Multifunctional nanomaterials for biosensing and bioimaging : insights from metal oxides and carbon-dots

Abstract

The investigations embodied in thesis entitled “MULTIFUNCTIONAL NANOMATERIALS FOR BIOSENSING AND BIOIMAGING: INSIGHTS FROM METAL OXIDES AND CARBON-DOTS” was initiated in January 2014 in the Discipline of Biosciences & Bio-Medical Engineering, Indian Institute of Technology Indore. The objectives of this thesis are to design and construct nanomaterials for biomedical and bio-sensing applications. The focal points of thesis are as follows- 1. Construction of single source molecular precursors derived ZnO nanoflowers for biological activities. 2. Development of peroxidase mimics CuO nanoparticle and its graphene nanosphere composite for colorimetric cholesterol detection. 3. Chemical synthesis of c-dots towards heavy metal ion sensing and bioimaging. 4. Green synthesis of c-dots for optical sensing of metal ion, anion and specific intracellular targeting. This thesiscomprises of eight chapters. It begins with a general introduction to the topic and literature review (Chapter 1), followed by use of ZnO nanoparticles and single source molecular precursors (SSMP’s) for antibacterial applications (Chapter 2), CuO:Graphene noanosphere composite for colorimetric cholesterol sensor (Chapter 3), and carbon-dots as sensors and bioimaging probes (Chapter 4-7). The thesis outlines the future perspective in the Chapter 8. The introductory chapter (Chapter 1) of this thesis illustrates the brief background and literature review of nanoparticle synthesis specifically metal oxide nanoparticles and c-dots. Furthermore, the bio-medical applications ofnanomaterials including antibacterial activities, optical bio-sensing and bioimaging is elaborated in sufficient details. Chapter 2, describes the synthesis of ZnO nanoflowers (ZnO-1 and ZnO-2) at room temperature using new structurally characterized single molecular precursors (Asymmetric dimer [Zn(hmp-H)2(H2O)(μ-Cl)Zn(μ-Cl)(Cl)3] (1) and symmetric dimer [Zn(hep-H)(μ-Cl)(Cl)]2 (2)). 1, 2 and ZnO-1 were explored for their potential to cause loss of viability to gram-negative bacteria Escherichia coli. 1 and 2 were found to be promising antibacterial agents, while the ZnO nanoflowers have shown a relative non-toxic nature. 1, 2 and ZnO-1 were further evaluated for DNA binding and cleavage behaviour. 1 and 2 have shown strong binding affinity towards CT-DNA as compare to ZnO-1. Additionally, all three of them have shown oxidative cleavage of pBluescript plasmid DNA in the presence of H2O2.The Chapter 3 highlights the challenge of faster and easy detection of cholesterol. The development of colorimetric sensor may lead to the fabrication of a ready to use sensing strip. This chapter demonstrates the use of a cytocompatible CuO:Graphene nanosphere (CuO:GNS) composite as a peroxidase mimic for detection of H2O2 and free cholesterol. The synthesized CuO:GNS composite was investigated systematically for structural, morphological and functional aspects. The proposed methodology involves detection of H2O2 produced during oxidation of free cholesterol in the presence of cholesterol oxidase. The nanocomposite sensor has shown excellent detection sensitivity for cholesterol and has demonstrated a linear response in the range of 0.1 mM - 0.8 mM with limit of detection (LOD) as low as 78 μM. This nanocomposite sensor also detected a very low concentration of H2O2 (0.01–0.1 mM) with LOD of 6.88 μM. An AND logic gate system based on CuO:GNS and Cholesterol input was also proposed. The CuO:GNS was found to have better cytocompatibility than standalone CuO.Carbon based nanomaterials are emerging as a desirable alternative to semiconducting quantum dots due to their unique optical properties and biocompatibility. The Chapter 4 demonstrates the investigation of highly fluorescent carbon nanoparticles (CNP) for biocompatibility, biosensing and bioimaging. The CNP were successfully employed as promising multicolor bioimaging probe in A375 and DU145 cell lines. Further, a “Turn off” mode has been established for detection of noble metal palladium (Pd2+) and heavy metal mercury (Hg2+) by quenching of fluorescence of CNP. The CNP sensor responded for a wide range of Pd2+ (5 μM-100 μM) and Hg2+ (1 μM-18 μM) detection with LOD of 58 nM for Pd2+ and 100 nM for Hg2+. The CNP sensor was employed for detection of Pd2+ and Hg2+ in real water samples and detection of