Protein assisted nanoparticle assembly and protein-nanocomposite fabrication

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Protein assisted nanoparticle assembly and protein-nanocomposite fabrication

Dissertation zur Erlangung des akademischen

Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von

Tuan Anh Pham

an der

Mathematisch - Naturwissenschaftliche Sektion Fachbereich Chemie

Tag der mündlichen Prüfung: 17. Oktober 2016 1. Referent: Prof. Dr. Helmut Cölfen

2. Referent: Prof. Dr. Stefan Mecking

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-372200

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Preface

Diese Arbeit ist in dem Zeitraum zwischen Juni 2012 und Januar 2016 entstanden und war im Rahmen des Teilprojekts B3 ‘Kodierte Selbstorganisation von Nanopartikeln und Mechanismen der nicht-klassischen Kristallisation‘. Die Arbeit wurde durch das Kompetenznetzwerk für Funktionelle Nanostrukturen finanziell unterstützt. Das Projekt wird durch die protein-kodierte Nanopartikelassemblierung inspiriert, welche zur Bildung von Überstrukturen bestehend aus geordneten und gegenseitig ausgerichteten Nanopartikeln führen kann. Ein bekanntes Beispiel derartiger Überstrukturen ist ein Mesokristall, beispielsweise im Seeigelstachel. Dieses Projekt wurde von den Forschungsgruppen aus Konstanz und Freiburg bearbeitet. Die Nanopartikelsynthese und die Untersuchung der Nanopartikelassemblierung wurden in Konstanz durchgeführt. Die Freiburger Arbeitsgruppe ist für die Proteinsynthese verantwortlich.

Das Projekt ist durch eine sehr enge Zusammenarbeit zwischen beiden Arbeitsgruppen ausgezeichnet. So hängen die Fortschritte in Konstanz direkt mit der erfolgreichen Proteinmodifikation in Freiburg und vice versa zusammen. So ergab sich eine kleine zeitliche Unterbrechung im Projektverlauf im Sommer 2014 aufgrund einer Personalumstellung in Freiburg.

Viele Menschen haben mich während meiner Doktorarbeit begleitet und unterstützt. Aus diesem Grund möchte ich an dieser Stelle eine herzliche Danksagung an sie ausrichten.

Zuerst, möchte ich mich bei Prof. Dr. Helmut Cölfen für sein Vertrauen in meine Person und meine Arbeit sowie seine Unterstützung und Betreuung bedanken. Die fruchtbaren Diskussionen mit ihm haben mir über die schwierigen Zeiten hinweg geholfen. Seine positive Einstellung und scharfsinnige Beobachtungen haben mir immer wieder gezeigt, die Arbeit mit Zuversicht anzugehen. Darüber hinaus, möchte ich mich für seine Bemühungen, mich bei schriftlichen Arbeiten trotzt der Jetlags und der langsamen Internetverbindungen zu unterstützen, bedanken. Ich weiß Helmut sowohl als Mentor, als auch als private Person sehr zu schätzen. Daher möchte ich ihm an dieser Stelle für die letzten drei und einhalb Jahre sagen:

‘‘Ich danke Dir‘‘.

Ich möchte mich genauso herzlich bei Prof. Dr. Stefan Mecking für die Übernahme des zweiten Gutachtens bedanken. Er hat sich immer die Zeit gefunden, meine Präsentationen im Rahmen der Jahresgespräche aufmerksam zu verfolgen. Dabei sind seine Ratschläge und Fragen immer sehr inspirierend und eröffnen neue Perspektive für die Arbeit. Seine Bereitschaft auf meinem Wunsch die Arbeit in absehbarer Zeit zu bewerten, weiß ich besonders zu schätzen, gerade auch aufgrund seines vollen Kalenders.

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Prof. Dr. Christine Peter gebührt mein Dank für die Übernahme des Prüfungsvorsitzendes bei der mündlichen Prüfung.

Einen besonderen Dank geht natürlich an Andreas Schreiber und Stefan Schiller aus Freiburg.

Die regelmäßige Lieferung von Proteinen und die endlose Proteinmodifikation durch Andreas Schreiber haben zum Erfolg der Arbeit und des Projektes beigetragen. Ich habe durch diese enge Zusammenarbeit und meinen Aufenthalt in Freiburg sehr viel dazu gelernt. Außerdem, möchte ich mich bei Marius Schmid für die angenehme Zusammenarbeit bei der AF4 Messung und der Simulation sowie bei Elena Sturm für die Simulation der Nanopartikelorientierung bedanken.

Des Weiteren stellen die Mitglieder der Arbeitsgruppe Cölfen, Gebauer und Sturm eine besondere Hilfe während meiner Doktorarbeit dar. Ich fühle mich sehr verbunden mit jeder von ihnen. Es war mir eine Freude mit ihnen zusammenzuarbeiten sowie mit ihnen ein Büro und den Laborräumen teilen zu dürfen. Sie bieten eine positive Umgebung um sich wissenschaftlich und menschlich weiterzuentwickeln. Dadurch sind wir mehr als Arbeitskollegen geworden. Die Mitarbeiterpraktikanten und wissenschaftliche Hilfskräfte haben viele synthetische Arbeiten und Messungen für mich übernommen und verdienen daher auch meinen herzlichen Dank.

Für spezielle Messungen möchte ich mich bei Dr. Marina Krumova für die Einführung und Hilfestellung bei TEM, Holger Reiner für die HRTEM-Aufnahmen, Dr. Markus Drechsler an der Uni Bayreuth für die KryoTEM-Aufnahmen, Matthias Hagner für die SEM Einführung und die Herstellung der FIB-Schnitte, Maria Siglreitmeier für die XRD Messung, Masoud Farhadi Khouzani für die Hilfe bei der IR Messung, Philipp Erler und seine Hiwis für die SQUID Messungen, Rose Rosenberg für die AUZ Einführung und Dirk Haffke für die technische Hilfestellung.

Und zum Schluss möchte ich mich bei meiner Familie und Freunden bedanken, die trotz der weiten Entfernung immer zu mir gehalten haben. Die viele Telefonate und Skype-Gespräche haben mir immer die Kraft gegeben weiterzumachen.

