Non-covalent π-π functionalized Gii-senseⓇ graphene foam for interleukin 10 impedimetric detection.

Monitoring Interleukin 10 (IL-10) is essential for understanding the vast responses of T-cells in cancer, autoimmunity, and internal homeostasis after physical stress. However, current diagnostic methods are complex and more focused on medical screening rather than point-of-care monitoring. Biosensors based on graphene's conductivity and flexibility are attractive to offer simple single-use and reduced handling. However, oxidation of its carbon lattice to develop functional moieties for biomolecule immobilization cuts down its electronic conductivity potential. In this work, the authors present a microfluidic lab-on-chip device for simple impedimetric monitoring of IL-10 based on graphene foam (GF) flexible electrodes. Graphene's structure was maintained by employing π-π non-covalent functionalization with pyrene carboxylic acid (PCA). Impedimetric measurements could be performed in low ionic strength phosphate-buffered saline (LI-PBS). The PCA-antibody modification showed to endure the incubation, measurement, and washing processes performed in the microfluidic device. Electrode modification and measurements were characterized by, electrochemical impedance spectroscopy (EIS), contact angle, and scanning electron microscopy. From the contact angle results, we found that the wettability of the graphene surface increased gradually after each modification step. Detection measurements performed in the 3D-printed microfluidic device showed a linear response between 10 fg/mL to 100 fg/mL with a limit of detection (LOD) of 7.89 fg/mL in artificial saliva. With these features, the device was used to quantify IL-10 samples by the standard addition method for 10 fg and 50 fg with recoveries between 82% and 99%. Specificity was evaluated towards interleukin 6, TNF-⍺ and bovine serum albumin.

Amperometric quantification based on serial dilution microfluidic systems.

This paper describes a microfluidic device fabricated in poly(dimethylsiloxane) that was employed to perform amperometric quantifications using on-chip calibration curves and on-chip standard addition methods. This device integrated a network of Au electrodes within a microfluidic structure designed for automatic preparation of a series of solutions containing an electroactive molecule at a concentration linearly decreasing. This device was first characterized by fluorescence microscopy and then evaluated with a model electroactive molecule such as Fe(CN(6))(4-). Operating a quantification in this microfluidic parallel approach rather than in batch mode allows a reduced analysis time to be achieved. Moreover, the microfluidic approach is compatible with the on-chip calibration of sensors simultaneously to the analysis, therefore preventing problems due to sensor response deviation with time. When using the on-chip calibration and on-chip standard addition method, we reached concentration estimation better than 5%. We also demonstrated that compared to the calibration curve approach, the standard addition mode is less complex to operate. Indeed, in this case, it is not necessary to take into account flow rate discrepancies as in the calibration approach.

Biopatterning of antibodies on poly(pyrrole)-nanowires using nanocontact printing: Surface characterization.

A highly performant patterning of antibodies using poly(pyrrole) nanowires (PPy-NWs) was devised on thermoplastic surfaces based on silane derivatives. The PPy-NWs were fabricated employing nanocontact printing and controlled chemical polymerization (nCP-CCP) on poly(ethylene terephthalate), cyclic olefin copolymer, poly(ethylene 2,6-naphthalate), and polyimide. The technique used a commercial compact disk as a template (mold) to produce nanopatterned polydimethylsiloxane stamps. The nanopatterned stamp was then employed to print PPy-NWs. The printing technique permits to control PPy-NW size and shape. The dimensions of the printed PPy-NWs were: 785 ±â€¯1.5 nm (width), 174 ±â€¯2.1 nm (height), and a separation between wires of 540 ±â€¯1.2 nm. The printing process and the surface properties of the PPy-NWs pattern were successfully characterized by scanning electron microscopy and atomic force microscopy. Biopatterning was completed by the chemical immobilization of the specific anti-human interleukin-10 monoclonal antibody on PPy-NW using gluteraldehyde. The biocomposite was tested using qualitative immunocytokine bioassay, which is of great importance for early stage cancer detection. For that purpose, fluorescent imaging was used to certify the immunodetection of the recombinant human interleukin-10. The biopatterning technology provides a simple, low cost and one step procedure. Undoubtedly, this new technology will impact and provide an alternative to the current techniques applied for bioengineering and nanopatterning.

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode.

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.

A fully integrated electrochemical biosensor platform fabrication process for cytokines detection.

Interleukin-1b (IL-1b) and interleukin-10 (IL-10) biomarkers are one of many antigens that are secreted in acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). In the present study, we have developed a fully integrated electrochemical biosensor platform for cytokine detection at minute concentrations. Using eight gold working microelectrodes (WEs) the design will increase the sensitivity of detection, decrease the time of measurements, and allow a simultaneous detection of varying cytokine biomarkers. The biosensor platform was fabricated onto silicon substrates using silicon technology. Monoclonal antibodies (mAb) of anti-human IL-1b and anti-human IL-10 were electroaddressed onto the gold WEs through functionalization with 4-carboxymethyl aryl diazonium (CMA). Cyclic voltammetry (CV) was applied during the WE functionalization process to characterize the gold WE surface properties. Finally, electrochemical impedance spectroscopy (EIS) characterized the modified gold WE. The biosensor platform was highly sensitive to the corresponding cytokines and no interference with other cytokines was observed. Both cytokines: IL-10 and IL-1b were detected within the range of 1pgmL-1 to 15pgmL-1. The present electrochemical biosensor platform is very promising for multi-detection of biomolecules which can dramatically decrease the time of analysis. This can provide data to clinicians and doctors concerning cytokines secretion at minute concentrations and the prediction of the first signs of inflammation after LVAD implantation.

Methylene blue phosphoramidite for DNA labelling.

We report the first synthesis of a methylene blue (MB) phosphoramidite derivative suitable for DNA solid-phase synthesis. The electrochemical and optical properties of the resulting MB modified oligonucleotides were confirmed. This new molecule is an important breakthrough in the design of new probes labelled with MB.