Rolled up microtubes for the capture, guidance and release of single spermatozoa

Hybride Mikroschwimmer, die einen biologischen Antrieb und eine künstlich hergestellte Mikrostruktur enthalten sind ein attraktiver Ansatz um kontrollierte Bewegung auf kleinstem Maßstab zu erreichen. In dieser Dissertation wird ein neuer hybrider Mikroschwimmer vorgestellt, der aus ferromagnetischen Nanomembranen besteht, die sich zu Mikroröhrchen aufrollen und in der Lage sind, einzelne Spermien einzufangen. Dieser Mikrobioroboter nutzt die starke Antriebskraft der Spermazelle um das magnetische Mikroröhrchen fortzubewegen. Die vorliegende Arbeit beschreibt, wie dieser Mikroschwimmer seine Bewegung vollzieht und wie verschiedene Faktoren wie Temperatur, Radius der Mikroröhrchen, Eindringtiefe der Spermien in das Röhrchen und Länge der Röhrchen einen Einfluss auf sein Verhalten haben. Richtungskontrolle wird durch externe magnetische Felder realisiert und es wird dargestellt, wie dies zur Trennung der Mikrobioroboter aus einer Mischung von Spermien und Mikroröhrchen genutzt werden kann. Weiterhin werden zwei Oberflächenmodifizierungsmethoden angewandt um die Kupplungseffizienz zwischen Mikroröhrchen und Spermien zu erhöhen. In diesen Methoden wird das extrazelluläre Protein Fibronektin auf die innere Röhrchenoberfläche aufgebracht und dient als Bindungsstoff für Spermien. Schließlich wird durch den Einbau temperatursensitiver Material in die Mikroröhrchen ein ferngesteuerter Freisetzungsmechanismus für die Spermazelle vorgestellt. Dabei falten sich die Mikroröhrchen bei kleinen Temperaturerhöhungen auf und setzen die Zelle frei. Diese Arbeit diskutiert letztendlich das Potential solch eines hybriden Mikroschwimmers für die Anwendung in assistierter Reproduktion.:TABLE OF CONTENTS SELBSTSTÄNDIGKEITSERKLÄRUNG 0 ABSTRACT 1 TABLE OF CONTENTS 3 1 MOTIVATION AND GOALS 5 1.1 MINIATURIZATION: “THERE IS PLENTY OF ROOM AT THE BOTTOM…” 5 1.2 SPERMBOTS: POTENTIAL IMPACT 7 2 BACKGROUND AND STATE-OF-THE-ART 11 2.1 MICROBIOROBOTICS 11 2.2 SPERM MORPHOLOGY AND THEIR JOURNEY TO THE EGG 15 2.3 INFERTILITY AND ASSISTED REPRODUCTION TECHNIQUES 19 2.4 SINGLE CELL RELEASE 22 2.5 STIMULI-RESPONSIVE MATERIALS 25 3 MATERIAL AND METHODS 29 3.1 ROLLED UP TECHNOLOGY 29 3.2 TREATMENT OF BOVINE SPERMATOZOA 32 3.2.1 Preparation of Spermbots 32 3.2.2 Speed Measurements 33 3.2.3 Separation On Chip 33 3.3 SURFACE MODIFICATION OF MICROTUBES 34 3.3.1 Surface Chemistry 35 3.3.2 Microcontact printing 39 3.4 POLYMER TUBE FABRICATION 44 3.4.1 Synthesis of photosensitive monomer 4-Acryloylbenzophenone 44 3.4.2 Synthesis of poly (N-isopropylacrylamide-co-Acryloylbenzophenone) 46 3.4.3 Photolithography of polymeric films 48 3.5 VIABILITY TESTS 51 4 RESULTS AND DISCUSSION 53 4.1 CHARACTERIZATION OF SPERMBOTS 55 4.2 TEMPERATURE INFLUENCE 60 4.3 MAGNETIC CONTROL 62 4.4 SEPARATION ON CHIP 68 4.5 EFFECT OF DECREASED MICROTUBE LENGTH 72 4.6 COUPLING EFFICIENCY 74 4.7 THERMORESPONSIVE POLYMERIC MICROTUBES FOR CELL RELEASE 80 4.8 SPERM VIABILITY TESTS 94 5 SUMMARY AND CONCLUSIONS 97 6 OUTLOOK 101 7 LIST OF FIGURES 107 8 LIST OF TABLES 113 9 ABBREVIATIONS 115 10 CURRICULUM VITAE 117 11 LIST OF PUBLICATIONS 119 JOURNAL ARTICLES 119 CONTRIBUTIONS TO