An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

Implementation of porous silicon technology for a fluidic flow-through optical sensor for pH measurements

This work presents an innovative integration of sensing and nano-scaled fluidic actuation in the combination of pH sensitive optical dye immobilization with the electro-osmotic phenomena in polar solvents like water for flow-through pH measurements. These flow-through measurements are performed in a flow-through sensing device (FTSD) configuration that is designed and fabricated at MTU. A relatively novel and interesting material, through-wafer mesoporous silica substrates with pore diameters of 20 -200 nm and pore depths of 500 µm are fabricated and implemented for electro-osmotic pumping and flow-through fluorescence sensing for the first time. Performance characteristics of macroporous silicon (> 500 µm) implemented for electro-osmotic pumping include, a very large flow effciency of 19.8 µLmin-1V-1 cm-2 and maximum pressure effciency of 86.6 Pa/V in comparison to mesoporous silica membranes with 2.8 µLmin-1V-1cm-2 flow effciency and a 92 Pa/V pressure effciency. The electrical current (I) of the EOP system for 60 V applied voltage utilizing macroporous silicon membranes is 1.02 x 10-6A with a power consumption of 61.74 x 10-6 watts. Optical measurements on mesoporous silica are performed spectroscopically from 300 nm to 1000 nm using ellipsometry, which includes, angularly resolved transmission and angularly resolved reflection measurements that extend into the infrared regime. Refractive index (n) values for oxidized and un-oxidized mesoporous silicon sample at 1000 nm are found to be 1.36 and 1.66. Fluorescence results and characterization confirm the successful pH measurement from ratiometric techniques. The sensitivity measured for fluorescein in buffer solution is 0.51 a.u./pH compared to sensitivity of ~ 0.2 a.u./pH in the case of fluorescein in porous silica template. Porous silica membranes are efficient templates for immobilization of optical dyes and represent a promising method to increase sensitivity for small variations in chemical properties. The FTSD represents a device topology suitable for application to long term monitoring of lakes and reservoirs. Unique and important contributions from this work include fabrication of a through-wafer mesoporous silica membrane that has been thoroughly characterized optically using ellipsometry. Mesoporous silica membranes are tested as a porous media in an electro-osmotic pump for generating high pressure capacities due to the nanometer pore sizes of the porous media. Further, dye immobilized mesoporous silica membranes along with macroporous silicon substrates are implemented for continuous pH measurements using fluorescence changes in a flow-through sensing device configuration. This novel integration and demonstration is completely based on silicon and implemented for the first time and can lead to miniaturized flow-through sensing systems based on MEMS technologies.

Micro-fabrication of bio-MEMS based force sensor to measure the force response of living cells

Understanding how a living cell behaves has become a very important topic in today’s research field. Hence, different sensors and testing devices have been designed to test the mechanical properties of these living cells. This thesis presents a method of micro-fabricating a bio-MEMS based force sensor which is used to measure the force response of living cells. Initially, the basic concepts of MEMS have been discussed and the different micro-fabrication techniques used to manufacture various MEMS devices have been described. There have been many MEMS based devices manufactured and employed for testing many nano-materials and bio-materials. Each of the MEMS based devices described in this thesis use a novel concept of testing the specimens. The different specimens tested are nano-tubes, nano-wires, thin film membranes and biological living cells. Hence, these different devices used for material testing and cell mechanics have been explained. The micro-fabrication techniques used to fabricate this force sensor has been described and the experiments preformed to successfully characterize each step in the fabrication have been explained. The fabrication of this force sensor is based on the facilities available at Michigan Technological University. There are some interesting and uncommon concepts in MEMS which have been observed during this fabrication. These concepts in MEMS which have been observed are shown in multiple SEM images.

