Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
1.
APL Bioeng ; 3(4): 046103, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31803860

ABSTRACT

Enhanced vascular permeability in the lungs can lead to pulmonary edema, impaired gas exchange, and ultimately respiratory failure. While oxygen delivery, mechanical ventilation, and pressure-reducing medications help alleviate these symptoms, they do not treat the underlying disease. Mechanical activation of transient receptor potential vanilloid 4 (TRPV4) ion channels contributes to the development of pulmonary vascular disease, and overexpression of the high homology (HH) domain of the TRPV4-associated transmembrane protein CD98 has been shown to inhibit this pathway. Here, we describe the development of an adeno-associated virus (AAV) vector encoding the CD98 HH domain in which the AAV serotypes and promoters have been optimized for efficient and specific delivery to pulmonary cells. AAV-mediated gene delivery of the CD98 HH domain inhibited TRPV4 mechanotransduction in a specific manner and protected against pulmonary vascular leakage in a human lung Alveolus-on-a-Chip model. As AAV has been used clinically to deliver other gene therapies, these data raise the possibility of using this type of targeted approach to develop mechanotherapeutics that target the TRPV4 pathway for treatment of pulmonary edema in the future.

2.
Nat Nanotechnol ; 14(12): 1143-1149, 2019 12.
Article in English | MEDLINE | ID: mdl-31712665

ABSTRACT

Affinity-based electrochemical detection in complex biological fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 month of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy preparation, stability and simplicity of this nanocomposite allow the generation of electrochemical biosensors that can operate in complex biological fluids such as blood plasma or serum.


Subject(s)
Biosensing Techniques/instrumentation , Gold/chemistry , Metal Nanoparticles/chemistry , Nanotubes, Carbon/chemistry , Serum Albumin, Bovine/chemistry , Antibodies/blood , Biofouling , Electrochemical Techniques/instrumentation , Electrodes , Equipment Design , Humans , Immobilized Proteins/chemistry , Models, Molecular , Nanotubes, Carbon/ultrastructure , Plasma/chemistry
3.
Lab Chip ; 19(3): 452-463, 2019 01 29.
Article in English | MEDLINE | ID: mdl-30632575

ABSTRACT

Here, we describe methods for combining impedance spectroscopy measurements with electrical simulation to reveal transepithelial barrier function and tissue structure of human intestinal epithelium cultured inside an organ-on-chip microfluidic culture device. When performing impedance spectroscopy measurements, electrical simulation enabled normalization of cell layer resistance of epithelium cultured statically in a gut-on-a-chip, which enabled determination of transepithelial electrical resistance (TEER) values that can be compared across device platforms. During culture under dynamic flow, the formation of intestinal villi was accompanied by characteristic changes in impedance spectra both measured experimentally and verified with simulation, and we demonstrate that changes in cell layer capacitance may serve as measures of villi differentiation. This method for combining impedance spectroscopy with simulation can be adapted to better monitor cell layer characteristics within any organ-on-chip in vitro and to enable direct quantitative TEER comparisons between organ-on-chip platforms which should help to advance research on organ function.


Subject(s)
Dielectric Spectroscopy/instrumentation , Intestinal Mucosa/cytology , Lab-On-A-Chip Devices , Caco-2 Cells , Electric Stimulation , Equipment Design , Humans , Intestinal Mucosa/metabolism
4.
Lab Chip ; 17(13): 2294-2302, 2017 06 27.
Article in English | MEDLINE | ID: mdl-28608907

ABSTRACT

Here we demonstrate that microfluidic cell culture devices, known as Organs-on-a-Chips can be fabricated with multifunctional, real-time, sensing capabilities by integrating both multi-electrode arrays (MEAs) and electrodes for transepithelial electrical resistance (TEER) measurements into the chips during their fabrication. To prove proof-of-concept, simultaneous measurements of cellular electrical activity and tissue barrier function were carried out in a dual channel, endothelialized, heart-on-a-chip device containing human cardiomyocytes and a channel-separating porous membrane covered with a primary human endothelial cell monolayer. These studies confirmed that the TEER-MEA chip can be used to simultaneously detect dynamic alterations of vascular permeability and cardiac function in the same chip when challenged with the inflammatory stimulus tumor necrosis factor alpha (TNF-α) or the cardiac targeting drug isoproterenol. Thus, this Organ Chip with integrated sensing capability may prove useful for real-time assessment of biological functions, as well as response to therapeutics.