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Table of content

1. Introduction and Scope of the thesis …..………... 1

2. Fundamentals 2.1 Nanoparticle assembly ………. 8

2.2 Nanoparticles with different physical properties 2.2.1 Superparamagnetic nanoparticles ……… 10

2.2.2 Surface plasmon resonance ……….. 15

2.2.3 Semiconductor nanoparticles ……….. 20

2.3 Protein structures 2.3.1 Hemolysin coregulated protein 1 – Hcp1 structure ……….. 24

2.3.2 Elastin like protein – ELP structure ……… 28

2.4 Analytical ultracentrifugation ……… 29

2.5 Asymmetrical flow-field-flow fractionation ………..………. 33

2.6 Hcp1/ELP – nanoparticle assembly ……… 35

3. ELP induced nanoparticle assembly 3.1 Introduction ……… 40

3.2 Experimental 3.2.1 Chemicals………. 41

3.2.2 Nanoparticle syntheses ……… 41

3.2.3 Protein preparation ………. 43

3.2.4 Sample preparation ……… 43

3.2.5 Analytical methods ………. 43

3.3 Results and Discussion ……….. 44

3.4 Conclusion ……….. 51

4. Template assisted nanoparticle assembly 4.1 Introduction ……….. 54

4.2 Experimental

4.2.1 Chemicals ……… 55

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4.2.2 Nanoparticle syntheses ……… 55

4.2.3 Protein preparation ………. 56

4.2.4 Sample preparation ……… 56

4.2.5 Analytical methods ………. 57

4.3 Results and Discussion ……….. 57

4.4 Conclusion ……….. 64

5. Hcp1 induced gold and silver nanoparticle assembly 5.1 Introduction ……….. 67

5.2 Experimental 5.2.1 Chemicals ……… 68

5.2.2 Nanoparticle syntheses ……… 68

5.2.3 Protein preparation ………. 69

5.2.4 Sample preparation ……… 69

5.2.5 Analytical methods ………. 70

5.3 Results and Discussion 5.3.1 Hcp1_cys3 induced assembly of 10 nm gold nanoparticles ……… 71

5.3.2 Hcp1_Q54C induced assembly of 10 nm gold nanoparticles ………... 84

5.3.3 Summary ………. 86

5.3.4 Hcp1_Q54C induced assembly of 4 nm gold nanoparticles ……… 87

5.3.5 Hcp1_cys3 induced assembly of 10 nm silver nanoparticles ….. 97

5.4 Conclusion ……… 101

6. Characterization of nanoparticle-protein hybrid structures using analytical ultracentrifugation and asymmetrical flow-field-flow fractionation 6.1 Introduction ……… 104

6.2 Experimental

6.2.1 Chemicals ……… 105

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6.2.2 Protein preparation ………. 106

6.2.3 Sample preparation ………. 106

6.2.4 Analytical methods ……….. 106

6.3 Results and Discussion ……… 107

6.4 Conclusion ……… 117

7. Hcp1 induced cadmium- and zinc sulfide nanoparticle assembly 7.1 Introduction ……… 120

7.2 Experimental 7.2.1 Chemicals ……… 121

7.2.2 Nanoparticle syntheses ………. 121

7.2.3 Protein preparation ………. 122

7.2.4 Sample preparation ………. 122

7.2.5 In-situ synthesis of nanoparticles inside of the Hcp1_Q54C protein cavity .……….. 123

7.2.6 Analytical methods ……….. 123

7.3 Results and Discussion ……… 124

8. Hcp1_cys3 induced magnetite and cobalt ferrite nanoparticle assembly and nanocomposite materials 8.1 Introduction ……… 131

8.2 Experimental 8.2.1 Chemicals ………... 132

8.2.2 Nanoparticle syntheses ………. 132

8.2.3 Protein preparation ……… 134

8.2.4 Sample preparation ………. 134

8.2.5 Analytical methods ……….. 135

8.3 Results and Discussion 8.3.1 Hcp1_cys3 induced assembly of magnetite nanoparticles …… 136

8.3.2 Hcp1_cys3 induced assembly of cobalt ferrite nanoparticles ………

………. 149

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8.4 Conclusion ……… 163

9. Conclusion and Outlook ……….……….……….. 166

10. List of figures ………. 169

11. List of schemes and tables ……… 178

Bibliography

Appendix

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VII

Summary

The trend of miniaturization requires increasingly smaller components with special properties, which can be fulfilled by nanoparticles of different inorganic materials. Furthermore, the assembly of nanoparticles provides a great opportunity to produce materials on different length scale. Nature as a very important inspiration source shows different examples of inorganic and organic assembly structures. The magnetotactic bacteria ‘synthesize’ nanoparticles e.g. iron oxides and order them in an assembly structure comprising of well-oriented nanoparticles for application as a natural compass. Here, the specific interactions of the proteins, which surround the iron oxide nanoparticle, are responsible for the linear nanoparticle arrangement in these bacteria. Another example of assembly structure is the tobacco mosaic virus, which shows a linear stacking of the coat proteins resulting in the rod-like morphology of this virus. In this case the defined protein interactions also contribute to the structure formation. This kind of specific interaction can also be found between the DNA strands leading to the double strand structure.

The formation of this DNA superstructure was successfully exploited to organize DNA- functionalized gold nanoparticles into structures of one- to three dimensions. Since bio macromolecules such as proteins and DNA show very specific and well-defined intermolecular interactions, they can be helpful to assemble nanoparticles in an ordered manner. Inspired by these examples, we provide here a route to utilize protein structures as a guiding component in the inorganic nanoparticle assembly to obtain structures with oriented inorganic building units and composite materials with enhanced properties.

The Hcp1 protein as the starting point of this work has a toroid shape with an inner diameter of 4 nm and outer diameter of 9 nm as well as a height of 4.4 nm. The Hcp1 structure is composed of six identical monomer subunits giving the toroid morphology. Two Hcp1 mutants with cysteine moieties on the upper and lower rim of the toroid, and inside of the toroid cavity are available for the investigation. In the first part of this work, the nanoparticle synthesis focuses on the materials with optical properties such as surface plasmon resonance (gold and silver nanoparticles), exciton absorption (cadmium sulfide and zinc sulfide quantum dots), and magnetic properties (superparamagnetic ferrite nanoparticles). At the same time, the nanoparticle size is chosen to be in the same range of the Hcp1 protein structure, to ensure the uniformity of the building units for the subsequent assembly process. After the Hcp1 attachment to the nanoparticle forming a nanoparticle-protein bioconjugate, the condition for an oriented assembly of the bioconjugates into larger structures up to micrometer length scale should be determined. This represents the most important and challenging part in this work considering the scarce knowledge about the Hcp1 protein induced nanoparticle assembly.

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Finally, the protein-nanocomposite materials from the assembly process are characterized regarding their optical and magnetic properties to establish a relation between the nanoparticle structure and the material properties. The takeaway from this work can be seen as the first step towards the formation of new protein-nanocomposite materials using a bio-inspired approach

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Zusammenfassung

Die zunehmende Tendenz der Miniaturisierung verlangt immer kleiner werdende Bauteile, die spezielle Eigenschaften benötigen. Diese Anforderung können viele anorganische Nanopartikel erfüllen. Darüber hinaus bietet der Mechanismus der Assemblierung eine hervorragende Möglichkeit, aus diesen Nanopartikeln Strukturen bzw. Materialien auf verschiedener Längenskala herzustellen. Die Natur als eine sehr wichtige Inspirationsquelle weist verschiedene Beispiele für anorganische und organische Assemblierungsstrukturen auf. Die magnetotaktische Bakterien ‘synthetisieren‘ Nanopartikel wie Eisenoxide und ordnen sie in einer Assemblierungsstruktur an, welche aus orientierten, anorganischen Nanopartikeln besteht, um diese als ein natürlicher Kompass zu verwenden. Dabei sind die spezifischen Wechselwirkungen zwischen den Proteinen, die die Eisenoxid-Nanopartikel umgeben, entscheidend für die lineare Nanopartikel-Anordnung in diesen Bakterien. Ein weiteres Beispiel ist das Tabakmosaikvirus mit einer stäbchen-artigen Struktur, welche durch eine lineare Assemblierung von Proteinen entsteht. In diesem Fall spielen die definierten Proteinwechselwirkungen eine wichtige Rolle bei der Strukturbildung. Derartige spezifische Interaktion ist auch bei den DNA Strängen zu finden, wodurch die DNA Helix-Struktur zustande kommt. Die Bildung von dieser DNA-Überstruktur wurde erfolgreich verwendet um ein- bis drei-dimensionale Assemblierungsstrukturen aus DNA funktionalisierten Gold Nanopartikeln herzustellen. Es ist nun ersichtlich, dass Bio- Makromoleküle wie Proteine und DNA wohl-definierte intermolekulare Wechselwirkungen aufweisen, die bei der geordneten Assemblierung von Nanopartikeln nützlich sein können.