Integration of room temperature single electron transistor with CMOS subsystem

The single electron transistor (SET) is a charge-based device that may complement the dominant metal-oxide-semiconductor field effect transistor (MOSFET) technology. As the cost of scaling MOSFET to smaller dimensions are rising and the the basic functionality of MOSFET is encountering numerous challenges at dimensions smaller than 10nm, the SET has shown the potential to become the next generation device which operates based on the tunneling of electrons. Since the electron transfer mechanism of a SET device is based on the non-dissipative electron tunneling effect, the power consumption of a SET device is extremely low, estimated to be on the order of 10^-18J. The objectives of this research are to demonstrate technologies that would enable the mass produce of SET devices that are operational at room temperature and to integrate these devices on top of an active complementary-MOSFET (CMOS) substrate. To achieve these goals, two fabrication techniques are considered in this work. The Focus Ion Beam (FIB) technique is used to fabricate the islands and the tunnel junctions of the SET device. A Ultra-Violet (UV) light based Nano-Imprint Lithography (NIL) call Step-and-Flash- Imprint Lithography (SFIL) is used to fabricate the interconnections of the SET devices. Combining these two techniques, a full array of SET devices are fabricated on a planar substrate. Test and characterization of the SET devices has shown consistent Coulomb blockade effect, an important single electron characteristic. To realize a room temperature operational SET device that function as a logic device to work along CMOS, it is important to know the device behavior at different temperatures. Based on the theory developed for a single island SET device, a thermal analysis is carried out on the multi-island SET device and the observation of changes in Coulomb blockade effect is presented. The results show that the multi-island SET device operation highly depends on temperature. The important parameters that determine the SET operation is the effective capacitance Ceff and tunneling resistance Rt . These two parameters lead to the tunneling rate of an electron in the SET device, Γ. To obtain an accurate model for SET operation, the effects of the deviation in dimensions, the trap states in the insulation, and the background charge effect have to be taken into consideration. The theoretical and experimental evidence for these non-ideal effects are presented in this work.

Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT) studies.

Abstract Inhalation of ambient air particles or engineered nanoparticles (NP) handled as powders, dispersions or sprays in industrial processes and contained in consumer products pose a potential and largely unknown risk for incidental exposure. For efficient, economical and ethically sound evaluation of health hazards by inhaled nanomaterials, animal-free and realistic in vitro test systems are desirable. The new Nano Aerosol Chamber for in-vitro Toxicity studies (NACIVT) has been developed and fully characterized regarding its performance. NACIVT features a computer-controlled temperature and humidity conditioning, preventing cellular stress during exposure and allowing long-term exposures. Airborne NP are deposited out of a continuous air stream simultaneously on up to 24 cell cultures on Transwell® inserts, allowing high-throughput screening. In NACIVT, polystyrene as well as silver particles were deposited uniformly and efficiently on all 24 Transwell® inserts. Particle-cell interaction studies confirmed that deposited particles reach the cell surface and can be taken up by cells. As demonstrated in control experiments, there was no evidence for any adverse effects on human bronchial epithelial cells (BEAS-2B) due to the exposure treatment in NACIVT. The new, fully integrated and transportable deposition chamber NACIVT provides a promising tool for reliable, acute and sub-acute dose-response studies of (nano)particles in air-exposed tissues cultured at the air-liquid interface.

Nano-mechanical transduction of polymer micro-cantilevers to detect bio-molecular interactions

Using variothermal polymer micro-injection molding, disposable arrays of eight polymer micro-cantilevers each 500 μm long, 100 μm wide and 25 μm thick were fabricated. The present study took advantage of an easy flow grade polypropylene. After gold coating for optical read-out and asymmetrical sensitization, the arrays were introduced into the Cantisens(®) Research system to perform mechanical and functional testing. We demonstrate that polypropylene cantilevers can be used as biosensors for medical purposes in the same manner as the established silicon ones to detect single-stranded DNA sequences and metal ions in real-time. A differential signal of 7 nm was detected for the hybridization of 1 μM complementary DNA sequences. For 100 nM copper ions the differential signal was found to be (36 ± 5) nm. Nano-mechanical sensing of medically relevant, nanometer-size species is essential for fast and efficient diagnosis.

