The SCP-RICM assay employs the measurable surface energy (or adhesive work W_adh) of a micrometer-sized polymeric sphere (soft colloidal probe, SCP) interacting with a glass chip using reflection interfer-ence contrast microscopy (RICM). Depending on those two interacting surfaces' nature and functional-ization, the SCP will deform, creating a contact area with the hard glass chip. This contact area is clearly distinguishable from the sphere’s interference ring pattern and can be measured. The adhesive surface energy W_adh can be calculated from the size of the contact area.
An immobilization can be overcome by choosing a two-component analyte-dependent interaction, here presented for the copper (Cu) detection.
The detection of Cu was chosen as a proof-of-concept system. However, detecting metal ions is an essential endeavor because, in excessive amounts, they present a severe threat to health and the environment. The copper-dependent interaction of the yeast chaperones yCox17 (also Cox17) and ySco1 (also Sco1) were chosen as the two-component analyte-dependent interaction. The chaperones partic-ipate in vivo in the formation of the electron transport chain of S. cerevisiae and interact in the mito-chondrial inner membrane to transfer one Cu(I) ion from Cox17 to Sco1.
It was necessary to immobilize one protein to the SCPs and one to the chip surface, to transfer the copper chaperones' interaction into the SCP-RICM assay core detection components.
The unique self-assembling characteristics of the class I hydrophobin Ccg-2 from N. crassa were used to immobilize one interaction partner to the chip surface. Class I hydrophobins are known for the formation of re-sistant and uniform layers at hydrophilic/hydrophobic interfaces.
Initial SCP-RICM assay measurements with Sco1Δ95_a-SCPs and the Cox17_c-chips indicate that copper detection using the proposed mechanism is possible (Figure 39-3). Measurements can be differentiated between 0 and 0.1 mM Cu(I) concentration in solution. Further screening of concentrations be-low 0.1 mM is still necessary. The presented proof-of-principle system for the indirect detection of copper shows copper-dependent behavior. These positive results give rise to many more options to use the SCP-RICM assay as an indirect detection system. The application range of the SCP-RICM assay could be enlarged for different analytes such as other heavy metals, bacteriophages, biomarkers, et cetera, and is relevant for fields from medicine to environmental monitoring.:TABLE OF CONTENT
Table of Content I
List of Figures VII
List of Tables IX
List of Abbreviations XI
1 Introduction 1
1.1 Biosensors 1
1.2 Analytical Detection Methods: Copper 2
1.3 SCP-RICM Assay 3
1.3.1 Sensor Chip Surface 4
1.3.2 Soft Colloidal Probes 5
1.3.3 Reflection Interference Contrast Microscopy 6
1.4 Hydrophobins 9
1.4.1 Structure and Functions of Hydrophobins 9
1.4.2 Ex vivo Applications of Hydrophobins 11
1.4.3 Class I Hydrophobin: Ccg-2 12
1.5 Mitochondrial Respiratory Chain 14
1.5.1 Copper Transport in Yeast 14
1.5.2 S. cerevisiae Sco1 protein 18
1.5.3 S. cerevisiae Cox17 protein 21
1.6 SCP-RICM Assay for Copper Detection 23
1.7 Aim of the Study 24
2 Materials and Methods 25
2.1 Laboratory Equipment 25
2.1.1 Devices 25
2.1.2 Chemicals 26
2.1.3 Consumables 28
2.1.4 Antibodies 29
2.1.5 Enzymes 30
2.1.6 Molecular Weight Standards 30
2.1.7 DNA Oligonucleotides 31
2.1.8 Plasmids and Vectors 32
2.2 Microorganisms 33
2.2.1 Strains 33
2.2.2 Cultivation of Microorganisms 34
2.2.3 Preparation of Electrocompetent E. coli Cells 36
2.2.4 Preparation of E. coli Glycerol Stocks 36
