Cryo-Electron Microscopy (Cryo-EM) enables high-resolution bio-molecular imaging but is limited by inconsistent vitrified ice thickness and quality during sample preparation. This thesis develops MEMS-based Electrical Impedance Spectroscopy (EIS) as a rapid, non-destructive method to extract quantitative thickness information from thin liquid and ice layers prior to vitrification. Numerical simulations in COMSOL Multiphysics, supported by analytical parallel-plate and equivalent-circuit models, demonstrate that measurable impedance variations emerge for dielectric layers in the 50–500 nm range under idealized conditions, with monotonic and near-linear relationships between impedance-derived metrics and layer thickness over selected frequency windows. Experimental validation using fabricated MEMS sensor chips by DENSsolutions and white-light interferometry for ground-truth thickness confirm that EIS measurements demonstrate qualitative agreement in impedance–thickness correlation for liquid water, as impedance measurements can be reliably calibrated to thickness with repeatable trends and nanometer-scale sensitivity. Overall, this study demonstrates that MEMS-based EIS is a feasible and scalable approach for quantitative pre-vitrification thickness monitoring in cryo-EM workflows, offering a pathway toward reduced trial-and error grid screening and improved reproducibility, while clearly identifying the remaining experimental and calibration challenges required for practical deployment. ... Read more

Early detection of plant diseases is crucial to minimize crop losses and reduce the usage of pesticides. Electronic Nose (E-Nose) detect volatile organic compounds (VOCs) emitted by stressed or diseased plants, and one such device is a pixelated capacitive sensor (PCS). We designed and built a setup to investigate the influence of light (wavelength and intensity) on the sensitivity of a functionalized PCS for VOCs detection. Our test results indicate that UV illumination, particularly at 375 nm, enhances the sensitivity of the PCS, with a 3-fold enhancement compared to dark conditions. The sensor showed fast saturation (<1 min) and recovery (<2 min) times, confirming the effectiveness of the chamber design for repeatable gas exposure. ... Read more

Abstract—Resonant mass sensors are emerging as innovative instruments for particle measurement, which utilize the resonance frequency shifts of oscillatory structures. Micro- and nano-pillars have been adopted as the resonators for the high sensitivity of this mass sensing technique. However, existing frequency measurement methods for micro- and nano-mechanical resonators are mostly based on piezoresistive readouts, which were challenged for the integration of such small structures as submicron pillars. In this paper, aluminum nitride surface acoustic wave (SAW) delay line devices were utilized to investigate the potential for measuring the resonance frequencies of several micro/nano-pillars simultaneously. SAW delay line devices for this resonance measuring application have been successfully designed, fabricated, and tested. Both two-photon polymerized (TPP) pillars and focused ion beam (FIB) deposited pillars have been incorporated into the delay line devices. COMSOL simulations in this work have demonstrated the feasibility of multi-resonance measurements. Small peaks, likely corresponding to pillar resonances, were detected and showed some correlation with the resonances. These results suggest that using SAW delay lines is a promising technique for multi-resonance detection of nanopillar arrays. This work paved the way for high-sensitivity
mass sensor development with effective SAW devices and methods of incorporating pillars. ... Read more

Subcellular biopsy (SCB) using atomic force microscopy (AFM) with a microfluidic cantilever enables the extraction of biological samples from living cells. Traditional cryo-electron microscopy (cryo-EM) sample preparation methods are unsuitable for the small volumes from SCB. This study presents a novel method to prepare cryo-EM samples directly from SCB samples, integrating dispensing, thickness measurement, and local temperature control to minimize evaporation. The method uses a microfluidic AFM cantilever for dispensing, a Mach-Zehnder interferometer for real-time thickness measurement, and plunge freezing for vitrification. The system successfully demonstrated dispensing picoliter droplets, with potential for femtoliter volumes. The temperature-controlled grid holder reduced evaporation rates, allowing sufficient time for real-time thickness measurement. The system has demonstrated the required transition speed and plunge velocity. Initial cryo-EM thickness measurements demonstrate the method's potential. In conclusion, a novel system for subcellular biopsy sample preparation for cryo-EM was designed, fabricated, and tested. It maintains picoliter sample volumes on a temperature-controlled EM grid, enabling real-time thickness measurement and immediate plunge freezing. ... Read more