Inspiriert von diesen Beispielen, wird diese Arbeit einen Weg aufzeigen, wie Proteinstrukturen die Assemblierung von Nanopartikeln steuern können um daraus Überstrukturen aus orientierten, anorganischen Bausteinen und Kompositmaterialien mit verbesserten Eigenschaften aufzubauen.

Das in dieser Arbeit verwendete Hcp1 Protein, welches der Ausgangspunkt dieser Arbeit darstellt, hat die Struktur eines Torus (Donut-ähnlich) mit einem inneren Durchmesser von 4 nm, einem Außendurchmesser von 9 nm und eine Höhe von 4.4 nm. Das Hcp1 Protein besteht aus sechs, identischen Monomer-Untereinheiten. Zwei Hcp1 Mutanten mit Cysteinmodifikationen am oberen und unteren Rand des Torus, und an der Innenseite der Torus-Kavität stehen dabei zur Verfügung. Im ersten Teil der Arbeit, steht die Synthese von Nanopartikeln mit optischen wie Oberflächenplasmonresonanz (Gold und Silber Nanopartikel), Exziton-Absorption (Cadmiumsulfid und Zinksulfid Quantenpunkte) sowie magnetischen Eigenschaften (superparamagnetische Ferrit Nanopartikel) im Vordergrund. Dabei liegt die

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Größe der synthetisierten Nanopartikeln im gleichen Bereich wie die Größe der Hcp1 Proteinstruktur, um eine Einheitlichkeit der Bausteine für die anschließende Assemblierung sicherzustellen. Die Nanopartikel und das Hcp1 Protein bilden Biokonjugate, welche durch die Anbindung des Proteins an die Nanopartikel entstehen. Die Bedingung für eine gerichtete Assemblierung von diesen Biokonjugaten zu größeren Strukturen, möglichst auf der Mikrometerskala, soll ermittelt werden. Angesichts der mangelnden Erkenntnisse über die Hcp1 gesteuerte Nanopartikelassemblierung stellt diese Untersuchung den wichtigsten und zugleich den anspruchsvollsten Teil der Arbeit dar. Zum Schluss werden die aus der Assemblierung entstandenen Protein-Nanokompositmaterialien hinsichtlich ihrer optischen und magnetischen Eigenschaften untersucht. Hierbei soll möglichst eine Beziehung zwischen der Struktur und den Materialeigenschaften hergestellt werden. Die aus dieser Arbeit neu gewonnenen Erkenntnisse können als Wegbereiter für die Bildung von neuen Protein-Nanokompositmaterialien durch einen bioinspirierten Ansatz angesehen werden.

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Chapter 1 – Introduction and Scope of the thesis

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Chapter 1 Introduction

In the past decades ‘nanoparticle’ (NP) becomes one of the most important keywords in the research field (over 200 000 hits on the Web of Science website in 2016), and in several industrial sectors. Their unique properties have attracted great interest in physics, chemistry and biology¹. Their unique properties resulted in a breakthrough of nanoparticle utilization in biosensing², imaging³, therapeutical applications⁴, electronic devices with high performance⁵as well as consumer products such as sun cream, deodorant and clothing⁶. The highly demanded nanoparticle system, thus, offers a tremendous potential for interdisciplinary research works leading to novel, multifunctional structures and materials, which will contribute to the design of new consumer products. Furthermore, the accumulation of nanoparticles in the form of superstructures and assembly structures offers the opportunity to create nanoparticle–based structures of different dimensions and sizes. These new structures containing well-ordered nanoparticles as building units display new collective properties due to the assembled nanoparticles, which are distinct from those of the single NPs. For example, nature exploits the directional alignment and arrangement of anisotropic or isotropic subunits, through which unique materials with enormous morphological diversities such as opal containing periodically packed silica spheres of nm-size range⁷, composite materials such as tooth and bone comprising of inorganic nano-needles and nano-platelets embedded in a soft organic matrix⁸, can be produced. The organization of the nanoparticles within these structures gives rise to the typical colored appearance of the natural opal depending on the viewing angle. The high toughness of the composite materials like bone and tooth emerges from the well-defined organization of the components in a composite in the form of interlaced bricks separated by soft layers leading to a better stress-redistribution and crack-stopping mechanisms. In contrary to the composite materials, the bare inorganic component itself is quite brittle. Also, artificial assembly structures of, for example, metal nanoparticles show electrical⁹, optical and magnetic¹⁰ properties that are different from those of the individual NPs and the corresponding bulk material. The above examples show that the assembly of nanoparticles in an ordered manner results in unique material properties, which exceed the property of single nanoparticles. In order to achieve these structures two synthetic routes of bottom-up and top-down approaches are used. In the top-down approach nanostructures can be produced by lithography techniques, which unfortunately are limited to structures above 20 nm and two-dimensional structures¹¹. In the bottom-up approach the self-assembly of structures of few nanometers can create one- to

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three-dimensional materials of nm- to mm-scale range¹². Especially the flexibility to obtain different materials of specifically designed morphologies and properties makes this approach very promising for the synthesis of advanced nanostructures. Thus, the obtained materials range from open, regular three-dimensional structures of mm-size¹³ to network structures of nanowires¹⁴, to magnetic m-sized opal structures by superparamagnetic magnetite NP assembly¹⁵ and photonic materials¹⁶. The self-assembly of nanoparticles in the bottom-up approach is divided into static and dynamic processes. The static self-assembly is defined as an energy consuming process, which includes NP systems located in their local equilibrium. The resulting structures are stable after the self-assembly. Therefore, the main research works also focus on this process due to a considerable library of available assembled structures and materials¹⁷. But not only artificial systems show nanoparticles assembly, nature also has famous examples of assembled nanostructures. One of the most prominent systems is the magnetotactic bacteria. The magnetotactic bacteria exhibit iron oxide, mostly magnetite (Fe3O4) NPs of sizes between 20 and 120 nm arranged in a single chain structure¹⁸. The NPs are formed in the magnetosome vesicles, which serve first of all as template for the mineralization of the inorganic material¹⁹. The NPs within a chain are iso-oriented to adjacent NPs along the crystallographic axis [1,1,1]. The linear NP arrangement maximizes the magnetic dipole moment compared to the single magnetosomes and thus, increases their magnetotaxis¹⁸. The magnetotaxis is defined as the ability of cells to orientate and migrate along the geomagnetic field line²⁰. The formation of those NP chains is not fully understood yet, but there are hints for the involvement of special proteins in the vesicle¹⁸. Another example of NP assembly is the chain-like structure of magnetite NPs in the forehead of salmon²¹. Both organisms use the NP assembly structures as a natural senor, which can respond to the magnetic field of the earth and help them to find their coordination, as shown in Fig. 1.