COLLECTED EDITIONS/PROCEEDINGS 121 12 ACKNOWLEDGEMENTS 123 13 REFERENCES 125The search for autonomously moving, highly functional and controllable microdevices is a purpose of current micro/nanobiotechnology research, especially in the area of biomedical applications, for which reason, biocompatible solutions are in demand. In this thesis, a novel type of hybrid microswimmer is fabricated by the combination of rolled up thin nanomembranes with bovine spermatozoa. The microbiorobot presented here uses the powerful motion of the sperm flagella as a propulsion source for the magnetic microtube. This work demonstrates how the microswimmer performs its motion and how several factors such as temperature, radius of the microtube, penetration of the cell inside the microtube and length of the tube have influence on its performance. Directional control mechanisms are offered by external magnetic fields and are presented to be useful for the on-chip separation of the microbiorobots from a mixture of cells and microtubes. Two surface modification methods are presented as means to improve the coupling efficiency between the microtubes and the sperm cells. By these surface functionalizations, the extracellular matrix protein fibronectin is attached on the inner microtube walls and serves as binding agent for the spermatozoa. Finally, a remote release mechanism for the sperm cells is demonstrated by the incorporation of thermoresponsive material into the microtubes, which makes them fold and unfold upon small temperature changes. This work discusses the potential of such microswimmers for the application in assisted reproduction techniques and gives an outlook on future perspectives.:TABLE OF CONTENTS SELBSTSTÄNDIGKEITSERKLÄRUNG 0 ABSTRACT 1 TABLE OF CONTENTS 3 1 MOTIVATION AND GOALS 5 1.1 MINIATURIZATION: “THERE IS PLENTY OF ROOM AT THE BOTTOM…” 5 1.2 SPERMBOTS: POTENTIAL IMPACT 7 2 BACKGROUND AND STATE-OF-THE-ART 11 2.1 MICROBIOROBOTICS 11 2.2 SPERM MORPHOLOGY AND THEIR JOURNEY TO THE EGG 15 2.3 INFERTILITY AND ASSISTED REPRODUCTION TECHNIQUES 19 2.4 SINGLE CELL RELEASE 22 2.5 STIMULI-RESPONSIVE MATERIALS 25 3 MATERIAL AND METHODS 29 3.1 ROLLED UP TECHNOLOGY 29 3.2 TREATMENT OF BOVINE SPERMATOZOA 32 3.2.1 Preparation of Spermbots 32 3.2.2 Speed Measurements 33 3.2.3 Separation On Chip 33 3.3 SURFACE MODIFICATION OF MICROTUBES 34 3.3.1 Surface Chemistry 35 3.3.2 Microcontact printing 39 3.4 POLYMER TUBE FABRICATION 44 3.4.1 Synthesis of photosensitive monomer 4-Acryloylbenzophenone 44 3.4.2 Synthesis of poly (N-isopropylacrylamide-co-Acryloylbenzophenone) 46 3.4.3 Photolithography of polymeric films 48 3.5 VIABILITY TESTS 51 4 RESULTS AND DISCUSSION 53 4.1 CHARACTERIZATION OF SPERMBOTS 55 4.2 TEMPERATURE INFLUENCE 60 4.3 MAGNETIC CONTROL 62 4.4 SEPARATION ON CHIP 68 4.5 EFFECT OF DECREASED MICROTUBE LENGTH 72 4.6 COUPLING EFFICIENCY 74 4.7 THERMORESPONSIVE POLYMERIC MICROTUBES FOR CELL RELEASE 80 4.8 SPERM VIABILITY TESTS 94 5 SUMMARY AND CONCLUSIONS 97 6 OUTLOOK 101 7 LIST OF FIGURES 107 8 LIST OF TABLES 113 9 ABBREVIATIONS 115 10 CURRICULUM VITAE 117 11 LIST OF PUBLICATIONS 119 JOURNAL ARTICLES 119 CONTRIBUTIONS TO COLLECTED EDITIONS/PROCEEDINGS 121 12 ACKNOWLEDGEMENTS 123 13 REFERENCES 12