This study presents the development of a pro grammable microfluidic device designed for precise hydrodynamic manipulation of particles and fluids. Utilising femtosecond laser ablation and cold oxygen plasma bonding, the device features highly accurate microfluidic inlets with minimal surface rough ness, ensuring consistent flow dynamics. Experimental validation confirmed the device’s ability to generate uniform flow fields and half Rankine bodies, demonstrating its potential for real time reconfigurability in various applications, including particle manipulation and flow field analysis. The research lays the groundwork for future advancements in microfluidic technology, emphasising high precision and structural integrity. ... Read more

Organ-on-chip (OoC) technology has transformed biomedical research by providing a platform to simulate physiological conditions for drug development and disease modeling. Incubator-free OoC systems offer notable advantages over traditional approaches, including enhanced adaptability and impermeability to gases. Nevertheless, achieving precise oxygen regulation remains a challenge in such systems. This thesis investigates the application of femtosecond (Fs) laser ablation to fabricate a glass microfluidic gas exchange system for accurate oxygen regulation in incubator-free OoCs.
Through successful engraving of fluidic channels and integration of an off-the-shelf optical oxygen sensor, this study highlights the efficacy of Fs laser technology in the rapid prototyping of intricate glass microfluidic devices. Despite encountering challenges such as dimensional losses and debris clogging, the study presents a functional gas exchanger prototype. Future research directions include optimization efforts, addressing issues like gas permeation through connectors, and testing under physiological conditions to further advance OoC technology. ... Read more

When designing mechanical components, they commonly undergo multiple modeling phases for stress determination using analytical or numerical methods like the Finite Element Method (FEM). This is followed by experimental validation performed via stress mapping to identify and account for possible mechanical failure within the design phase. Among the experimental stress mapping techniques, mechanosensing is gaining rapidly increasing attention by research and industry. Mechanosensing is a chemistry-based technique that utilizes molecules called mechanophores. When mechanophores are embedded in transparent polymers, they act as mechanical probes sensing stress/strain throughout the polymer by emitting fluorescence under deformation. While studies have shown the capabilities of mechanophores as stress/strain probes qualitatively, it is currently not known how the mechanophore activation is correlated with the stress/strain-based quantities from a solid mechanics perspective. This study addresses this problem from a phenomenological viewpoint to fill the research area gap. In this work, using a uniaxial tensile testor, experiments are conducted on a Polydimethylsiloxane (PDMS) with a mechanophore spiropyran embedded homogeneously in the bulk of the polymer. The fluorescence data captured in the tests is correlated with the numerically obtained continuum mechanics stress/strain quantities. These correlations will be useful in giving directions towards research in the fundamental understanding of the mechanics of mechanophores, thereby bridging the gap between chemistry and mechanics of mechanosensors. This will pave the way towards optical-only in-situ measurements of stress-strain behavior. ... Read more

This study investigates the processes of cell membrane puncturing and the factors influencing these processes through force spectroscopy experiments. A custom-designed tall and sharp tip was fabricated on a commercially available microcantilever using two-photon polymerization (2PP). Force spectroscopy curves were measured at several locations on the cell while the tip was inserted inside the cell. A cell membrane insertion event, identified as a ”force drop” in force spectroscopy curves, is often hardly visible or does not occur. In this study, the effects of probe tip diameter, cantilever spring constant, insertion velocity, and cell height of mouse preosteoblast cells were systematically analysed for their influence on how often the force drops occur and how clearly they are visible. Data processing algorithms were employed to process thousands of force spectroscopy curves, enabling large sample statistical analysis using multiple linear regression. The results demonstrate that selecting cells with maximum height and size increases force drop occurrence from 10% to 40%. Furthermore, while decreasing the tip diameter from 2 to 0.7 µm did not affect the occurrence rate, it increased force drop visibility from ∼250 to ∼600 pico-Newton (pN). Additionally, cantilevers with a spring constant of 0.2 N/m achieved higher force drop occurrence (40%) and visibility (∼300 pN), compared to more flexible cantilevers with a spring constant of 0.02 N/m (20% occurrence, ∼40 pN visibility). These findings provide valuable insights for optimizing experimental parameters in cell membrane mechanics research. ... Read more

