Title Page 2

ABSTRACT 5

Contents 8

Chapter 1. Introduction 19

1.1. Functional hybrid nanostructures: Advanced strategies for sensing applications 19

1.1.1. Synthesis of hybrid nanostructured materials 21

1.1.2. Hybrid nanostructure-based sensing platforms 24

1.2. Nanozymes: Promising alternatives for enzyme-based sensing approaches 28

1.2.1. Nanozymes classification 29

1.2.2. Strategies in nanozyme-based sensing applications 30

1.3. Overview of this dissertation 32

References 36

Chapter 2. An Efficient Surface Enhanced Raman Scattering (SERS)-Based Sensor for Detecting Thiol-Containing Molecules Using Silver-Polydopamine-Copper Hybrid Nanoflowers 40

2.1. Introduction 40

2.2. Experiment 42

2.2.1. Synthesis of silver-polydopamine-copper phosphate nanoflowers (Ag-PDA-Cu NFs) 42

2.2.2. Characterizations 42

2.2.3. Construction of a SERS chip based on Ag-PDA-Cu NFs and PDA-Cu NFs 43

2.2.4. SERS detection of thiol-containing molecules 43

2.3. Results and Discussion 44

2.3.1. Fabrication of Ag-PDA-Cu NFs and their SERS availability 44

2.3.2. Characterization of Ag-PDA-Cu NFs 46

2.3.3. Sensing properties of Ag-PDA-Cu NFs for thiocholine 53

2.3.4. Sensing properties of Ag-PDA-Cu NFs for benzenthiol 58

2.4. Conclusion 62

References 63

Chapter 3. Electrochemistry-Based Strategy for Simultaneous Detection of Dopamine and Epinephrine Utilizing Highly Conductive Peroxidase-Mimicking Ce-MoS₂ Nanoflowers 66

3.1. Introduction 66

3.2. Experiment 69

3.2.1. Preparation of MOS₂ and Ce-MoS₂ NFs 69

3.2.2. Evaluation of peroxidase-like activity of Ce-MoS₂ NFs 70

3.2.3. Fabrication of Ce-MoS₂ NFs/screen-printed electrode (SPE) and electrochemical analysis 71

3.3. Results and Discussion 71

3.3.1. Structure and morphology of synthesized Ce-MoS₂ NFs 71

3.3.2. Peroxidase-like activity of Ce-MoS₂ NFs 76

3.3.3. Electrochemical performance of Ce-MoS₂ NFs 78

3.3.4. Electrochemical simultaneous detection of dopamine and epinephrine 82

3.4. Conclusion 84

References 86

Chapter 4. Paper-Based Colorimetric Sensing Platform for Detecting Neurotransmitters Using Copper Single-Atom Aerogels 88

4.1. Introduction 88

4.2. Experiment 92

4.2.1. Preparation of nitrogen-rich carbon dots (CDs) 92

4.2.2. Preparation of CDs-induced copper single-atom aerogels (Cu-SANs) 92

4.2.3. Characterizations of Cu-SANs 92

4.2.4. Enzymatic assays 93

4.2.5. Colorimetric detection of neurotransmitters using Cu-SANs 95

4.2.6. Detection of neurotransmitters by Cu-SANs-incorporated paper-based microfluidic device 96

4.3. Results and Discussion 97

4.3.1. Characterizations of Cu-SANs 97

4.3.2. Evaluation of enzymatic activities of CuSANs Paroxidase-like activity 102

4.3.3. Colorimetric detection of neurotransmitters using Cu-SANs Detection of acethylcholine employing peroxidase-like property of Cu-SANs 113

4.3.4. Detection of neurotransmitters on Cu-SANs-incorporated paper-based chip Paper-based chip design and optimization 118

4.4. Conclusion 124

References 125

Chapter 5. A Novel Carboxylesterase-Mimicking Cobalt MOFs for Dual-Mode Sensing of Carbaryl 130

5.1. Introduction 130

5.2. Experiment 132

5.2.1. Synthesis of cobalt MOFs (ZIF-67) 132

5.2.2. Characterizations of ZIF-67 133

5.2.3. Carboxylesterase-like activity of ZIF-67 133

5.2.4. Carbaryl detection using ZIF-67 134

5.3. Results and Discussion 135

5.3.1. Characterizations of ZIF-67 135

5.3.2. Evaluation of enzymatic activities of ZIF-67 140

5.3.3. Colorimetric detection of carbaryl using ZIF-67 146

5.4. Conclusion 150

References 151

Chapter 6. Overall conclusion and future aspects 154

6.1. Overall conclusion 154

6.2. Future aspects 155

CURRICULUM VITAE 160

Table 2.1. Comparison of detection limit value of various SERS probes for thiol groups... 62