Drug-Loaded IRONSperm clusters: modeling, wireless actuation, and ultrasound imaging

Individual biohybrid microrobots have the potential to perform biomedical in vivo tasks such as remote-controlled drug and cell delivery and minimally invasive surgery. This work demonstrates the formation of biohybrid sperm-templated clusters under the influence of an external magnetic field and essential functionalities for wireless actuation and drug delivery. Ferromagnetic nanoparticles are electrostatically assembled around dead sperm cells, and the resulting nanoparticle-coated cells are magnetically assembled into three-dimensional biohybrid clusters. The aim of this clustering is threefold: First, to enable rolling locomotion on a nearby solid boundary using a rotating magnetic field; second, to allow for noninvasive localization; third, to load the cells inside the cluster with drugs for targeted therapy. A magneto-hydrodynamic model captures the rotational response of the clusters in a viscous fluid, and predicts an upper bound for their step-out frequency, which is independent of their volume or aspect ratio. Below the step-out frequency, the rolling velocity of the clusters increases nonlinearly with their perimeter and actuation frequency. During rolling locomotion, the clusters are localized using ultrasound images at a relatively large distance, which makes these biohybrid clusters promising for deep-tissue applications. Finally, we show that the estimated drug load scales with the number of cells in the cluster and can be retained for more than 10 h. The aggregation of microrobots enables them to collectively roll in a predictable way in response to an external rotating magnetic field, and enhances ultrasound detectability and drug loading capacity compared to the individual microrobots. The favorable features of biohybrid microrobot clusters place emphasis on the importance of the investigation and development of collective microrobots and their potential for in vivo applications

Precise Localization and Control of Catalytic Janus Micromotors Using Weak Magnetic Fields

We experimentally demonstrate the precise localization of spherical Pt-Silica Janus micromotors (diameter 5 μm) under the influence of controlled magnetic fields. First, we control the motion of the Janus micromotors in two-dimensional (2D) space. The control system achieves precise localization within an average region-of-convergence of 7 μm. Second, we show that these micromotors provide sufficient propulsion force, allowing them to overcome drag and gravitational forces and move both downwards and upwards. This propulsion is studied by moving the micromotors in three-dimensional (3D) space. The micromotors move downwards and upwards at average speeds of 19.1 μm/s and 9.8 μm/s, respectively. Moreover, our closed-loop control system achieves localization in 3D space within an average region-of-convergence of 6.3 μm in diameter. The precise motion control and localization of the Janus micromotors in 2D and 3D spaces provides broad possibilities for nanotechnology applications

Rethinking the Language Learner in the Post-method Era The Question of Identity

AbstractTaking into consideration the requirements of postmethod era which, according to Kumaravadivelu (2001), called for particular stances of language teachers towards the role of language learning theories in practice and putting the language learner within the frameworks of such theories as competition model, this paper attempts to verify and substantiate the claim that the language learner does not have a particular identity. This substantiation draws on two interrelated issues: (1) as people concerned with language education, teachers are responsible for practice which is mediated by different theories in the postmethod era, and (2) each theory allows for looking at the learner from one perspective according to which only partial understanding of the learner is within reach (only partial theoretical resource to pave the grounds for effective learning opportunities is available). Furthermore, since depending upon a particular theory there might be an understanding of the learner differnet from and sometimes even the opposite of that formed on the basis of another theory, and because in the postmethod era practice in general and moment by moment instances of practice in particular are quite likely to be subjected to opposing theories, it is concluded that the foreign language learner does not have a particular identity