Fig. 1: A) Magnetospirillum magneticum AMB-1 bacteria produce magnetosomes with encapsulated magnetite NPs. The MamA protein belongs to the magnetosome associated proteins and is postulated to be involved in the magnetosome alignment²². B) A magnetite NP- assisted natural navigation system used by salmon during migration.²¹

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But somehow nature and scientist share a similar interest of utilization of proteins to trigger and control NP assembly resulting in advanced structures with specifically designed properties. The main advantages of proteins over synthetic macromolecules are the precise sizes/molecular masses, morphologies and the complex opportunity of structural and chemical modification.

The natural NP assembly systems demonstrated that nature is the perfect inspiration for scientists and engineers. Therefore, in the last 30 years biotechnology gained in importance due to the fundamental interest in manipulating and controlling of biological systems. The obtained structures have the potential to revolutionize the industrial production of medicine, chemicals, foods and energy source. The research works focus on macromolecules such as DNAs and RNAs as well as protein-based viruses with highly controlled self-assembly mechanism of their building units such as DNA strands or coat proteins to the programmed target structures under appropriate physical conditions. The encoded information for this assembly process is located for example, in the DNA or RNA sequences due to the specific binding patterns of the complementary base pairs. On the other hand, virus structures are obtained by stacking of proteins in an ordered, symmetrical manner giving the defined rod-like or globular end structures. The most well-known examples are the rod-like tobacco mosaic virus (TMV) and M13 bacteriophage (M13) as well as the globular cowpea chlorotic mosaic virus (CCMV)²³. The self-assembly bases on the supramolecular interactions between the building units. Thus, the head-to-tail assembly of wild type TMV reportedly is a product of the complementary hydrophobic interactions between the dipolar ends of the helical structure²³. The synthetic chemistry in biotechnology is used to alter and re-program DNA and protein structures to create a unique set of biochemical functions. The systematic chemical modifications add new

‘programmable’ functions, sequences and/or multiple triggers to these structures leading to architectures of increasing complexities. As a result, DNA and RNA self-assembly structures with lattice^24,25, tube^26,25 and 3D²⁷ morphology can be obtained. Therefore, nucleic acids are a very prominent candidate to guide the assembly of NPs, e.g. DNA-gold NP bioconjugates, into superlattice structures²⁸, as shown in Fig. 2 A. The driving force for this process are the programmable recognition and hybridization interactions between DNA strands. In the virus mediated nanoparticle assembly the wild-type virus structures are modified with NP nucleating and binding moieties. The resulting rod-like or ring virus structures function as a template for the ordering of nanoparticles such as zinc sulfide and gold NPs on their surface^29,30 or as guiding component in the NP assembly leading to micelle-like or spherical, and closely packed lamellar assembly structures of zinc sulfide and iron oxide NPs^31,32 (Fig. 2 B-C).

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Fig. 2: A) Au-DNA conjugates assemble into bcc (blue circle) and fcc (green circle) superlattice depending on the DNA sequence length and composition²⁸. B) Rotavirus structural proteins forming nanotubes are used as template for the self-assembly of Au-NPs³³. C) A schematic diagram of the micelle-like structures, in which ZnS-NP aggregates are surrounded by A7 viruses and the corresponding TEM image shows that 100 to 150 NPs formed aggregates³¹.

The consequent question is now ‘Can we combine our knowledges of NP self-assembly and the biomolecular coding by protein modification in one concept to exploit new nanoparticle-based protein assembly structures?’ This question is also the aim of this thesis. The results of the thesis will show that the above question can be answered with a satisfied ‘YES’.

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Scope of the thesis

The inorganic nanoparticle assembly induced by protein structure as a guiding element and connecting unit leading to inorganic-protein assembly structures and protein-nanocomposite materials with optical (surface plasmon resonance), magnetic (superparamagnetic) and semiconductor (absorption, photoluminescence) properties is the main focus of the thesis.

Therefore, two protein structures are investigated. The Hcp1 structure with a toroid shape of defined dimension provides thiol moieties at geometrically designed positions. These encoded nanoparticle binding sites should allow a particle binding on selected protein surfaces and thus, a controlled assembly of the single nanoparticle-protein conjugates into larger structures. The assembly condition should be optimized to obtain oriented nanoparticle assembly in the form of a for example chain structure. The formation mechanism of these assembly structures is also of great interest. Therefore, several techniques such as spectroscopy, microscopy and analytical ultracentrifugation are used to investigate the single nanoparticle-Hcp1 bioconjugate in a quantitative and qualitative manner as well as the assembly process. The characterizations of the nanoparticle-Hcp1 structures and the protein-nanocomposite materials are important to establish a relationship between the structures and the material properties. A second protein structure, ELP, is used to point out the importance of a rigid protein structure for a defined nanoparticle assembly, since ELP is a more flexible structure than the Hcp1 structure. The ELP structure shows a coil-like structure at room temperature and a more linear beta-sheet structure at high temperature. Here, thiol- and magnetite-adhering moieties are also incorporated into the ELP structure to imitate the binding sites in the Hcp1 structure. To create a small selection of hybrid materials with different physical properties as mentioned before, six nanoparticle systems including gold, silver, magnetite, cobalt ferrite, cadmium sulfide and zinc sulfide are synthesized. First of all, the nanoparticle synthesis should focus on the size control.

The size of the nanoparticles should be in the same size range of the Hcp1 structure, since it can be assumed that uniform building units will contribute to a defined overall morphology of the end-assembly structures. A nanoparticle surface modification in terms of ligand exchange is also conducted depending on the reaction media.

