated onto the electrode junctions of the devices through various electropolymerization methods The application of alternating potential directly combined nanowire synthesis and device integration, eliminating the ohmic contact 124 problems when integrating pre-synthesized conducting polymer nanowires with electrode junction devices Detailed studies highlighted the important distinction between the intrinsic and surface negative charge effects on the FET characteristics of PEDOT nanowires The modulation of negative charges within the polymer’s intrinsic matrix caused a reduction to the polymer conductance, while the negative charges on the PEDOT nanowire surface created a negative potential field that increased its conductance The surface charge phenomenon was used in the application of DNA detection, achieving a detection limit of 10 nM DNA The coupling of the PEDOT nanowires and a suitable microjunction device, connected to a potentiostat via simple chip fixtures demonstrated the application of a DNA sensing transducer based on the PEDOT conducting polymer This direct detection system did not require the DNA targets to be pre-labeled and represented a significant step to the development of a more efficient, but yet simplified, DNA detection system Overall, the research examined two types of materials-based biosensing systems, the fluorescence detection system based on the GSH-capped QDs and the electrical detection system based on PEDOT integrated on microelectrode devices The mechanistic behaviors of QDs and CPs as transducing materials were investigated, and both systems were demonstrated as practical detection systems that could potentially be applied as important tools for environmental sensing and clinical diagnostics 125 Future Work In the GSH-capped QD-based system, the only ions that strongly interfered with the Pb2+ detection were Ag + and Cu2+ because they also showed similar quenching effect as Pb2+ at similar concentration levels This remained the limitation of the GSH-QD detection system, despite its higher sensitivity and more selective response as compared to other QD-based metal ion detection.1, The Cu2+ interference was most likely associated with different mechanism, such as metal cluster incorporation or coordination-mediated aggregation.3 It could possibly be eliminated with improved peptide coating Future work could also examine alternative peptides that could bind specifically with selected important biological targets For industrial and environmental applications of our QD system, the samples would need to be pretreated to avoid interference from Ag+ and Cu2+ ions A miniaturized fluorescence detection device could be designed and used to conduct the detection experiments This would enable the analysis to be conducted on-site IME device coated with poly(C4-EDOT-COOH) was demonstrated as a pHsensitive resistive sensor that produced a large amperometric response upon pH manipulation The pH change corresponded with the pKa value of the monomer’s carboxylic acid functional group A possible future direction of this research could be in the development of real-time pH sensors based on functionalized PEDOT coated IME devices The synthesis of functionalized EDOT monomers with varied pKa values and further co-polymerization studies would have to be performed In 126 particular, the electrical characteristics of poly(EDOT-NH2) would be of great interest EDOT-NH2 monomer was successfully synthesized by my colleagues, but could not be electropolymerized onto the IME devices Future efforts could be devoted towards overcoming this challenge Currently, we have only looked at the effects of negative charge perturbation on PEDOT’s electrical behavior The study of positive charge influence on the polymer would be a natural progression of this research In the application of PEDOT nanowires towards ‘label-free’ DNA detection, a major limitation was the detection consistency due to the morphological variation in the nanowires Future improvements could be made to the fabrication consistency of the microjunction devices The electropolymerization conditions could be further developed to produce more consistent nanowires The device’s sensitivity could be improved by using a more optimized gate voltage (Vg) Alternative bio-conjugation methods such as “click chemistry”4 could be employed, and sensing applications could be extended by utilizing bio-recognition elements such as aptamers,5 antibodies,6, and novel synthetic protein receptors via click chemistry.4, Reference Chen, Y.; Rosenzweig, Z., Luminescent CdS quantum dots as selective ion probes Anal Chem 2002, 74, 5132-5138 Gattas-Asfura, K M.; Leblanc, R M., Peptide-coated CdS quantum dots for the optical detection of copper(II) and silver(I) Chem Commun 2003, 26842685 Wuelfing, W P.; Zamborini, F P.; Templeton, A C.; Wen, X.; Yoon, H.; Murray, R M., Monolayer-protected clusters: Molecular precursors to metal films Chem Mater 2001, 13, 87-95 127 Kolb, H C.; Finn, M G.; Sharpless, K B., Click chemistry: Diverse chemical function from a few good reactions Angew Chem Int Ed Engl 2001, 40, 2004-2021 Jayasena, S D., Aptamers: An emerging class of molecules that rival antibodies in diagnostics Clin Chem 1999, 45, 1628-1650 Rachkov, A E.; Rozhko, M I.; Sergeyeva, T A.; Piletsky, S A., Method and apparatus for the detection of the binding reaction of immunoglobulins Sens Actuators B 1994, 19, 610-613 Sergeyeva, T A.; Lavrik, N V.; Piletsky, S A.; Rachkov, A E.; El'skaya, A V., Polyaniline label-based conductometric sensor for IgG detection Sens Actuators B 1996, 34, 283-288 Speers, A E.; Cravatt, B F., Profiling enzyme activities in vivo using click chemistry methods Chem Bio 2004, 11, 535-546 128 ... 0.5, and (e) 1.0 mM of Pb2+ and (f) 1.0 mM of Ca2+ 33 Figure 2-8 DLS data of µM of ZnCdSe (λmax = 469 nm) treated with (a) 0, (b) 0.1, (c) 0.25, (d) 0.5, and (e) 1.0 mM of Pb2+ and (f) 1.0 mM of. .. excited at 345 nm ml of µM of ZnCdSe (λmax = 469 nm) was added to ml of 0.1, 0.25, 0.5 and 1.0 mM of Pb2+ ions ml of deionized water and 1.0 mM of Ca2+ were used as controls sets of samples were prepared... of Figures xi Chapter : Introduction to Materials- based Biosensors and Literature Review Research Abstract Background Information Biosensors Based on Advanced