Cancer metastasis, the spread of cancer cells from a primary tumor to distant organs, is the leading cause of cancer-related deaths. During metastasis, cancer cells undergo significant changes in their mechanical properties, including alterations in cell elasticity. Cancer cells generally exhibit higher elasticity than normal cells, a key feature that may contribute to their ability to migrate and invade other tissues. Although extensive research has focused on cell-substrate interactions, these studies do not fully replicate the physiological environment, where cells are frequently in direct contact with each other. In this study, Atomic Force Microscopy (AFM) in force spectroscopy mode, utilizing a micro-sized cantilever with a hemispherical tip, was used to quantify the Young’s modulus (E) of MCF-7 breast cancer cells in two configurations: cell-substrate and cell-cell. The resulting force-indentation curves were analyzed and fitted to the Hertz contact model to determine the Young’s modulus. The results showed that the Young’s modulus in the cell-cell configuration was Ecc = 205.8 ± 50.88 Pa, while in the cell-substrate configuration, it was Ecs = 187.95 ± 78.26 Pa. These findings suggest that MCF-7 cells are slightly less elastic in the cell-cell configuration. This challenges the expectation of a more significant difference between the two configurations and highlights the importance of considering biological variability and experimental conditions when interpreting cell mechanical properties. ... Read more

Micropumps are essential for providing controlled fluid dynamics in Organ-on-a-Chip (OoC) devices. Additive manufacturing builds up prototypes in several hours with a free-geometry advantage. Therefore, this master's thesis investigates the utilization of additive manufacturing to produce a micropump with a flowrate in the range of several $\mu l/min$ for OoC applications. A ball valve-based piezoelectric micropump was fabricated with a mSLA 3D printer. This micropump generates unidirectional flow through the reciprocating motion of the piezoelectric actuator and the movement of a ball within the conical channel. The virtual mass due to the inertia of the fluid inside the chamber shifts the resonance frequency of the piezoelectric actuator from the 1600 Hz to 43 Hz. The maximum flow rate of $26.5 \mu l/min$ was generated when the applied sinusoidal voltage was 240 Vpp at 5Hz and the maximum back pressure of 36.5 mbar was obtained under this power supply. These results confirm that additive manufacturing provides a promising option for miniature pump manufacturing. ... Read more

Photonics biosensors convert biomolecular interactions into quantifiable optical signals for biomedical analysis, which enable continuous monitoring of health indicators. Among them the microring resonator has a good sensing performance and a very broad application prospect. This thesis studies sensing with microfluidic integrated microring resonator photonic microchips.
This thesis adopts finite element method to simulate the optical behavior of waveguides and reactions in the microfluidic channel. The microfluidic channel was designed and prepared and then integrated to the photonic chip. The optical performance parameters of the micro ring resonator were tested by using a high-precision optical test system. The sensing performance of waveguide microring was studied using different aqueous solution as the detection object. The feasibility and effectiveness of the optical waveguide chip sensing have been preliminary verified.

... Read more

A stage heater for inverted optical microscopy was developed as an add-on to TU Delft portable integrated microfluidic platform. The platform was modified to gain additional functionality and portability. The stage heater was designed to work with custom PDMS microfluidic chips and commercial polymer chips which highlights the adaptability of the setup. The on-off temperature control was evaluated via an external thermocouple sensor, and the standard deviation of temperature in the cell chamber did not exceed 0.3 °C. An intermittent flow mode (flow for 1 minute at 10 µl/min, pause for 10 minutes) was introduced to match the requirements of an experiment involving T-cell and tumour spheroid interaction. The stage heater was deemed compatible with fluorescent and transmission imaging modes for up to 20x magnifications when equipped with a PET ITO-coated bottom heater with a window underneath the cell chamber. An increase in T-cell activity was observed with enabled heating. The process of T-cells attacking a spheroid via a time series of images was captured. ... Read more

Much research is conducted on the biomechanical properties of cells, particularly in cancer research. Cell stiffness and cell adhesion are two properties often studied, because they are important for the functioning of a cell. To study these properties, atomic force microscopy(AFM) is often used, using nanoindentation for stiffness analysis and fluid force microscopy(FluidFM) for adhesion analysis. However, these techniques often involve different cantilever probes, which complicates the measurement of both properties in a single cell. To make it possible to study cell stiffness and cell adhesion of a single cell, a multifunctional microfluidic AFM cantilever is developed in this research. This cantilever is equipped with a blunted pyramid-shaped tip for nanoindentation enabling the measurement of cell stiffness and imaging. Moreover, it features a channel running through its length, with an aperture on the tip, facilitating fluid force microscopy for determining cell adhesion. The multifunctional cantilever is fabricated using a multiscale 3d-printing technique, where stereo lithography is used for the larger parts, and two-photon polymerisation is used for the cantilever. To demonstrate the capabilities of the cantilever, tests are conducted on various substrates. Nanoindentation is demonstrated on PDMS, hydrogel and endothelial cells to determine the Young’s modulus of these materials. Fluid force microscopy is showcased by examining prostate cancer cells (PCA-3), removing the cells from the substrate while measuring the adhesion forces. The imaging capabilities of the cantilever were also demonstrated by generating a height map and a Young’s modulus map through quantitative imaging of a hydrogel spheroid. This research shows that combining the properties of nanoindentation and FluidFM cantilevers into a single cantilever is possible. Integrating these functionalities into a single cantilever makes doing more mechanical measurements on a single cell possible. ... Read more

