Elisabet Prats-Alfonso

Iridium oxide pH sensor for biomedical applications. Case urea-urease in real urine samples.

This work demonstrates the implementation of iridium oxide films (IROF) grown on silicon-based thin-film platinum microelectrodes, their utilization as a pH sensor, and their successful formatting into a urea pH sensor. In this context, Pt electrodes were fabricated on Silicon by using standard photolithography and lift-off procedures and IROF thin films were growth by a dynamic oxidation electrodeposition method (AEIROF). The AEIROF pH sensor reported showed a super-Nerstian (72.9±0.9mV/pH) response between pH 3 and 11, with residual standard deviation of both repeatability and reproducibility below 5%, and resolution of 0.03 pH units. For their application as urea pH sensors, AEIROF electrodes were reversibly modified with urease-coated magnetic microparticles (MP) using a magnet. The urea pH sensor provided fast detection of urea between 78μM and 20mM in saline solution, in sample volumes of just 50μL. The applicability to urea determination in real urine samples is discussed.

Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects

UNLABELLED Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell-adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm(2)), with a threshold value of 4.0 pmol/cm(2) for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis. FROM THE CLINICAL EDITOR The authors observed by AFM nonlinear dependence of cell adhesion on RGD gradient surfaces with different surface densities. The nonlinear characteristics may be explained by non-homogeneous RGD surface distribution at the nanometer scale, conditioned by the stochastic nature of the hydrolysis process.

NMe Amide as a Synthetic Surrogate for the Thioester Moiety in Thiocoraline

Bridged N-methyl amides are used as isosteres for depsi and thiodepsi bonds in thiocoraline. The introduction of NMe-amides in bridges mimics the thioester bonds without imposing steric hindrance and allows conservation of the hydrogen bonding map of the natural product. NMe-azathiocoraline was constructed by solid-phase N-methylation of the side chain of diaminopropionic acid (Dap). The three consecutive N-methyl amino acids could be coupled in good yields by using HATU/HOAt/DIEA in DMF, and the final octapeptide was also obtained on solid phase following a 4 + 4 fragment coupling approach. NMe-azathiocoraline (NMA) displayed nanomolar activity in the same order as the natural product and the same mode of action. In fact, modeling of NMe-azathiocoraline bonded to a TCGA sequence showed how the methyl groups remained further away from the DNA strand without changing the recognition pattern of thiocoraline. Moreover, NMe-azathiocoraline displayed an increased stability in human serum as compared to the parent natural product. This approach could be used in other depsipeptides and side chain to side chain cyclic peptides.

Cancer Prognostics by Direct Detection of p53-Antibodies on Gold Surfaces by Impedance Measurements

The identification and measurement of biomarkers is critical to a broad range of methods that diagnose and monitor many diseases. Serum auto-antibodies are rapidly becoming interesting targets because of their biological and medical relevance. This paper describes a highly sensitive, label-free approach for the detection of p53-antibodies, a prognostic indicator in ovarian cancer as well as a biomarker in the early stages of other cancers. This approach uses impedance measurements on gold microelectrodes to measure antibody concentrations at the picomolar level in undiluted serum samples. The biosensor shows high selectivity as a result of the optimization of the epitopes responsible for the detection of p53-antibodies and was validated by several techniques including microcontact printing, self-assembled-monolayer desorption ionization (SAMDI) mass spectrometry, and adhesion pull-off force by atomic force microscopy (AFM). This transduction method will lead to fast and accurate diagnostic tools for the early detection of cancer and other diseases.

Mesopattern of immobilised bone morphogenetic protein-2 created by microcontact printing and dip-pen nanolithography influence C2C12 cell fate

Dip-pen nanolithography and microcontact printing were used to fabricate mesopatterned substrates for cell differentiation experiments. A biotin–thiol was patterned on gold substrates and subsequently functionalised with streptavidin and biotinylated bone morphogenetic protein-2 (BMP-2). The feasibility of mesopatterned substrates containing immobilised BMP-2 was proven by obtaining similar differentiation outcomes compared to the growth factor in solution. Therefore, these substrates might be suitable for replacing conventional experiments with BMP-2 in solution.

Chapter 2:Development of Microelectrode-based Biosensors for Biomedical Analysis

This chapter describes the most recent methods and technologies used in the development of microfabricated electrochemical devices for biomedical analysis. The chapter is structured in three main sections: the first part introduces the main aspects underpinning the design and development of miniaturized biosensing devices. The second part describes the fabrication of microelectrode detection systems, with a focus on biosensors in environment and biomedical analysis. This section covers the main aspects of microelectrode fabrication and packaging, and surface functionalization. Finally, representative applications in the field of biomedical electroanalysis, with an emphasis on the detection of cardiovascular disease biomarkers, is described and discussed. The aim is to illustrate the importance and potential of microelectrode-based detection systems in this area, which can be extrapolated to other cases.

Flexible microfluidic bio-lab-on-a-chip multi-sensor platform for electrochemical measurements

A novel polymeric microfluidic multi-sensor system has been developed using rapid prototyping techniques and low-cost materials which permits the dynamic online monitoring of several cell culture parameters in a wide range of biomedical applications. The multi-sensor is made of polymeric flexible substrate with integrated gold microband electrodes for electrochemical measurements of Dissolved Oxygen (DO) and Hydrogen (H+), Sodium (Na+) and Potassium (K+) cations. The platform integrates several working electrodes (WE), pseudoreferences (pRE) and a counter electrode (CE) for electrochemical detection. The bio-lab-on-a-chip allows the detection of changes in real time, with rapid response, allowing to measure small volumes and fast cell metabolisms changes. The reliable analytical performance of the platform in in-vitro conditions has been demonstrated. DO was detected with activated gold electrode, and H+, Na+ and K+ were detected with the developed ion-selective microelectrodes (μISE) using electropolymerized polypyrrole (PPy) film as a contact layer, allowing a good performance of the sensor.