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Chapter 2 Fundamentals

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Chapter 2 – Fundamentals

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2.1 Nanoparticle assembly

In his book Helmut Cölfen dedicates a complete chapter to the investigation of the formation conditions of the so-called mesocrystals, which are defined as a highly ordered assembly structure of nanocrystals. Here, the nanoparticle alignment is based on capillary forces, hydrophobic forces, interface energies, additive coding of nanoparticles as well as physical fields such as mechanical, magnetic and electrical fields³⁴. Since the inner anisotropy of the NPs often is not sufficient enough to trigger the self-assembly process due to the small energy differences between different surfaces and therefore, an intensification of NP anisotropy is required to induce the self-assembly leading to the alignment of the NPs into a crystallographic order. This requirement can be fulfilled with the ’additive coding concept’. This concept involves the

‘coding’ of NP surfaces by hydrophobic/hydrophilic or cationic/anionic molecules and the amplification of the nanoparticle anisotropy by the binding or adsorption of these additives on a specific NP surface leading to subsequent directed assembly. The additives are the anisotropic components dictating the nanoparticle assembly structure. The self-assembly and assembly guiding component in the additive coding concept mostly are organic components leading to discrete hybrid structures up to the mesoscale. The general strategies for the additive induced NP assembly will be the nanoscale incarceration, supramolecular wrapping, nanostructure templating, unitary nano-objects, extended nanostructures and mesocrystal formation³⁵. The organic components vary from synthetic materials such as polymers e.g. block- to dendrimer- polymers, organic molecules e.g. porphyrin, polysaccharides, peptides, amphiphiles to bio- macromolecules such as capsid-like proteins or viral coat proteins, enzymes, DNA, microtubules, collagen fibrils, viruses e.g. rod-shaped tobacco mosaic virus. In Fig. 3 A-B two examples of the nanoparticle coding with additives are shown. The latex NPs and gold nanorods are coded with additives at certain surfaces (pols, tips). Under appropriate conditions the interactions between the additives on the coded surface become stronger and an anisotropic nanoparticle assembly occurs resulting in chain and ring structures of NPs.

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Fig. 3: A) Schematic representation of self-assembly of latex particles³⁶: Spherical particles are

‘coded’ with hydrophobic moieties on the poles (black area) and charged in the equator section (white). B) Gold nanorods carry a double layer of CTAB along the longitudinal side and polystyrene molecules at both ends and assemble into rings (left SEM image) and chains (right SEM image) structures³⁷.

In this thesis the focus will be on the works of protein induced inorganic NP assemblies. The general concept of protein induced NP assembly falls into two strategies of template-assisted and template-free assembly. The template-assisted strategy comprises the nano-patterning of the biomolecular template with pre-synthesized NPs or the modification of the template surface with nucleation and growth induced sequences for the in-situ synthesis of NPs. The end- structures on the nano- to mesoscale can be one- to three-dimensional, as shown in Fig. 2 B.

The advantage of this strategy is the establishment of several protein based templates with rod, sheet and tubulin morphology^38,39. The wide structure variation and chemical stability of these structures enable the synthesis of semiconductor (e.g. cadmium selenide, cadmium- and zinc sulfide)^40,41,42 , metal (gold, silver)43,44,45,33

, magnetic (iron oxide, cobalt)^46,47,48 hybrid structures.

The resulting materials have great potential in biosensors, electronic device and magnetic

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storage applications. On the other hand, the template-free protein induced nanoparticle assembly follows the additive coding concept, since the protein is considered as the coding additive initiating the anisotropic nanoparticle assembly. However, the template-free assembly is more difficult to achieve, since specific interactions in terms of chemical and spatial interplay of proteins and nanoparticles have to be ensured. Furthermore, fundamental colloidal effects such as surface charges at different pH, Debye screening length at different ionic strengths, solvent properties also have to be taken into account, because most assembly reactions take place in aqueous media. Therefore, the template-free NP assembly is the more sophisticated strategy, which attracts less attention in terms of material fabrication. Anyhow, examples for this strategy are Au-NP assembly with insulin or lysozyme49,50,51,52,53,54

, Fe3O4- or Fe2O3-NPs with apoferritin^55,56,57 as well as CdSe- and AgS-NP assembly into cluster structure with virus capsid protein⁵⁸. In the case of Au-NPs the non-covalent protein interactions including hydrophobic, Van-der-Waals interaction are responsible for the NP assembly. These interactions can be tuned by the addition of salt. For the assembly of Fe3O4 and AgS NP the electrostatic interaction of the proteins bound to NP is the key parameter, which is controlled by the pH value in the solution.

For this reason, this thesis wants to contribute to the research progress of the template-free assembly of inorganic NPs induced by protein structures.

2.2 Nanoparticles with different physical properties 2.2.1 Superparamagnetic nanoparticles

Every material holds magnetic properties to a certain extent depending on the molecular composition and temperature. The response of a magnetic material in an external magnetic field H is defined as the magnetic induction B, which is the sum of the magnetic moment of an atom (Equ. 1 and 2)⁵⁹.

𝐵 = 𝜇₀(𝐻 + 𝑀) (1)

𝜇₀-permeability of a material, 𝑀 – magnetization.

The magnetization is the total sum of the magnetic moments per volume unit or mass of the material.

𝑀 =_{𝑣𝑜𝑙𝑢𝑚𝑒}^𝑚 =_{𝑚𝑎𝑠𝑠}^𝑚 (2)

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The magnetic susceptibility χ in Equ. 3 is a material-specific value, which is related to the magnetization of the material in a magnetic field strength H.

𝑀 = 𝜒𝐻 (3) In general, materials are divided regarding their electronic structures into two classes, the diamagnetic and paramagnetic systems, as shown in Scheme 1. The diamagnetic materials show fully occupied electron shell leading to the cancellation of singular magnetic moments of the electrons and thus, the loss of an overall magnetic moment. Paramagnetic materials do not have a complete electronic configuration. Therefore, the magnetic moments do not compensate each other leading to a permanent, overall magnetic moment. In an external magnetic field the randomly oriented magnetic moments in a paramagnetic material will align along the magnetic field lines and a magnetization can be detected. The magnetization remains as long as the magnetic field is present.

Scheme 1: Electron configuration of beryllium and aluminum as a typical diamagnetic and paramagnetic material, respectively.

The ferro-, ferri-, and antiferro-magnetism are cooperative magnetic phenomena, which occur due to the interaction between the electron spins in a paramagnetic substance below certain temperatures (Fig. 4). Thus, it results in the parallel alignment of all spins in one domain, the so- called Weiss’s domain. The Néels- and Curie temperatures indicate the high temperature transition of antiferromagnetism and ferromagnetism into paramagnetism. The antiferromagnetism is characterized by the antiparallel alignment of adjacent electron spins of the same magnetic moment in two adjacent domains leading to zero net-magnetization. The ferrimagnetic materials also show antiparallel spin alignment. But the spins have different magnetic moments leading to a partial compensation of magnetization. Therefore, this special class of magnetic material outwardly exhibits ferromagnetic behavior. The ferrimagnetism is often observed for ferrite materials, which include magnetite and cobalt ferrite. More structural details and origins of ferrimagnetism will be given in the following chapter. The parallel alignment of the spins in one domain is typical for a ferromagnetic system. Similar to paramagnetism the single domains are randomly oriented in a ferromagnetic material. The exposure to a magnetic field will trigger the alignment of all domains and the magnetization of

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the materials. But contrary to the paramagnetism, the ferromagnetism is identified by characteristics in the hysteric magnetization curve such as the remanence magnetization Mr, which is the remaining magnetization of the material after turning-off of the magnetic field, the saturation magnetization Ms, which is the maximum magnetization that cannot be exceeded even at very high magnetic field and the coercive field Hc, which is the required magnetic field to bring the magnetization to zero, as shown in Fig. 6 C.