Single photon detectors are an important optical sensing tool in many industries. However, these highly efficient detectors suffer from variations in the size of an optical cavity when cooling down to 3K. An active positioning system is therefore required to correct the relative position of the fibre to the detector, so an optimal and reproducible cavity size can be achieved, thereby maximizing their efficiency. The literature study performed on cryogenic precision actuators showed that a stepper motor in combination with a motion reduction mechanism is the most feasible design. A stepper motor and a precision screw were two fundamental components of the preliminary design, so their lubrication was removed and were proven to work cryogenically. The rest of the design was based around these fundamental components. Room temperature tests were done to show the functionality of the design and it showed a positioning resolution between 10 nm and 30nm with just a 100nm loss of motion when unloading and loading the system. Cryogenic tests achieved similar results and it showed that the reflected power of a detector was reduced from 8.8% to 3.6%, indicating that the maximum achievable detector efficiency increased from 91.2% to 96.4%. ... Read more

An Organ-on-a-Chip (OoC) is a microfluidic device that mimics an organ function on a chip. The goal of the OoC technology is to create disease models of various organs and use them to minimise animal testing. One of the important parameters to maintain the cells/tissues on the OoC is the temperature, typically at 37 °C. Usually, cell culture incubators are used to maintain the temperature of cells on the OoC. However, it is inconvenient for external flow control instrumentation to interface with OoC inside the incubator, and other temperatures being not possible. Therefore, an on-chip temperature controller is needed. In this report, a temperature control system for an OoC is designed, implemented and tested. To cover a wide temperature range, multiple peltier elements are chosen to confine the temperature control only at the region close to cell chambers in the OoC. A resistive temperature detector (RTD) is used as feedback to control the temperature by a proportional-integral-derivative (PID) controller. To avoid any heat loss beyond the Organ-on-a-Chip, a 3D-printed polylactic acid (PLA) holder with good thermal insulating properties is used. The holder is designed for four chips to accommodate control chips along with test chips. A dedicated PCB with control electronics is designed and implemented to control the temperature on all four chips powered by a battery. A temperature uniformity of +/- 0.7 °C of 19 mm at 68 μL/min and 23 mm at 10 μL/min along a total microfluidic channel length of 40 mm (from inlet to outlet), a width of 5 mm, and a depth of 0.3 mm before compression, was achieved. The response time to recover from changes in the temperature is approximately 2 minutes. With a power consumption of 0.84 Watts per chip to maintain 37 °C, four chips can function on a 20000 mAh battery for a minimum of 4.5 hours (only cooling at a minimum of 18 °C) and a maximum of 8.3 hours (only heating at 37 °C) before recharge. The temperature control system was tested on Hepatocytes and Cholangiocytes for liver-on-a-chip applications, which showed that the oxygen disappearance rate is 9.87 times faster when maintained at 37 °C compared to room temperature. ... Read more

Microvalves are useful components for several microfluidic applications in which they control the fluid flow in a microfluidic system. Most microvalves to date are made by using silicon micro-machining, which is a complex manufacturing process, or soft lithography using Polydimethylsiloxane (PDMS) which has a high gas permeability. Next to that most microvalves in literature have small actuation forces resulting in small pressure ranges and large leakages at low pressure levels. In this paper, a normally open microvalve which is fabricated by only using 3D printing techniques with a bio-compatible resin is presented, making it more easy and accessible to manufacture. The novelty is the integrated micro-channels, membrane and microfluidic connections in a single 3D printed piece. The utilized actuator is a commercially available piezo stack, which has a displacement of 34 µm at 150V. Due to its large actuation force the microvalve can modulate fluids from -600 to 600 mbar, with measured flow-rate levels between 0-90 µl/min and projected flow-rate levels between 0-410 µl/min. In fully closed state the leakage-rate of the microvalve is 1.67 µl/min at 600 mbar, with a static power consumption of 442.5 mW. Subsequently it is shown that after using higher clamping torques (> 0.7 Nm) the microvalve can operate leakage free up to 1.5 bar. Additionally a 3-to-1 fluid selector is designed using three microvalves, which can be integrated into a portable microfluidic platform for Organ-on-Chip (OoC) applications. ... Read more