Table 3.1. Comparison of kinetic parameters of Ce-MoS₂ NFs-based peroxidase-like... 78

Table 3.2. Comparison of electroanalytical performances of dopamine and epinephrine ... 84

Table 4.1. Kinetic parameters of peroxidase activity of Cu-SANs, other copper-based... 105

Table 4.2. Kinetic parameters of laccase activity of Cu-SANs, other laccase mimics, and... 110

Table 4.3. Comparison for the sensitivity assay of this acetylcholine sensing system and... 116

Table 4.4. Comparison for the sensitivity assay of this epinephrine sensing system and... 118

Table 5.1. Comparison of kinetic parameters of ZIF-67, natural CES, and other reported... 144

Table 5.2. Detection precision of ZIF-67-mediated dual sensing platform toward carbaryl... 150

Hybrid nanomaterials have emerged as transformative tools in the realm of biosensing, offering unprecedented enhancements in sensitivity, selectivity, and multifunctionality. These innovative materials facilitate the development of sophisticated biosensors capable of detecting a wide range of analytes with high precision, making them invaluable in fields such as medical diagnostics, environmental monitoring, and food safety. Inspired from the significant advancements and future prospects of using hybrid nanomaterials in biosensing applications, this dissertation focusses on development of different hybrid nanostructures with unique physical, chemical, and biological behaviors which were further employed for integrating into sensing platforms, aiming to enhance the diagnostic efficiency.

In the first study, a Surface-Enhanced Raman Scattering (SERS)-based chip was developed using silver nanoparticle-incorporated polydopamine (PDA)-copper hybrid nanoflowers for the ultrasensitive detection of thiol-containing molecules. The SERS signal, a cornerstone of this detection method, was generated through the intricate interaction between the silver nanoparticles and the sulfur anions present in the thiol groups of the target molecules. This interaction significantly amplified the Raman signal, enabling the precise and ultrasensitive detection of even trace amounts of thiol-containing analytes. The hybrid nature of the nanoflowers played a crucial role in this process, as it facilitated a more effective and uniform distribution of AgNPs, enhancing the overall SERS performance.

The next study presents the synthesis of Ce-MoS₂ nanoflowers, which exhibit remarkable conductivity and peroxidase-like activity (POD). These uniquely prepared nanoflowers are designed to catalyze the reduction of hydrogen peroxide (H₂O₂) as well as the oxidation of epinephrine (EP) and dopamine (DA). The incorporation of cerium into the MoS₂ structure enhances the material's catalytic properties and electrical conductivity, making it highly effective for electrochemical applications. By applying this Ce-MoS₂ material onto the surface of a screen-printed electrode, a highly sensitive platform for monitoring and discriminating between the analytes EP and DA was fabricated. The modified electrode demonstrated excellent performance in catalyzing the redox reactions of both EP and DA, allowing for their effective and simultaneous detection.

Another study focused on the development of aerogel-based copper single-atom nanozymes (Cu-SANs) exhibiting dual enzymatic activities of peroxidase and laccase. Leveraging the abundant single-atom active sites, Cu-SANs demonstrated superior catalytic performance in bothenzymatic reactions. This dual functionality was applied for the sensitive and selective detection of acetylcholine (ACh) and epinephrine (Epi). ACh was quantified using the peroxidase activity of Cu-SANs combined with acetylcholinesterase and choline oxidase, while Epi was directly monitored through the laccase activity of Cu-SANs. The study also integrated these nanozymes into a paper-based microfluidic device, enabling simultaneous detection of ACh and Epi via distinct colorimetric responses, showcasing the practical potential of Cu-SANs in developing portable and efficient diagnostic tools.

Lastly, ZIF-67 was synthesized and identified as a novel carboxylesterase mimic. This material was straightforwardly prepared using a co-precipitation method involving a cobalt precursor and 2-methylimidazole organic linkers. The enzymatic activity of ZIF-67 was rigorously confirmed through the hydrolysis of p-nitrophenol acetate (p-NPA) and p-nitrophenol butylate (p-NPB). Utilizing this unique property, ZIF-67 enabled the efficient detection of carbaryl, a pesticide, through dual sensing modes - colorimetry and fluorometry. The reaction between carbaryl and ZIF-67 produced 1-naphthol as a by-product, which exhibited intrinsic fluorescence and interacted with the chromogenic indicator DDQ, forming a complex that displayed a red-brown color and strong absorbance at 390 nm. This dual-mode sensing capability not only underscores the versatility and efficiency of ZIF-67 but also highlights its potential for practical applications in environmental monitoring and pesticide detection. The successful synthesis and application of ZIF-67 demonstrate the significant promise of metal-organic frameworks in the development of advanced biosensing technologies.