Publications by authors named "Pedro Novo"

10 Publications

  • Page 1 of 1

Coupling Miniaturized Free-Flow Electrophoresis to Mass Spectrometry a Multi-Emitter ESI Interface.

Anal Chem 2021 05 3;93(19):7204-7209. Epub 2021 May 3.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Bunsen-Kirchhoff-Str. 11, 44139 Dortmund, Germany.

We present a novel multi-emitter electrospray ionization (ESI) interface for the coupling of microfluidic free-flow electrophoresis (μFFE) with mass spectrometry (MS). The effluents of the μFFE outlets are analyzed in near real-time, allowing a direct optimization of the electrophoretic separation and an online monitoring of qualitative sample compositions. The short measurement time of just a few seconds for all outlets even enables a reasonable time-dependent monitoring. As a proof of concept, we employ the multi-emitter ESI interface for the continuous identification of analytes at 15 μFFE outlets MS to optimize the μFFE separation of important players of cellular respiration . The results indicate great potential of the presented system in downstream processing control, for example, for the monitoring and purification of products in continuous-flow microreactors.
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http://dx.doi.org/10.1021/acs.analchem.1c00200DOI Listing
May 2021

Multiplexed Online Monitoring of Microfluidic Free-Flow Electrophoresis via Mass Spectrometry.

Anal Chem 2020 05 24;92(9):6764-6769. Epub 2020 Apr 24.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany.

Free-flow electrophoresis is a tool for the continuous fractionation of electrically charged analytes. In this study, we introduce a novel method to couple microchip-based free-flow electrophoresis with mass spectrometry. The successive connection of multiple microchip outlets to the electrospray ionization source of a mass spectrometer is automated using a multiposition valve. With this novel setup, it is possible to continuously fractionate and collect compounds while simultaneously monitoring the process online with mass spectrometry. The functionality of the method is demonstrated by the successful separation and identification of the biomolecules AMP, ATP, and CoA, which are fundamental for numerous biochemical processes in every organism.
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http://dx.doi.org/10.1021/acs.analchem.0c00996DOI Listing
May 2020

Flow rate independent gradient generator and application in microfluidic free-flow electrophoresis.

Anal Chim Acta 2018 Dec 22;1044:77-85. Epub 2018 May 22.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany. Electronic address:

Microfluidic gradient generators have been employed in several works in the literature. However, these are typically application specific and especially limited in the range of flow rates that result in the required concentration gradient outputs. Here, a flow rate independent gradient generator designed as a modified Christmas tree-like microfluidic channel network including micromixers at each channel branch is demonstrated. The device was characterized theoretically, modeled using finite element analysis and tested experimentally. Input flow rates up to 200 μl/min, resulting in a maximum speed of about 333 mm/s, for the generation of linear and mirrored linear gradients were demonstrated. As an application example, the gradient generator was monolithically integrated with microfluidic free-flow electrophoresis for the separation/concentration of fluorophores using a novel E-field gradient free-flow electrophoresis mode. The separation of fluorophores, having different charge stages, showed concentration factors of up to 10 fold. In addition, an extended theoretical description of the realizable concentration gradients and the electric field gradient is presented as supplementary information.
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http://dx.doi.org/10.1016/j.aca.2018.04.066DOI Listing
December 2018

Integration of polycarbonate membranes in microfluidic free-flow electrophoresis.

Analyst 2017 Nov;142(22):4228-4239

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany.

A general difficulty in the miniaturization of free-flow electrophoresis relates to the need to separate electrodes and separation bed compartments. This is usually performed by using membranes, which are either difficult to fabricate and integrate into microfluidic channels, or not stable over time. Here, we propose the use of track-etched polycarbonate membranes. Fabrication of the miniaturized device and integration of the membrane was simple, reproducible and allows for long shelf times. Furthermore, the membranes were resistant to high pressure values (up to 10 Pa), and contributed negligible electrical resistance, allowing setting of electric fields at the separation bed with high efficiency. A second microfluidic device was connected to the microfluidic free-flow electrophoresis chip via tubing, ensured flow stability over time and was used as a chip-to-world interface to a 96 well plate. We demonstrated microfluidic free-flow zone- and field-stacking electrophoresis, and isoelectric focusing proof-of-principle experiments, using fluorescent analytes and monitoring via fluorescence microscopy. Furthermore, the separation of a mixture of 7 proteins was performed in microfluidic free-flow zone electrophoresis mode. Subsequent analysis via protein mass spectrometry of the collected fractions revealed separation of the protein mixture, indicating a wide range of applications in the characterization of proteins and biosimilars.
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http://dx.doi.org/10.1039/c7an01514cDOI Listing
November 2017

Current advances and challenges in microfluidic free-flow electrophoresis-A critical review.