Fig. 4: The cooperative, magnetic phenomena in ferromagnetic (A), anti-ferromagnetic (B) and ferrimagnetic materials (C).

After the introduction of magnetism of bulk materials, the fundamental knowledge was provided to understand the changes of magnetism on the nanometer scale (Fig. 5 A-C). The two most important finite-size effects are the magnetic single domain and the superparamagnetism.

Magnetic bulk materials consist of multi Weiss’s domains, which are separated by the domain walls (Fig. 5 A). With decreasing size the energy to maintain the domain wall becomes too large for the material, which is energetically unfavorable for the system leading to dissolution of the walls (Fig. 5 B). The size limit for the formation of single domains in NP depends on the material and is displayed in Fig. 6.

Fig. 5: A) Weiss domains in a bulk material, which are separated by the domain walls. B) Single domains in NPs of decreasing sizes. C) In the superparamagnetic regime, the individual particle dipoles are randomly oriented (indicated by the black double-arrow) at temperatures above the blocking temperature TB and are quasi static at temperatures below TB.

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The second nanoscale magnetism effect is the superparamagnetism, which is caused by further size reduction of the single domain NPs (Fig. 5 C). In the superparamagnetic system the overall magnetic moment of a particle can change its direction. The magnetic anisotropy energy per particle E() in Equ. 4 expresses the required energy to hold the moment along a certain direction⁵⁹.

𝐸(𝜃) = 𝐾_𝑒𝑓𝑓𝑉𝑠𝑖𝑛²𝜃 (4)

𝜃 - angle between the magnetization and the easy axis, 𝑉 - particle volume.

Easy axis is the direction inside a crystal, along which already small applied magnetic field is sufficiently enough to reach the saturation magnetization. The energy barrier for a magnetization change between two equivalent directions is 𝐾_𝑒𝑓𝑓𝑉. For superparamagnetic NPs at room temperature the thermal energy of 𝑘_𝐵𝑇 exceedes this energy barrier leading to a continuous flipping of the magnetic moments. The system outwardly exhibits paramagnetism, including a sigmodal instead hysteric magnetization curve without a remanence magnetization and coercive field, as shown in Fig. 6 D. Another characteristic for superparamagnetism is the relaxation time  of the magnetic moment in a particle, which is described by Néel-Brown in Equ. 5.⁵⁹

𝜏 = 𝜏₀𝑒𝑥𝑝 [^𝐾_𝑘^𝑒𝑓𝑓^𝑉

𝐵𝑇 ] (5) Regarding to this equation the superparamagnetism is related to the measurement time of the technique 𝜏_𝑚. If the measurement time is larger than the relaxation time of the NPs (𝜏 ≪ 𝜏_𝑚), the magnetic moment will change the direction too fast in the time window of the measurement. The system is now in a paramagnetic state. For the 𝜏 ≫ 𝜏_𝑚 case, the direction change is slow and becomes quasi static. The nanoparticle is situated in a ‘blocked state’

showing properties of ferromagnetic nanoparticle. The temperature, which separates these two magnetic orders, is called the blocking temperature TB. The TB can be calculated by taking the large time window of the measurement, e.g. 100 s for the magnetometer into account, as shown in Equ. 6. It has to be mentioned that the blocking temperature depends on the anisotropy constant and size of NP, the magnetic field and the experimental conditions.

𝑇_𝐵 =𝐾_𝑒𝑓𝑓𝑉

30𝑘_𝐵 (6)

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Fig. 6: A): Size-dependent transition from superparamagnetic state to single domain state for different magnetic NPs. B) The magnetic response (coercivity or coercive field) rises drastically upon entering the single domain regime⁶⁰. Schematic illustration of magnetic measurement shows a typical hysteresis loop of an array of single domain ferromagnetic nanoparticles (C) and a typical curve for a superparamagnetic material (D) at room temperature.⁶¹

Ferrite materials

The ferrite materials usually have the composition MeÎII (MeÎI, MeÎII) O4 with a statistical occupation of the metal ions positions. MeÎI can be metal ions such as Mg²⁺, Mn²⁺, Fe²⁺, Co²⁺, Zn²⁺, as well as the combination of these ions e.g. Mn²⁺/Zn²⁺, and MeÎII is often the trivalent Fe cation⁶². Most of these ferrites show an inverse spinel crystal structure with a face-centered- cubic unit cell. In this unit cell half of the Fe³⁺atoms occupy the tetrahedral sites and the other half of the Fe³⁺ atoms as well as the Fe²⁺ atoms occupy the octahedral sites (Fig. 7 A). In the

A B

C D

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defected inverse spinel structure some octahedral sites are not filled, as known for maghemite (-Fe2O3) with Fe²⁺ vacancies on the octahedral sites. The magnetization arises from the net magnetic moment of all metal ions in the sublattices. The spins of cations in the octahedral sites compensate pairwise with the cations with antiparallel spin orientation in the adjacent tetrahedral sites⁶². The compensation is only partial, because the amount of atoms in the octahedral sites is twice as much as the atoms in the tetrahedral sites. Thus, the over-all magnetic moment is larger than zero and spontaneous magnetization can occur by the parallel alignment of the magnetic moments to the applied magnetic field HO, similar as for ferromagnetic materials. Magnetite with the composition FeFe2O4 (or Fe3O4) and cobalt ferrite CoFe2O4 have a net magnetic moment of 4 B and 3 B (B – Bohr’s magneton)⁶³. The net magnetic moments in a magnetic field HO and the resulting magnetization of some ferrite bulk materials are shown in Fig. 7 B revealing the influence of the Me^II cations on the magnetic properties.

Fig. 7: A) Inverse spinel structure of ferrite materials⁶⁴. B) Ferrimagnetic spin structure indicates the alternate ferrimagnetism due to the different magnetic moments of the cations (Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺) in ferrites yielding different magnetization values²¹.

2.2.2 Surface plasmon resonance

Noble metal nanoparticles have a long tradition in the human history and science. The first appearance of gold nanomaterial goes back to the Roman time with the famous example of the Lycurgus cup, and reaches down to the post-modern time with the ‘Purple of Cassius’ pigment.

In both examples NPs of gold and silver material are embedded in glasses. The research on gold

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nanostructures was firstly initiated by Michael Faraday in middle of the 19^th century⁶⁵. His appreciation for the red nanoparticle solution attracted attention of other researchers such as Gustav Mie or Maxwell Garnett. Their works lay the theoretical foundation for the explanation of the optical properties of metal NPs in an isolated/non-interacting or aggregated state. In general, nanoparticles comprise a large amount of atoms or molecules bonded together and can be dispersed in a solution or gas, as well as placed in a solid medium. With increasing size the energy levels in NP continuously fuse into the quasi-continuous band structure of a bulk material. Materials of nanometer scale show dramatic volume and surface change leading to their electronic properties, crystallographic structures and optical properties. The origin of the optical response of noble metal NPs such as Cu, Au and Ag is the surface plasmon oscillation. In Fig. 8 A a simplified scheme of the interaction between the NPs and the incoming light and their optical responses is shown. The electric field of the light wave induces a polarization of the conduction electrons with regards to the nearly immobile (heavier) ionic core in the nanoparticle. Thus, the negative charges compared to the fixed positive core move in the external electromagnetic field resulting in a charge difference at the particle boundaries. At the same time this movement generates a restoring force in the system, which gives rise to the formation of a dipolar oscillation of the electrons, also called the surface plasmon oscillation.