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

Improved Space Object Orbit Determination Using CMOS Detectors

CMOS-sensors, or in general Active Pixel Sensors (APS), are rapidly replacing CCDs in the consumer camera market. Due to significant technological advances during the past years these devices start to compete with CCDs also for demanding scientific imaging applications, in particular in the astronomy community. CMOS detectors offer a series of inherent advantages compared to CCDs, due to the structure of their basic pixel cells, which each contains their own amplifier and readout electronics. The most prominent advantages for space object observations are the extremely fast and flexible readout capabilities, feasibility for electronic shuttering and precise epoch registration,and the potential to perform image processing operations on-chip and in real-time. Here, the major challenges and design drivers for ground-based and space-based optical observation strategies for objects in Earth orbit have been analyzed. CMOS detector characteristics were critically evaluated and compared with the established CCD technology, especially with respect to the above mentioned observations. Finally, we simulated several observation scenarios for ground- and space-based sensor by assuming different observation and sensor properties. We will introduce the analyzed end-to-end simulations of the ground- and spacebased strategies in order to investigate the orbit determination accuracy and its sensitivity which may result from different values for the frame-rate, pixel scale, astrometric and epoch registration accuracies. Two cases were simulated, a survey assuming a ground-based sensor to observe objects in LEO for surveillance applications, and a statistical survey with a space-based sensor orbiting in LEO observing small-size debris in LEO. The ground-based LEO survey uses a dynamical fence close to the Earth shadow a few hours after sunset. For the space-based scenario a sensor in a sun-synchronous LEO orbit, always pointing in the anti-sun direction to achieve optimum illumination conditions for small LEO debris was simulated.

Uptake efficiency of surface modified gold nanoparticles does not correlate with functional changes and cytokine secretion in human dendritic cells in vitro.

Engineering nanoparticles (NPs) for immune modulation require a thorough understanding of their interaction(s) with cells. Gold NPs (AuNPs) were coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA) or a mixture of both with either positive or negative surface charge to investigate uptake and cell response in monocyte-derived dendritic cells (MDDCs). Inductively coupled plasma optical emission spectrometry and transmission electron microscopy were used to confirm the presence of Au inside MDDCs. Cell viability, (pro-)inflammatory responses, MDDC phenotype, activation markers, antigen uptake and processing were analyzed. Cell death was only observed for PVA-NH2 AuNPs at the highest concentration. MDDCs internalize AuNPs, however, surface modification influenced uptake. Though limited uptake was observed for PEG-COOH AuNPs, a significant tumor necrosis factor-alpha release was induced. In contrast, (PEG+PVA)-NH2 and PVA-NH2 AuNPs were internalized to a higher extent and caused interleukin-1beta secretion. None of the AuNPs caused changes in MDDC phenotype, activation or immunological properties.

Alginate-coated chitosan nanogels differentially modulate class-A and class-B CpG-ODN targeting of dendritic cells and intracellular delivery.

UNLABELLED CpG-oligodeoxynucleotides (CpG-ODNs) interact with dendritic cells (DCs), but evidence is less clear for CpG-ODN admixed with or incorporated into vaccine delivery vehicles. We loaded alginate-coated chitosan-nanogels (Ng) with class-A or class-B CpG-ODN, and compared with the same CpG-ODNs free or admixed with empty Ng. Experiments were performed on both porcine and human blood DC subpopulations. Encapsulation of class-A CpG-ODN (loading into Ng) strongly reduced the CpG-ODN uptake and intracellular trafficking in the cytosol; this was associated with a marked deficiency in IFN-α induction. In contrast, encapsulation of class-B CpG-ODN increased its uptake and did not influence consistently intracellular trafficking into the nucleus. The choice of CpG-ODN class as adjuvant is thus critical in terms of how it will behave with nanoparticulate vaccine delivery vehicles. The latter can have distinctive modulatory influences on the CpG-ODN, which would require definition for different CpG-ODN and delivery vehicles prior to vaccine formulation. FROM THE CLINICAL EDITOR This basic science study investigates the role of class-A and class-B CpG-oligodeoxynucleotides loaded into alginate-coated chitosan nanogels, demonstrating differential effects between the two classes as related to the use of these nanoformulations as vaccine delivery vehicles.