2.3 Protein Design 37
2.4 Molecular Cloning Methods 38
2.4.1 Vector Template Preparation 38
2.4.2 Agarose Gel Electrophoresis 40
2.4.3 DNA Extraction from Agarose Gels 41
2.4.4 Polymerase Chain Reaction 41
2.4.5 DNA Restriction Digest 42
2.4.6 DNA Dialysis 43
2.4.7 Ligation of DNA Fragments 43
2.4.8 Isolation of DNA from E. coli 44
2.4.9 DNA Sequencing 45
2.4.10 Transformation of E. coli via Electroporation 45
2.5 Protein Detection and Quantification 46
2.5.1 SDS PAGE 46
2.5.2 Coomassie Staining 50
2.5.3 Western Blot Analysis 51
2.5.4 Immunological Detection 51
2.5.5 Protein Quantification: Lowry Assay 52
2.5.6 Protein Quantification: Bradford Assay 53
2.5.7 Protein Quantification: NanoDrop Measurement 53
2.6 Protein Purification and Storage 54
2.6.1 Expression Analysis of Recombinant Proteins 54
2.6.2 Solubility Analysis 54
2.6.3 Protein Purification by Ni2+ Affinity Chromatography 55
2.6.4 Quantification of Purified Proteins 64
2.6.5 Dialysis of Purified Proteins 65
2.7 Glass Surface Functionalization 65
2.7.1 Glass Surface Preparation 66
2.7.2 Hydrophobin and Fusion Protein-Based Coating 66
2.7.3 Contact Angle Measurement 67
2.7.4 DRoPS Test 67
2.7.5 Atomic Force Microscopy 67
2.8 SCP Functionalization 68
2.8.1 Functionalization of SCPs with Proteins 68
2.8.2 Validation of SCP Functionalization with FITC Staining 69
2.9 SCP-RICM Assay and Its Analysis 69
3 Results 73
3.1 Generation of Recombinant Fusion Proteins 73
3.1.1 Sco1 and Sco1∆95 73
3.1.2 Cox17 84
3.1.3 Ccg-2 88
3.1.4 Overview: Optimization of Expression and Purification of Recombinant Proteins 90
3.2 His-Tag Cleavage 92
3.3 Chip Surface Functionalization 94
3.3.1 Optimization of the Glass Chip Preparation 94
3.3.2 Macroscopic Properties of the Functionalized Chip Surface 95
3.3.3 AFM Measurements 102
3.3.4 Theoretical Package of Hydrophobin Ccg-2 on the Chip Surface 103
3.4 SCP Functionalization 104
3.4.1 SCP Functionalization and FITC Staining 104
3.4.2 Theoretical Package of Proteins on SCPs 106
3.5 SCP-RICM Assay 107
4 Discussion and Further Prospectives 113
4.1 Discussion: SCP-RICM Assay and Protein-Protein Interaction 113
4.2 Outlook and Further Prospects 119
4.2.1 Heterologous Protein Expression and Purification: Methods, Cleavage and Refolding 119
4.2.2 Further Analysis of Chip Surface Functionalization 124
4.2.3 Alternative Chip Surface Functionalization Methods 126
4.2.4 SCP-RICM Assay: Data Acquisition and Evaluation 128
4.2.5 SCP-RICM Assay: Copper Detection 130
4.2.6 Exploiting the SCP-RICM Assay using Protein-Protein Interactions 131
4.2.7 Exploiting the SCP-RICM Assay with Alternative Interactions 133
5 Summary 137
6 Bibliography 141
7 Appendix 165
7.1 Sequences of Protein Constructs 165
7.1.1 Sequences of the Protein Construct Cox17_a 165
7.1.2 Sequences of the Hydrophobin-Cox17 Fusion Protein Cox17_b 165
7.1.3 Sequences of the Hydrophobin-Cox17 Fusion Protein Construct Cox17_c 166
7.1.4 Sequences of the Protein Construct Sco1_a and Sco1Δ95_a 167
7.1.5 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_b and Sco1Δ95_b 169
7.1.6 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_c and Sco1Δ95_c 171
7.1.7 Sequences of the Hydrophobin Ccg-2 173
7.2 pET-28b(+): Plasmid Map 173
7.3 Nickel Removal During Dialysis 175
7.4 DGR Assay 176
7.5 SCP diameter 179
Acknowledgements 181
Declaration of Authorship 183
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:75771 |
| Date | 24 August 2021 |
| Creators | Hannusch, Lisa |
| Contributors | Rödel, Gerhard, Ostermann, Kai, Pompe, Tilo, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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