Subject(s)
Cell Culture Techniques/instrumentation , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/instrumentation , Cell Line , Electric Impedance , Electrodes , Equipment Design , Human Umbilical Vein Endothelial Cells , Humans , Microfluidic Analytical Techniques/methods
5.
Lab Chip ; 17(13): 2264-2271, 2017 06 27.
Article in English | MEDLINE | ID: mdl-28598479

ABSTRACT

Trans-epithelial electrical resistance (TEER) is broadly used as an experimental readout and a quality control assay for measuring the integrity of epithelial monolayers cultured under static conditions in vitro, however, there is no standard methodology for its application to microfluidic organ-on-a-chip (organ chip) cultures. Here, we describe a new microfluidic organ chip design that contains embedded electrodes, and we demonstrate its utility for assessing formation and disruption of barrier function both within a human lung airway chip lined by a fully differentiated mucociliary human airway epithelium and in a human gut chip lined by intestinal epithelial cells. These chips with integrated electrodes enable real-time, non-invasive monitoring of TEER and can be applied to measure barrier function in virtually any type of cultured cell.


Subject(s)
Electric Impedance , Epithelial Cells , Lab-On-A-Chip Devices , Models, Biological , Organ Culture Techniques/instrumentation , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/physiology , Epithelium/physiology , Equipment Design , Humans
6.
Anal Chem ; 89(6): 3378-3385, 2017 03 21.
Article in English | MEDLINE | ID: mdl-28211676

ABSTRACT

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.


Subject(s)
Biosensing Techniques , Breast Neoplasms/genetics , Electrochemical Techniques , Neoplastic Cells, Circulating/pathology , RNA, Messenger/genetics , Single-Cell Analysis , Breast Neoplasms/pathology , Female , Genetic Profile , Humans
7.
Biosens Bioelectron ; 73: 130-137, 2015 Nov 15.
Article in English | MEDLINE | ID: mdl-26056956

ABSTRACT

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts.


Subject(s)
Biosensing Techniques/methods , DNA/analysis , Biosensing Techniques/instrumentation , Click Chemistry , Computer Systems , DNA Probes , DNA, Bacterial/analysis , DNA, Bacterial/genetics , DNA-Directed DNA Polymerase , Enzymes, Immobilized , Equipment Design , Francisella tularensis/genetics , Immobilized Nucleic Acids , Nanotechnology , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization , Recombinases , Silicon , Solid-Phase Synthesis Techniques
8.
Biosens Bioelectron ; 54: 674-8, 2014 Apr 15.
Article in English | MEDLINE | ID: mdl-24334283

ABSTRACT

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 µL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 µL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.


Subject(s)
DNA, Bacterial/genetics , Francisella tularensis/genetics , Polymerase Chain Reaction/instrumentation , Base Sequence , DNA Probes/genetics , DNA, Bacterial/metabolism , Equipment Design , Humans , Limit of Detection , Molecular Sequence Data , Polymerase Chain Reaction/methods , Recombinases/metabolism , Tularemia/microbiology
9.
Macromol Rapid Commun ; 32(18): 1405-10, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21800393

ABSTRACT

Short DNA oligonucleotide branches are incorporated into acrylamide brushes via surface initiated atom transfer radical polymerization in an attempt to increase DNA surface density by building three-dimensional molecular architectures. ATR-FTIR as well as hybridization studies followed by SPR confirm the incorporation of the DNA sequences into the polymer backbone. MALDI-TOF analysis further suggests that six acrylamide monomer units are typically separating DNA branches present on a single brushes approximately 26 units long. This new approach offers a promising alternative to SAM-based nucleic acid and aptamer sensors and could enable the realization of more complex soft materials of controlled architecture capable of both recognition and signaling by including additional optically or electrochemically active moieties.


Subject(s)
Acrylic Resins/chemistry , BRCA1 Protein/genetics , Breast Neoplasms/therapy , DNA/chemistry , Genetic Therapy/instrumentation , Oligonucleotides/chemistry , Breast Neoplasms/genetics , Exons , Female , Humans , Polymerization
10.
Biosens Bioelectron ; 26(9): 3962-6, 2011 May 15.
Article in English | MEDLINE | ID: mdl-21444197

