Noemi Rozlosnik

Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ1–40 using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ1–42 were compatible with the conclusions for Aβ1–40 even though the low solubility of Aβ1–42 precluded examination under the same conditions as for the Aβ1–40. Experiments with Aβ1–16 and Aβ1–28 showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ1–40 depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

Comparative study on aptamers as recognition elements for antibiotics in a label-free all-polymer biosensor

We present an all-polymer electrochemical microfluidic biosensor using Topas(®) as substrate and a conductive polymer bilayer as electrode material. The conductive bilayer consists of tosylate doped poly(3,4-ethylenedioxythiophene) (PEDOT:TsO) and the hydroxymethyl derivative PEDOT-OH:TsO, which was covalently functionalized with two aptamer probes with affinity to ampicillin or kanamycin A, respectively. Using electrochemical impedance spectroscopy (EIS) we were able to detect ampicillin in a concentration range from 100pM to 1μM and kanamycin A from 10nM to 1mM. The obtained EIS spectra were fitted with an equivalent circuit model successfully explaining the impedance signal. Real samples from regular ultra-high temperature treated low-fat milk spiked with ampicillin were successfully tested to assess the functionality of the sensor with real samples. In conclusion, we have demonstrated the applicability of the newly developed platform for real time, label-free and selective impedimetric detection of commonly used antibiotics. Additionally it was possible to detect ampicillin in a milk sample at a concentration below the allowed maximum residue limit (MRL) in the European Union.

Injection molded chips with integrated conducting polymer electrodes for electroporation of cells

We present the design-concept for an all polymer injection molded single use microfluidic device. The fabricated devices comprise integrated conducting polymer electrodes and Luer fitting ports to allow for liquid and electrical access. A case study of low voltage electroporation of biological cells in suspension is presented. The working principle of the electroporation device is based on a focusing of the electric field by means of a constriction in the flow channel for the cells. We demonstrate the use of AC voltage for electroporation by applying a 1 kHz, ±50 V square pulse train to the electrodes and show delivery of polynucleotide fluorescent dye in 46% of human acute monocytic leukemia cells passing the constriction.

High sensitivity point-of-care device for direct virus diagnostics

Influenza infections are associated with high morbidity and mortality, carry the risk of pandemics, and pose a considerable economic burden worldwide. To improve the management of the illness, it is essential with accurate and fast point-of-care diagnostic tools for use in the field or at the patients bedside. Conventional diagnostic methods are time consuming, expensive and require specialized laboratory facilities. We present a highly sensitive, highly specific, and low cost platform to test for acute virus infections in less than 15 min, employing influenza A virus (H1N1) as an example of its usability. An all polymer microfluidic system with a functionalized conductive polymer (PEDOT-OH:TsO) microelectrode array was developed and exploited for label free and real time electrochemical detection of intact influenza A virus (H1N1) particles. DNA aptamers with affinity for influenza A virus (H1N1) were linked covalently to the conductive polymer microelectrodes in the microfluidic channel. Based on changes in the impedance when virions were captured by immobilized probes, we could detect clinically relevant concentrations of influenza A virus (H1N1) in saliva. This is a new, stable and very sensitive point-of-care platform for detection and diagnostics of intact virus particles.

Polymer based biosensor for rapid electrochemical detection of virus infection of human cells

The demand in the field of medical diagnostics for simple, cost efficient and disposable devices is growing. Here, we present a label free, all-polymer electrochemical biosensor for detection of acute viral disease. The dynamics of a viral infection in human cell culture was investigated in a micro fluidic system on conductive polymer PEDOT:TsO microelectrodes by electrochemical impedance spectroscopy and video time lapse microscopy. Employing this sensitive, real time electrochemical technique, we could measure the immediate cell response to cytomegalovirus, and detect an infection within 3h, which is several hours before the cytopathic effect is apparent with conventional imaging techniques. Atomic force microscopy and scanning ion conductance microscopy imaging consolidate the electrochemical measurements by demonstrating early virus induced changes in cell morphology of apparent programmed cell death.

