Investigation of Bifurcation Effect on Various Microfluidic Designs for Blood Separation.

In this project, a microfluidic device for blood separation will be designed and tested in order to separate plasma from whole blood for diagnostic purposes. The design will be based on previously implemented designs that will be further discussed in the next sections. When designing microfluidic devices, it is essential to consider the different physical phenomena that arise from switching from the macro scale to the micro scale. Parameters such as the Reynolds number and the forces affecting the fluid must be studied in order to produce a suitable and effective design. Finite element methods have been implemented prior to the production of the microfluidic devices. Various geometries/designs have been tested using Fluent ANSYS software. Later on, the successful design was fabricated using micromachining on an acrylic substrate and was tested using simulated blood through of a syringe pump.

Sensitivity Enhancement of Point-of-Care for Cardiac Markers Detection using Micro-Impedimetric Immunosensor Arrays.

This paper describes the development and characterisation of a novel, electrical impedance spectroscopy-based (EIS) immunosensor array for point-of-care applications. EIS is a highly sensitive, label-free, real time technique suitable for single use, point-of-care cardiac marker detection devices. However, the underlying source of the observed change in EIS immunoassay response has not been well characterised or understood. A full understanding of the relationship between target binding and impedance response would significantly advance biosensor design and most probably increase detection limit sensitivity. The development of micro-/nano- structured electrodes for multi-frequency EIS procedure propose substantial benefits over classical macro-structured systems.Countless manipulations of electrode features and inter-electrode spacing will enhance the electrode surface area, increase the charge-transfer resistance and reduce the double-layer capacitance. These in turn give rise to improved signal-to-noise ratios, therefore affording greater sensitivity, lower detection limits and faster detection times.The sensor sensitivity range was within that required for human myoglobin determination, following acute myocardial infarction (heart attack). Real-time MyAb-MyAg interaction monitoring, permitted the determination of the binding events in less than one minute.

Detection of Human Chorionic Gonadotropin (hCG) Hormone using Digital Lateral Flow Immunoassay.

The main goal of this work was to establish a hybrid device incorporating an electrochemical-based transducer on a conventional lateral flow assay strip in order to perform an on-chip fast testing method for the detection of various bio-analyses. In this context, the expected development of the digital lateral-flow immunoassay to be considered a reliable low-cost instrument improves the future of the very simple and flexible approach oflateral-flow assays. It is anticipated to achieve a digital quantitative lateral-flow immunoassay by exploring the electrochemical transducers alongwith recognition elements for digitization of commercially available rapid tests. As a preliminary step, the described technique will be validated using two standard electrochemical measurements (amperometric and impedimetric) across two electrodes fixed onto the surface of LFA strip. The LFA strips were prepared at the factory for pregnancy tests and modified by adding two parallel copper electrodes at the lab. These strips were proven by in-vitro experiments to be reusable lasting for 20-30 multiple days. Further on, the detection of hCG Ab-Ag interaction using these strips was performed. Two different types of measurements, namely amperometric and impedimetric, were used which yielded similar results to those reported in literature with screen-printed micro-electrodes. In addition, different concentrations of NaCl and hCG Ag solution were investigated. However, the expected linear concentration response was obtained. A promising proof-of-concept have been achieved through this study. Further studies are needed to complete the development of fully printed disposable electrochemical devices that are able to either display a digital result directly or transmit data to a mobile phone using RFID/NFC.

A Brain Machine Interface for command based control of a wheelchair using conditioning of oscillatory brain activity.

In this research a new method of wheelchair control using a Brain Computer Interface (BCI) is proposed, in an attempt to bridge the gap between in-lab and real life applications, we believe it would provide a high level control over the BCI instead of the normal low level commands. It is anticipated to emphasis on mu rhythm to provide the control signals. The wheelchair is equipped with a mapping system, which scans the area and provides a map containing information about the user's current location and next possible destinations, then provides an optimized list of possible trajectories to reach the destination. The paradigm allows users to control the interface using motor imagery and issue commands to switch between possible trajectories and then confirm the choice. Commands trigger the motion of the wheelchair to the intended destination using a user selected path with speed up to 0.5 m/s. The interface also allows the user to interact with different robots through a common robotic system. Evaluation results indicate that this paradigm is indeed usable and could lead to promising outcomes.

Artificial neural networks for dihedral angles prediction in enzyme loops: a novel approach.

Structure prediction of proteins is considered a limiting step and determining factor in drug development and in the introduction of new therapies. Since the 3D structures of proteins determine their functionalities, prediction of dihedral angles remains an open and important problem in bioinformatics, as well as a major step in discovering tertiary structures. This work presents a method that predicts values of the dihedral angles φ and ψ for enzyme loops based on data derived from amino acid sequences. The prediction of dihedral angles is implemented through a neural network based mining mechanism. The amino acid sequence data represents 6342 enzyme loop chains with 18,882 residues. The initial neural network input was a selection of 115 features and the outputs were the predicted dihedral angles φ and ψ. The simulation results yielded a 0.64 Pearson's correlation coefficient. After feature selection through determining insignificant features, the input feature vector size was reduced to 45, while maintaining close to identical performance.

Flexible, polymer-supported synthesis of sphingosine derivatives provides ceramides with enhanced biological activity.

A polymer-supported route for the synthesis of sphingosine derivatives is presented based on the C-acylation of polymeric phosphoranylidene acetates with an Fmoc-protected amino acid. The approach enables the flexible variation of the sphingosine tail through a deprotection-decarboxylation sequence followed by E-selective Wittig olefination cleavage. d-Erythro-sphingosine analogs have been synthesized by diastereoselective reduction of the keto group employing LiAlH(O-tBu)3 as reducing agent. The effect of ceramides and keto-ceramides on the proliferation of three cancer cell lines HEP G-2, PC-12 and HL-60 was investigated and a ceramide containing an aromatic sphingosine tail was identified as being most active.

Soluble peptidyl phosphoranes for metal-free, stereoselective ligations in organic and aqueous solution.

Protocols for solid-phase syntheses of soluble peptidyl phosphoranes are presented. Various supported phosphoranylidene acetates were prepared on Rink amide or via alkylation of trialkyl- and triarylphosphines with bromoacetyl Wang ester. C-Acylation was conducted racemization-free with activated Fmoc-amino acids, followed by SPPS (solid-phase peptide synthesis). Acidic conditions released decarboxylated peptidyl phosphoranes into solution. The protocol allowed for the electronic variation of peptidyl phosphoranes which were investigated in ligation reactions with azides in organic and aqueous solvents.

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease.

SARS coronavirus main protease (SARS-CoV M(pro)) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M(pro). Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M(pro) requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M(pro) in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M(pro), with K(i) values in the µM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2=Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M(pro) in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M(pro), with K(i)=2.24±0.58 µM. These results show that the stringent substrate specificity of the SARS-CoV M(pro) with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.

An efficient method for the synthesis of peptide aldehyde libraries employed in the discovery of reversible SARS coronavirus main protease (SARS-CoV Mpro) inhibitors.

A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidation of amino alcohols with Dess-Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV M(pro)(also known as 3CL(pro)), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepared by this method and investigated in inhibition assays against SARS-CoV M(pro). Several potent inhibitors were found with IC(50) values in the low micromolar range. An IC(50) of 7.5 muM was found for AcNSTSQ-H and AcESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV M(pro) in a noncanonical binding mode.