ABSTRACT

Biosensor read out signals can be enhanced by carefully designing the transducer surfaces to achieve an optimal interaction between the recognition elements immobilised and the targeted analyte. This is particularly evident in the case of genosensors, where spacing and orientation of immobilised DNA capture probes need to be controlled to maximise subsequent surface hybridisation with the target sequence and achieve high binding signals. Addressing this goal, we present a novel approach based on the surface nanostructuring of glassy carbon electrodes (GCEs) towards the development of highly sensitive electrochemical genosensors. Gold nanoparticles were sequentially electrochemically nucleated on glassy carbon electrodes to form dense arrays of randomly distributed gold nanodomains. The number density of the electronucleated nanoparticles could be increased by repeatedly alternating between a short electronucleation step and the subsequent insulation of the nucleated nanoparticles with thiolated DNA probes. This approach allowed for the creation of highly structured surfaces whilst preventing aggregation of nanoparticles. The performances of planar gold electrodes and that of the nanopatterned surfaces prepared following several rounds of deposition were compared for the amperometric detection of DNA. Three rounds of deposition exhibited the highest sensitivity (44.89 nA × nM(-1)), with a dynamic detection range spanning from 0.53 nM to 25 nM of the targeted sequence, i.e. one order of magnitude lower than that obtained for the planar gold electrodes. The use of the nanostructured surface we report here may find application not only in DNA biosensors but also for any sensing application requiring highly sensitive measurements.


Subject(s)
Biosensing Techniques , DNA/isolation & purification , Metal Nanoparticles/chemistry , Carbon , DNA/chemistry , DNA Probes/chemistry , Electrochemistry , Electrodes , Gold/chemistry , Limit of Detection , Nucleic Acid Hybridization , Surface Properties
11.
Anal Bioanal Chem ; 397(6): 2325-34, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20526769

ABSTRACT

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP-HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L(-1), with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.


Subject(s)
Breast Neoplasms/genetics , Nucleic Acid Amplification Techniques/methods , RNA, Messenger/analysis , Biotin , Breast Neoplasms/pathology , Cell Line, Tumor , Colorimetry/methods , Female , Humans , Limit of Detection , Molecular Probe Techniques , Oligonucleotide Probes , Streptavidin
12.
Biosens Bioelectron ; 23(12): 1769-75, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18378439

ABSTRACT

The possibility to assess several functional polymeric materials in parallel in a microchip format could find a wide range of applications in sensing, combinatorial and high-throughput screening. However several factors, inherent to the nature of material polymerisation have limited such development. We here report an innovative fabrication approach for the elaboration of polymer microarrays bearing polymer dots typically 300 microm in diameter fabricated in situ on a glass cover slip via CO(2) laser pulse initiated polymerisation, as well as initial results on the identification of a suitable monomer composition for the molecular imprinting of dansyl-L-phenylalanine as a proof-of-concept example. A combination of methacrylic acid and 2-vinylpyridine showed the largest affinity to dansyl-L-phenylalanine which agreed with the existing literature and the results were further confirmed by HPLC. Finally, a sensor chip bearing both non-imprinted as well as imprinted polymers was also prepared in order to prove the suitability of this fabrication approach for the elaboration of MIP based sensors. The assay consisted in a simple dip-and-read step and the sensing system was able to discriminate between the l and d enantiomers of dansylphenylalanine with an imprinting factor of 1.6.


Subject(s)
Microarray Analysis/instrumentation , Microarray Analysis/methods , Polymers/chemistry , Polymers/radiation effects , Equipment Design , Equipment Failure Analysis , Infrared Rays , Lasers , Surface Properties
13.
Anal Bioanal Chem ; 382(4): 947-56, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15940451

ABSTRACT

This article reviews the progress and developments achieved in the past five years (2000-2005) in the application of optical analytical techniques to the evaluation of molecularly imprinted polymer (MIP) characteristics. The MIP binding efficiency, recognition processes and selectivity have been intensively studied by optical means due to the general high sensitivity and simplicity of the utilisation of optical techniques. In addition, recent progress in the covalent linkage of MIPs to optical transducers has allowed for the realisation of highly efficient and robust optical MIP-based molecular recognition sensors. The review provides insight into the various approaches to the optical interrogation of MIPs, and is organised according to the type of optical technique employed (fluorescence, UV/Vis and infrared spectroscopy, surface plasmon resonance, chemiluminescence, refractive interference spectroscopy and Raman scattering) and the detailed strategies applied. The review also covers the recent progress achieved in the area of optical sensors based on MIPs.


Subject(s)
Biosensing Techniques/methods , Polymers/chemistry , Luminescent Measurements/methods , Optics and Photonics , Sensitivity and Specificity , Spectrophotometry, Infrared/methods , Spectrophotometry, Ultraviolet/methods , Surface Plasmon Resonance/methods , Surface Properties
SELECTION OF CITATIONS
SEARCH DETAIL
...