The surface plasmon resonance (SPR) denotes the coherent excitation of all conduction electrons, which equates with an in-phase oscillation, at certain field strength leading to the light absorption and the formation of the SPR band. The part “surface” bases on the fact that the main effect producing the restoring force is the surface polarization. Thus, the surface plays a very important role for the observation of SPR and, therefore, shifts the resonance to optical frequencies. The coherent excitation can be observed for example in UV-Vis spectroscopy with the typical SPR band of a peak maximum at 520 nm for spherical Au-NPs of size around 5-20 nm (Fig. 8 B).

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Fig. 8: A) Electromagnetic wave of light interacts with the electrons in the conducting band of a NP leading to the oscillation of the electron cloud. B) If the wavelength of the incoming light matches the resonance frequencies of the NP, the resulting UV-Vis spectra will show the SPR peaks depending on the morphology of the NPs, e.g. sphere or rod⁶⁶.

The Mie theory explains the size dependency of the optical response of the Au-NPs⁶⁷. It is possible to correlate the wavelength shift of the SPR peak with the NP size. Mie assumption requires a spherical morphology and radius of 2R << of the NP (R - NP radius, - wavelength of light). The Au-NPs can be presumed in the quasi-static state similar to the electrostatic theory. In this approximation the wavelength-dependent dielectric constant of a metal particle  and the surrounding medium m can be applied to calculate the electric field inside the NP 𝐸₀ (Equ. 7). Furthermore, additional phase shifts, also called the retardation effects, of the electrodynamic field are negligible in the quasi-static regime.

𝐸_𝑖 = 𝐸₀ 3𝜀_𝑚

𝜀 + 2𝜀_𝑚 (7)

𝐸_𝑖 - electric field of the incident electromagnetic wave.

A spherical Au-NP exposed to an electromagnetic field shows charge polarization of the conduction electrons on the surface leading to Equ. 8 with , as polarizability of Au-NP.

𝛼 = 4𝜋𝜀₀𝑅³ 𝜀 − 𝜀_𝑚

𝜀 + 2𝜀_𝑚 (8)

R – particle radius, 𝜀 and 𝜀_𝑚 – dielectric constant of the metallic NP and the surrounding medium.

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Since the electro-magnetic excitation is not valid in this approximation, the 𝜀 value can be translated into the wavelength dependent value (𝑤). 𝜀(𝑤) contains a real and an imaginary part and is given in Equ. 9

𝜀 = 𝜀₁(𝑤) + 𝑖𝜀₂(𝑤) (9)

Under the consideration of 𝜀_𝑚 as a real constant, we can observe a field enhancement and a polarization of the NP with a minimal value of the 𝜀 + 2𝜀_𝑚 term in Equ. 7 and 8. The condition for the wavelength dependent plasma resonance will be:

[𝜀₁(𝑤) + 2𝜀_𝑚]²+ [𝜀₂(𝑤)]²= 𝒎𝒊𝒏𝒊𝒎𝒖𝒎 (10)

In the quasi-static regime of NPs with 2R << scattering can be neglected, therefore, the absorbance of the dipole is the only contribution in the extinction of NP and can be expressed in Equ. 11.

𝜎_{𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛}= 9𝑤

𝑐 𝜀_𝑚^3/2𝑉 𝜀₂(𝑤)

[𝜀₁(𝑤) + 2𝜀_𝑚]²+ [𝜀₂(𝑤)]² (11)

𝜎_{𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛} – extinction coefficient, 𝑉 – particle volume, 𝑤 – angular frequency of the exciting light, 𝑐 – velocity of light.

As a consequence, the resonance condition is fulfilled when 𝜀₁(𝑤) = −2𝜀_𝑚 , if 𝜀₂(𝑤) is small or weakly wavelength-dependent. Equation 11 shows clearly that the absorbance of the Au-NP depends on the size, expressed by the particle volume and the dielectric constant. Based on this expression, the absorbance spectra of Au-NPs can be calculated at given function and particle radius. Since the surface controls the boundary conditions for the polarizability of the metal, the nature and the environment of the nanoparticle surface are essential for the surface plasmon resonance. The nature of the surface is related on the one hand to the surface atoms and stabilizing ligands and on the other hand to the dispersing solvents. So Ag-NPs show a red-shift of the SPR peak in solvents with decreasing dielectric constants⁶⁸. In this work Au- and Ag-NPs of radii from 2 to 6 nm were used. Anyhow, we want to point out the retarding effect of larger NPs, since they also show interesting optical responses. For example, Au-NPs of diameter larger than 20 nm show a distinct red-shift and broadening of the SPR peaks⁶⁹. In this size range higher modes of the resonance become more important compared to the usual dipole resonance, which can be found at lower energies. Thus, firstly the incident light cannot polarize the NP homogenously and secondly the SPR peak red-shifts with increasing sizes⁷⁰. The assumption of spherical particle morphology in the Mie theory displays an incomplete image of real NP systems. The deviation from spherical shape also leads to altered optical responses because the

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longitudinal and transversal dipoles have not the same resonance. For instance, Au nanorods have a broadened, longitudinal and a transversal plasmon absorption corresponding to the oscillation of the electrons along and perpendicular to the long axis of the rods⁷¹, as shown in Fig. 8 B.

All the above mentioned optical responses are related to the isolated, non-interacting metallic NPs, where the particle-particle distance is larger than five times the particle radius (d > 5R). But in case of densely packed NPs the surface plasmon oscillation will change drastically, because the single NPs are in close contact (d ≤ 5R) and electronically coupled to each other⁷². The results are complicated UV-Vis spectra comprising of the red-shifted SPR peaks⁷³, as well as new particle-particle peaks. The new SPR peaks of different widths and at different wavelengths are observed depending on the morphology of the Au-NP aggregates, as shown in Fig. 9 A-B⁷⁴. This band can be regarded as the longitudinal resonance absorption similar to the nanorods in case of chain-like aggregation of individual spherical Au-NPs. Maxwell Garnett pioneered in his work from 1904 the investigation of strong interparticle coupling⁷⁵. His theory is only valid for the quasi-static limit (2R << ) and very small interparticle distances. Furthermore, this approximation can be generalized to various particle shapes. The aggregated structures of single NPs are characterized by their correlation length of spatial order, volume fraction, geometric ordering, etc. Therefore, the exact knowledge of statistical distribution of the positions and pairwise distances of all particles are strongly required. The Maxwell-Garnett theory bases on the Clausius-Mosotti equation, which describes a metal NP assembly embedded in an inert medium by an effective complex dielectric constant 𝜀_𝑒𝑓𝑓 (Equ. 12)⁷⁶.