Liulin silicon semiconductor spectrometers as cosmic ray monitors at the high mountain observatories Jungfraujoch and Lomnický stit

Currently, most cosmic ray data are obtained by detectors on satellites, aircraft, high-altitude balloons and ground (neutron monitors). In our work, we examined whether Liulin semiconductor spectrometers (simple silicon planar diode detectors with spectrometric properties) located at high mountain observatories could contribute new information to the monitoring of cosmic rays by analyzing data from selected solar events between 2005 and 2013. The decision thresholds and detection limits of these detectors placed at Jungfraujoch (Switzerland; 3475 m a.s.l.; vertical cut-off rigidity 4.5 GV) and Lomnicky stıt (Slovakia; 2633 m a.s.l.; vertical cut-off rigidity 3.84 GV) highmountain observatories were determined. The data showed that only the strongest variations of the cosmic ray flux in this period were detectable. The main limitation in the performance of these detectors is their small sensitive volume and low sensitivity of the PIN photodiode to neutrons.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Cellular Trafficking of Single and Multistage Vectors

Nanomedicine is an innovative field of science which has recently generated many drug delivery platforms with exciting results. The great potential of these strategies rely on the unique characteristics of the devices at the nano-scale in terms of long time circulation in the blood stream, selective accumulation at the lesions sites, increased solubility in aqueous solutions, etc. Herein we report on a new drug delivery system known as a multistage system which is comprised of non-spherical, mesoporous silicon particles loaded with second stage nanoparticles. The rationally designed particle shape, the possibility to modulate the surface properties and the degree of porosity allow these carriers to be optimized for vascular targeting and to overcome the numerous biological barriers found in drug delivery. In this study we investigated the intra and inter cellular trafficking of the multistage system in endothelial cells bringing evidence of its bio-compatibility as well as its ability to perform multiple intra and inter cellular tasks. Once internalized in cells, the multi-particle construct is able to dissociate, localizing in different subcellular compartments which can be targeted for exocytosis. In particular the second stage nanoparticles were found to be secreted in microvesicles which can act as mediators of transfer of particles across the endothelium and between different endothelial and cancer cells.

Alleviation of solar ultraviolet radiation (UVR)-induced photoinhibition in diatom Chaetoceros curvisetus by ocean acidification

The study aimed to unravel the interaction between ocean acidification and solar ultraviolet radiation (UVR) in Chaetoceros curvisetus. Chaetoceros curvisetus cells were acclimated to high CO2 (HC, 1000 ppmv) and low CO2 concentration (control, LC, 380 ppmv) for 14 days. Cell density, specific growth rate and chlorophyll were measured. The acclimated cells were then exposed to PAB (photosynthetically active radiation (PAR) + UV-A + UV-B), PA (PAR + UV-A) or P (PAR) for 60 min. Photochemical efficiency (phi PSII), relative electron transport rate (rETR) and the recovery of ?PSII were determined. HC induced higher cell density and specific growth rate compared with LC. However, no difference was found in chlorophyll between HC and LC. Moreover, phi PSII and rETRs were higher under HC than LC in response to solar UVR. P exposure led to faster recovery of phi PSII, both under HC and LC, than PA and PAB exposure. It appeared that harmful effects of UVR on C. curvisetus could be counteracted by ocean acidification simulated by high CO2 when the effect of climate change is not beyond the tolerance of cells.