Anal Chim Acta 2017 Oct 23;991:9-29. Epub 2017 Aug 23.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., 44227, Otto-Hahn-Str. 6b, Dortmund, Germany. Electronic address:

The research field on microfluidic free-flow electrophoresis has developed vast amounts of devices, methods, applications and raised new questions, often in analogy to conventional techniques from which it derives. Most efforts have been employed on device development and a myriad of architectures and fabrication techniques have been reported using simple proof-of-principle separations. As technological aspects reach a quite mature state, researchers' new challenges include the development of protocols for the separation of complex mixtures, as required in the fields of application. The success of this effort is extremely dependent on the capability to transfer the device's fabrication to an industrial setting as well as to ensure interfacing simplicity, namely at the solutions' supply and collection, and actuation such as electric potential application and temperature control. Other advanced applications such as direct interfacing to downstream systems such as mass spectrometry, integration of sensing and feedback controls will require further development in the laboratory. In this review we provide an overview on the field, from basic concepts, through advanced developments both in the theoretical and experimental arenas, and addressing the above details. A comprehensive survey of designs, materials and applications is presented with particular highlights to most recent developments, namely the integration of electrodes, flow control and hyphenation of microfluidic free-flow electrophoresis with other techniques.
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http://dx.doi.org/10.1016/j.aca.2017.08.017DOI Listing
October 2017

Lab-on-chip systems for integrated bioanalyses.

Essays Biochem 2016 06;60(1):121-31

Instituto de Engenharia de Sistemas E Computadores-Microsistemas e Nanotecnologias (INESC MN) and IN-Institute of Nanoscience and Nanotechnology, Rua Alves Redol, 9, 1000-029 Lisbon, Portugal.

Biomolecular detection systems based on microfluidics are often called lab-on-chip systems. To fully benefit from the miniaturization resulting from microfluidics, one aims to develop 'from sample-to-answer' analytical systems, in which the input is a raw or minimally processed biological, food/feed or environmental sample and the output is a quantitative or qualitative assessment of one or more analytes of interest. In general, such systems will require the integration of several steps or operations to perform their function. This review will discuss these stages of operation, including fluidic handling, which assures that the desired fluid arrives at a specific location at the right time and under the appropriate flow conditions; molecular recognition, which allows the capture of specific analytes at precise locations on the chip; transduction of the molecular recognition event into a measurable signal; sample preparation upstream from analyte capture; and signal amplification procedures to increase sensitivity. Seamless integration of the different stages is required to achieve a point-of-care/point-of-use lab-on-chip device that allows analyte detection at the relevant sensitivity ranges, with a competitive analysis time and cost.
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http://dx.doi.org/10.1042/EBC20150013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986467PMC
June 2016

High spatial and temporal resolution cell manipulation techniques in microchannels.

Analyst 2016 Mar;141(6):1888-905

Protein Dynamics Group, Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany.

The advent of microfluidics has enabled thorough control of cell manipulation experiments in so called lab on chips. Lab on chips foster the integration of actuation and detection systems, and require minute sample and reagent amounts. Typically employed microfluidic structures have similar dimensions as cells, enabling precise spatial and temporal control of individual cells and their local environments. Several strategies for high spatio-temporal control of cells in microfluidics have been reported in recent years, namely methods relying on careful design of the microfluidic structures (e.g. pinched flow), by integration of actuators (e.g. electrodes or magnets for dielectro-, acousto- and magneto-phoresis), or integrations thereof. This review presents the recent developments of cell experiments in microfluidics divided into two parts: an introduction to spatial control of cells in microchannels followed by special emphasis in the high temporal control of cell-stimulus reaction and quenching. In the end, the present state of the art is discussed in line with future perspectives and challenges for translating these devices into routine applications.
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http://dx.doi.org/10.1039/c6an00027dDOI Listing
March 2016

Integrated fluorescence detection of labeled biomolecules using a prism-like PDMS microfluidic chip and lateral light excitation.

Lab Chip 2014 Jun 8;14(12):1991-5. Epub 2014 May 8.

INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

Microfabricated amorphous silicon photodiodes were integrated with prism-like PDMS microfluidics for the detection and quantification of fluorescence signals. The PDMS device was fabricated with optical quality surfaces and beveled sides. A 405 nm laser beam perpendicular to the lateral sides of the microfluidic device excites the fluorophores in the microchannel at an angle of 70° to the normal to the microchannel/photodiode surface. This configuration, which makes use of the total internal reflection of the excitation beam and the isotropy of the fluorescence emission, minimizes the intensity of excitation light that reaches the integrated photodetector. A difference of two orders of magnitude was achieved in the reduction of the detection noise level as compared with a normally incident excitation configuration. A limit-of-detection of 5.6 × 10(10) antibodies per square centimeter was achieved using antibodies labeled with a model organic fluorophore. Furthermore, the results using the lateral excitation scheme are in good proportionality agreement with those by fluorescence quantification using wide-field fluorescence microscopy.
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http://dx.doi.org/10.1039/c4lc00241eDOI Listing
June 2014

Control of sequential fluid delivery in a fully autonomous capillary microfluidic device.

Lab Chip 2013 Feb;13(4):641-5

INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

Microfluidics and miniaturization of biosensors are fundamental for the development of point-of-care (PoC) diagnostic and analytical tools with the potential of decreasing reagent consumption and time of analysis while increasing portability. However, interfacing microfluidics with fluid control systems is still a limiting factor in practical implementation. We demonstrate an innovative capillary microfluidic design that allows sequential insertion of controlled volumes of liquids into a microfluidic channel with general applicability. The system requires only the placing of liquids at the corresponding inlets. Subsequently, the different solutions flow inside the microfluidic device sequentially and autonomously without the use of valves using integrated capillary pumps. The capillary microfluidic system is demonstrated with a model immunoassay.
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http://dx.doi.org/10.1039/c2lc41083dDOI Listing
February 2013

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

Lab Chip 2011 Dec 20;11(23):4063-71. Epub 2011 Oct 20.

INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.
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http://dx.doi.org/10.1039/c1lc20362bDOI Listing
December 2011
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