leftover palladium catalyst in a model reaction system. Also, the CNP was successfully employed as intracellular mercury and palladium sensor using confocal microscopy.Encouraged by the sensing and bio ncouraged by the sensing and bio ncouraged by the sensing and bio ncouraged by the sensing and bio ncouraged by the sensing and bioncouraged by the sensing and bioncouraged by the sensing and bioncouraged by the sensing and bio ncouraged by the sensing and bioncouraged by the sensing and bio ncouraged by the sensing and bio ncouraged by the sensing and bio ncouraged by the sensing and bioncouraged by the sensing and bioncouraged by the sensing and bio ncouraged by the sensing and bio -imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from imaging results from CNP CNP, green route , green route , green route , green route , green route , green route , green route , green route for synthesis of for synthesis of for synthesis of for synthesis of for synthesis of for synthesis of carbon nanomaterialcarbon nanomaterialcarbon nanomaterial carbon nanomaterial carbon nanomaterial carbon nanomaterial carbon nanomaterial carbon nanomaterial was explored. was explored. was explored.was explored.was explored. was explored. In In Chapter 5, Sugarcane derived carbon-based nanomaterials are developed as a cytocompatible alternative to semiconducting quantum dots for bioimaging and fluorescence-based pH sensing. This chapter demonstrates microwave based one step quick synthesis of fluorescent carbon material (FCM) having three variants: (i) un-doped fluorescent carbon material (UFCM) (ii) nitrogen doped FCM (N@FCM), and (iii) nitrogen & phosphorus co-doped FCM (N-P@FCM) using sugarcane extract as a carbon source. The N-P@FCM shows highest quantum yield among the three. The N-P@FCM was explored for alkaline pH sensing and it shows a quenching of fluorescence in the pH range 09-14. The sensing behaviour shows reversibility and high selectivity. Further, the sensor was also investigated for their biocompatibility and hence employed as a promising multicolour probe for cancer cell imaging. The generality in cell imaging was investigated by flow cytometry.The Chapter 6 shows the use of Rosa indica derived heteroatom i.e. nitrogen and sulfur co-doped rose carbon dot (N-S@RCD) for dual fluorescence based sensing of multi-analytes. The limitation of versatility in green precursors is addressed by exploring formation of carbon dots with respect to the contents of green precursor i.e. Rosa indica. The synthesis mechanism is elucidated by analyzing the precursors and intermediates at different interval. Moreover, The N-S@RCD shows interesting fluorescence turn “off-on-off” response towards S2- and Au3+ with limit of detection (LOD) 92.4 nM and 63.1 nM, respectively. To the best of our knowledge, this is the first report on direct “Turn-on” of c-dots with S2- ions without any intermediate c-dot–quencher complex. Further, the bioimaging and flow cytometry studies revealed the potential of live cell imaging, intracellular sensing and cytocompatibility of N-S@RCD. The hemocompatibility of N-S@RCD was also investigated for potential in vivo applications. Moreover, the “off-on-off” fluorescence behaviour was used to construct molecular logic gates which simulate single input logic “YES” and multi-input “INHIBIT” logic system.In an attempt to find a universally identical biomass for c-dot synthesis, the use of cell lines in envisioned in Chapter 7. The cervical cancer cell line HeLa was employed for c-dot synthesis as a uniform and expeditious regenerative green source and presented as a proof of concept for using cell lines as carbon precursors. The area of future development in c-dot based bio imaging probes is selective cellular targeting which is scarcely reported. The HeLa derived whole cell carbon-dot (WC-CD) shows exciting specific subcellular staining of endoplasmic reticulum adding another feather in the cap of cellular precursors for c-dot synthesis. The ER-tracking WC-CD shows generality over large population, high specificity and good compatibility for counter staining with other organelle tracker. Moreover, WC-CD also shows excellent staining capability towards 3D tumor spheroidwhich simulate in vivo environment. This work is expected to outset the attention towards cell line based precursors and selective cellular targeting using c-dots. The Chapter 8 outlines the future perspective of this work.