Ultrathin, Ultrasmooth Gold Layer on Dielectrics without the Use of Additional Metallic Adhesion Layers

With advances in the plasmonics and metamaterials research field, it has become more and more important to fabricate thin and smooth Au metal films in a reliable way. Here, by thin films we mean that their average height is below 10 nm and their average roughness is below 5% of the total thickness. In this article, we investigated the use of amino- and mercaptosilanes to increase the adhesion of Au on Si wafers, thus obtaining a smooth and thin layer. This method does not include the use of other metals to improve the adhesion of gold, like Ti or Cr, since they would reduce the optical characteristics of the structure. Our results show that layers having 6 nm thickness and below 0.3 nm roughness can be reproducibly obtained using aminosilanes. Layers having a nominal thickness of 5 nm have a yield of 58%; thus, this thickness is the limit for the process that we investigated.

Plasma Surface Modification of Glass-Fibre-Reinforced Polyester Enhanced by Ultrasonic Irradiation

During atmospheric pressure plasma treatment, reactive species generated in the plasma diffuse through a boundary gas layer which is adsorbed at the material surface. Many of the reactive species become inactivated before reaching the surface due to their short lifetime. The efficiency of plasma treatment can be highly enhanced by simultaneous high-power ultrasonic irradiation of the treating surface, because the delivered acoustic energy can reduce the thickness of the boundary gas layer. Here surfaces of glass-fibre-reinforced polyester (GFRP) plates were treated using an atmospheric pressure dielectric barrier discharge in helium with ultrasonic irradiation, particularly for the adhesion improvement. The ultrasound was irradiated through a powered mesh electrode using a high-power gas-jet ultrasonic generator. The discharge mode changed from glow to filamentary by the ultrasonic irradiation. The surface characterizations were performed using contact angle measurements, X-ray photoelectron spectroscopy (XPS) and atomic force mictroscopy (AFM). O/C ratios at the GFRP surfaces before the treatments, after 30-s plasma treatment, and after 30-s plasma treatment with ultrasonic irradiation were 0.295, 0.385 and 0.447, respectively. This indicated that the plasma treatment oxidized and roughened the GFRP surface, and the ultrasonic irradiation further enhanced the oxidation. It is concluded that plasma treatment efficiency for adhesion improvement of GFRPs is enhanced by the ultrasonic irradiation.

Cell-Based Biosensors: Electrical Sensing in Microfluidic Devices.

Cell-based biosensors provide new horizons for medical diagnostics by adopting complex recognition elements such as mammalian cells in microfluidic devices that are simple, cost efficient and disposable. This combination renders possible a new range of applications in the fields of diagnostics and personalized medicine. The review looks at the most recent developments in cell-based biosensing microfluidic systems with electrical and electrochemical transduction, and relevance to medical diagnostics.

Modification of Glassy Carbon Surfaces by Atmospheric Pressure Cold Plasma Torch

The effect of plasma treatment on glassy carbon (GC) surfaces was studied with adhesion improvement in mind. A newly constructed remote plasma source was used to treat GC plates. Pure He and a dilute NH3/He mixture were used as feed gases. Optical emission spectroscopy was performed for plasma torch diagnostics. The treatment resulted in surface etching, substantially enhanced by NH3, as well as a roughening of the surface as measured by atomic force microscopy. Furthermore, the treated area showed an increased wettability indicating the addition of polar functional groups to the surface. X-ray photoelectron spectroscopy confirmed the introduction of several oxygen and nitrogen containing surface functional groups. The adhesion to epoxy was dramatically improved after exposure to either plasma, the effect being largest when NH3 was present in the feed gas.

Microfluidic device to study cell transmigration under physiological shear stress conditions

The development of new drug therapies relies on studies of cell transmigration in in vitro systems. Migration has traditionally been studied using two methods, the Boyden chamber and a shear flow chamber assay. Though, commonly applied in cell transmigration studies, they are far from imitating a natural migration process. Here we describe a novel in vitro cell transmigration microfluidic assay, which mimicks physiological shear flow conditions in blood vessels. The device was designed to incorporate the principles of both the Boyden chamber and the shear flow chamber assay, i.e. migration through the membrane under flow conditions. The 3D environment of migrating cells is imitated by injecting cell adhesion proteins to coat the membrane in the device. We tested the developed device with Jurkat cells migration towards medium supplemented with serum, and with chemokine induced lymphocytes migration. The applied continuous flow of cell suspension and chemoattractant ensures that the concentration gradient is maintained in time and space. The cell adhesion proteins used to enhance cell migration in the device were fibronectin and VCAM-1. We successfully observed a multistep transmigration process by means of the developed microfluidic migration assay. The presented device is inexpensive, easy to fabricate and disposable, having a potential to be applied in basic research as well as in the drug development process.