𝜀_𝑒𝑓𝑓− 𝜀_𝑚

𝜀_𝑓𝑓+ 𝜅𝜀_𝑚 = 𝑓 𝜀 − 𝜀_𝑚

𝜀 + 𝜅𝜀_𝑚 (12)

𝜀 – dielectric constant of NP, 𝜀_𝑚 – dielectric constant of the medium, 𝜅 – screening parameter, 𝑓– volume fraction of the NPs in assembly structure with 𝑓 = 𝑉_{𝑎𝑠𝑠𝑒𝑚𝑏𝑙𝑦}⁄𝑉_{𝑠𝑎𝑚𝑝𝑙𝑒} .

The dielectric constants are listed in literature and the volume fraction 𝑓_𝑚 is known from the sample synthesis. The shape-dependent screening parameter 𝜅 has a value of two for spherical, one for rod-like NPs aligned parallel to the direction of the incoming light and approaches infinity for flat discs aligned perpendicular to the direction of light⁷⁶. At the given values the 𝜀_𝑒𝑓𝑓 can be calculated. At the end, absorption spectra of the metal NPs in a transparent non- interacting medium can be computed, as the dielectric constant is related to the optical refractive index 𝑛_𝑐 and the absorption coefficient 𝑘_𝑐⁷⁷.

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Fig. 9: A) The UV-Vis spectra of layers of Au-NPs (a= 1 layer and e = 5 layers) deposited on a glass substrate show dramatic change of the SPR peaks. The Au-NPs are coupled by an organic structure (purple square), as shown in the below scheme. B) The UV-Vis spectra of separated Au-NPs (a) and chain-like Au-NPs aggregate (b), as shown in the below scheme. C) The electron micrograph of the chain-like aggregate of (b) in B.⁷⁴

2.2.3 Semiconductor nanoparticles

Semiconductor NPs have attracted great attention due to the remarkable optical properties related to their absorption and emission features. A semiconductor material exposed to excitation shows a transition of electrons in the valence band into the unoccupied conduction band yielding an electron hole in the valence band. The electron-hole pair, which is attracted to each other by electrostatic Coulomb force, is termed as an exciton. The energy to overcome the Coulomb force of the exciton is equal to the bandgap of the semiconductor material.

Semiconductor NPs contain a finite number of atoms bonded together leading to the splitting of the energy levels into discrete states of different energies. The quantum confinement of the excitons within the NP and the dimensionality of nanoscale size lead to the terms of quantum dot (QD), quantum wire (QW) and quantum wells (QWE)⁷⁸. For a simple description, the electron–hole pair can be considered as an analog to a hydrogen atom, where the hole is a proton. Therefore, the classic Bohr model can be applied to estimate the average distance between electron and hole in a semiconductor NP also known as the exciton Bohr radius 𝑎_𝐵^𝑒𝑥 (Equ. 13)⁷⁹.

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𝑒² [ 1

𝑚_𝑒+ 1

𝑚_ℎ] (13)

ħ – reduced Planck‘s constant, e – elementary charge,  – dielectric constant of the semiconductor material, 𝑚_𝑒, 𝑚_ℎ – effective mass of electron and hole.

Thus, an exciton Bohr radius of 3 nm and 2.5 nm can be determined for cadmium sulfide (CdS)⁸⁰ and zinc sulfide (ZnS)⁸¹ QDs. When the QD size is similar to the exciton Bohr radius of the semiconductor material, the properties of the exciton will strongly depend on the space available for the electron-hole pair. Therefore, 𝑎_𝐵^𝑒𝑥 becomes a threshold value and the QDs can show size-dependent optical properties below this value. The formation of the band structure with discrete energy states within this size range is also known as the ‘quantum size effect’.

To explain the exciton-related properties, one can use the linear combination of atomic orbitals (AO) approach, as shown in Fig. 10. In a diatomic molecule the atomic orbitals interact with each other forming highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO). With increasing number of atoms more atomic orbitals will be included in the linear combination leading to more molecular orbitals of discrete energy levels. If an infinite number of atoms is used to form an extended bulk material, the distances between the energy levels will ‘infinitely’ decrease, which yields continuous bands from the combination of the atomic orbitals. In the case of QDs the distance between the energy levels and the bandgap becomes larger compared to the solid material due to the small number of atoms. Therefore, QDs can be placed in between the molecule and bulk material. Anyhow, the band gap of QDs closely resembles the bandgap of the bulk semiconductor for sufficiently large QD size and the optical features such as exciton absorption, photoluminescence disappear indicating the transition to the continuous band structure.

Fig. 10: The evolution of the electronic structure from atoms and molecules to semiconductor QDs and bulk material indicates the change of energy levels and the decreasing bandgaps with increasing sizes.

HOMO LUMO

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The effective mass approximation (EMA) was firstly introduced by Efros⁸² and later extended by Brus⁸³ with including of the particle in the box model. This approach quantitatively describes the size-dependency of the QD band gaps. A particle of a mass m is placed in a box of length L and is surrounded by infinite potential energy of the box walls preventing the particle from escaping.

The particle in the box itself does not possess any potential energy. The solution of the Schrödinger equation for the particle in the box model gives rise to the wavenumber related energy levels (Equ. 14).

𝐸_𝑛= ℏ²𝜋²

2𝑚𝐿²𝑛² (14)

𝑛 – positive integer.

This model is adapted to the QD system with exciton of the effective masses of electron and hole in a potential well with infinite potential. The result is the relationship between the bandgap 𝐸_𝑔 and QD radius in Equ. 15.

𝐸_𝑔 =ℏ²𝜋²

2𝜇𝑅²−1.8𝑒²

𝜀𝑅 + 𝐸_{𝑔,𝑏𝑢𝑙𝑘} =ℏ²𝜋² 2𝑅² [ 1

𝑚_𝑒+ 1

𝑚_𝐿] −1.8𝑒²

𝜀𝑅 + 𝐸_{𝑔,𝑏𝑢𝑙𝑘} (15)

𝜇 – reduced mass of electron and hole 𝑅 – QD radius

𝑚_𝑒, 𝑚_𝑙 – effective mass of electron and hole

𝐸_{𝑔,𝐵𝑢𝑙𝑘} – bandgap of the semiconductor bulk material

The first term of Equ. 15 represents the energy from the quantum confinement effect, which shifts the Eg to higher value as R^-2, while the second term is the Coulomb interaction of the excitons shifting Eg to lower energy as R^-1. Thus, the bandgap of the semiconductor NP will increase with decreasing QD size. Fig. 11 A shows the size-dependency of bandgaps for ZnO-, CdS-, GaAs- and InSb-QDs of increasing sizes. In this thesis cadmium sulfide (CdS) and zinc sulfide (ZnS) QDs were synthesized and used for the experiments with the Hcp1 protein structures. CdS and ZnS belong to the II-VI class of semiconductor compounds and exist in the wurtzite and zinc blende crystal structure (Fig. 11 B-C). The increasing bandgaps from ZnS to CdS yield absorption and emission properties in the UV and visible range (Table 1).