The processes that take place within cells are complex. Single-cell analysis is a method that is employed to gain further understanding of the working mechanisms on a single-cellular level. The controlled transport of substances through the cell membrane is important for studying the behaviour and responses of single living cells. Femtopipettes are used as a means to accurately target and sample cells with high viability rates. Different actuation principles exist for femtopipettes, but pressure actuation is identified as the most versatile and straightforward method. However, the volume dosing resolution of pressure driven femtopipettes lags behind other actuation methods. Throughout literature, the minimum reported dose is identified as 100 femtolitre (1 fL= 10^-15 L), achieved by applying a pressure pulse to a femtopipette. In this work, two new concepts are proposed and researched with the goal of increasing the volume dosing resolution of pressure driven femtopipettes. A multi-scale 3D printing strategy was employed where functional femtopipettes were successfully printed using two-photon-polymerization (2PP). The first concept incorporates a physical barrier in the form of a flexible membrane into the femtopipette. It was expected that the volume actuated by the deformation of the membrane could be controlled and calibrated. The deformation of 2PP printed membranes was characterized and the achieved volume displacement was well in the desired range of 100 fL. However, tests with liquid dosing did not succeed in achieving this same range, and difficulties were experienced with reproducibility. The second concept exploited the phenomenon of capillarity by incorporating axisymmetrical phaseguides as a means to control the position of the liquid-air meniscus. Consecutive geometrical steps were created that allowed for discrete and robust control over the liquid portion within the femtopipette. Step sizes of 10 picolitre (1 pL= 10^-12 L), 200 fL and 60 fL were fabricated and successfully tested, breaching beyond the current state of the art. This concept thus demonstrates both the level of customization and the unprecedented volume dosing resolution that was achieved. ... Read more

Electret transducers utilize the electric field generated by an electret, a dielectric with a quasi-permanent embedded charge, to induce charge on an electrode. When the electret is moved relative to the electrode, the induced charge magnitude on the electrode changes, generating a current that can be used to convert mechanical energy into electrical energy. Micro-electret transducers are promising alternatives to conventional electromagnetic transducers for small-scale energy harvesting as they can achieve a high voltage output at low frequencies, can be miniaturized effectively, and can be manufactured using microfabrication methods.

One-dimensional electrostatic models have been developed to predict the power output of electret transducers. However, for micro-electret transducers, fringing fields play a large role in the electrostatic domain. To be able to more accurately predict the output characteristics of micro-electret transducers, a two-dimensional (2D) electrostatic model is proposed. To verify the 2D model, a novel micro-electret transducer is designed. The novel electret design allows the micro-electret transducer to embed charges of only one polarity, increasing the power output of the electret transducer.

The novel 2D model more accurately predicts the power output characteristics of the micro-electret transducer with the voltage output deviating 57%, compared with 317% by the conventional model predictions. Furthermore, the novel unipolar micro-electret transducer achieves double the power output and better charge stability compared with conventional electret transducers. ... Read more

Any resonance based sensing method such as AFM or mass sensing using microfluidic cantilevers require high frequency stability. For mass measurement, a stable frequency allows for a lower mass resolution. One of the most commonly used methods to actuate these resonators is using piezoacoustics. A major downside of this method is the presence of spurious resonances, which corrugate the frequency response and can potentially degrade mass sensitivity. One way get rid of these spurious peaks is by
actuating photothermally (using laser light). This thesis is focused around designing and building a photothermal AFM to explore this. Additionally, some experiments are performed comparing piezoacoustic actuation to photothermal actuation in terms of the aforementioned frequency stability and
spurious peaks. ... Read more

Facioscapulohumeral muscle dystrophy (FSHD) is the third most common muscle disease in the world. No cure has been found for FSHD, and current treatments focus on alleviating the symptoms. The disease is caused by a genetic error in 1/1000 - 1/200 of the nuclei in a multinucleated skeletal muscle cell. Studying that specific nucleus, by removing it from the cell, perform transcriptomics on it and determining the cell viability after the nucleus removal, can provide important information about FSHD.

In this work, a multiscale 3D printing approach was optimized to fabricate a microfluidic atomic force microscopy (AFM) cantilever that can remove a nucleus from a living cell. With the device mounted on an AFM system, cell experiments were performed, which showed that the nucleus can be removed from a cell using 3D printed microfluidic cantilevers. The printing methods can be used to fabricate various types of suspended microfluidic devices to perform single-cell biopsy and biophysical characterization of single-cells. ... Read more