Publications by authors named "João Pedro Conde"

10 Publications

  • Page 1 of 1

Microfluidic device for multiplexed detection of fungal infection biomarkers in grape cultivars.

Analyst 2021 Jan;145(24):7973-7984

Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN), Portugal.

Early diagnosis of fungal infections, which have seen an increase due to different environmental factors, is essential to an appropriate treatment of the plant by avoiding proliferation of the pathogen without excessive fungicide applications. In this work, we propose a microfluidic based approach to a multiplexed, point-of-need detection system capable of identifying infected grape cultivars. The system relies on the simultaneous detection of three plant hormones: salicylic, azelaic and jasmonic acids with a total assay time under 7 minutes, with LODs of 15 μM, 10 μM and 4.4 nM respectively. The three detection assays are based on optical transduction, with the detection of salicylic and azelaic acids using transmission measurements, while the detection of jasmonic acid is a fluorescence-based assay. The molecular recognition event for each metabolite is different: nanoparticle conjugation for salicylic acid, enzymatic reaction for azelaic acid and antibody-antigen recognition for jasmonic acid. In this work, two cultivars, Trincadeira and Carignan, presented infections with two fungal pathogens, Botrytis cinerea and Erysiphe necator. The grapes were tested using the microfluidic system alongside the benchmark techniques such as, high-performance liquid chromatography and enzyme-linked immunosorbent assay. The microfluidic system was not only capable of distinguishing infected from healthy samples, but also capable of distinguishing between different infection types.
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http://dx.doi.org/10.1039/d0an01753aDOI Listing
January 2021

Microfluidic bioreactors for enzymatic synthesis in packed-bed reactors-Multi-step reactions and upscaling.

J Biotechnol 2020 Nov 23;323:24-32. Epub 2020 Jul 23.

Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN), Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

Enzymatic synthesis of biochemical commodities is of upmost importance as it represents a greener alternative to traditional chemical synthesis and provides easier downstream processing strategies compared to fermentation-based processes. A microfluidic system used to optimize the enzymatic production of both levodopa (L-DOPA) and dopamine in both single-step and multistep-reaction sequences with yield of approximately 30 % for L-DOPA production and 70 % for dopamine production is presented. The system for L-DOPA production was then up-scaled (780-fold increase) to a milliliter scale system by maintaining similar mass transport properties resulting in the same yield, space-time yield and biocatalyst yield as its microscale counterpart. The results obtained for yield and biocatalyst yield (351.7 mg mg h) were similar to what is reported in the literature for similar systems, however the space-time yield (0.806 mg L h) was smaller. This work demonstrates a microfluidic bioreactor that can be used for complex optimizations that can be performed rapidly while reducing the consumption of reagents by immobilizing the catalyst on a carrier which can then be used in a packed-bed reactor, thus extending the enzyme life span.
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http://dx.doi.org/10.1016/j.jbiotec.2020.07.016DOI Listing
November 2020

Top-Down Fabricated Silicon Nanowire Arrays for Field-Effect Detection of Prostate-Specific Antigen.

ACS Omega 2018 Aug 1;3(8):8471-8482. Epub 2018 Aug 1.

Department of Informatics and Microsystem Technology, University of Applied Sciences Kaiserslautern, Amerikastrasse 1, 66482 Zweibrücken, Germany.

Highly sensitive electrical detection of biomarkers for the early stage screening of cancer is desired for future, ultrafast diagnostic platforms. In the case of prostate cancer (PCa), the prostate-specific antigen (PSA) is of prime interest and its detection in combination with other PCa-relevant biomarkers in a multiplex approach is advised. Toward this goal, we demonstrate the label-free, potentiometric detection of PSA with silicon nanowire ion-sensitive field-effect transistor (Si NW-ISFET) arrays. To realize the field-effect detection, we utilized the DNA aptamer-receptors specific for PSA, which were covalently and site-specifically immobilized on Si NW-ISFETs. The platform was used for quantitative detection of PSA and the change in threshold voltage of the Si NW-ISEFTs was correlated with the concentration of PSA. Concentration-dependent measurements were done in a wide range of 1 pg/mL to 1 μg/mL, which covers the clinical range of interest. To confirm the PSA-DNA aptamer binding on the Si NW surfaces, a sandwich-immunoassay based on chemiluminescence was implemented. The electrical approach using the Si NW-ISFET platform shows a lower limit of detection and a wide dynamic range of the assay. In future, our platform should be utilized to detect multiple biomarkers in one assay to obtain more reliable information about cancer-related diseases.
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http://dx.doi.org/10.1021/acsomega.8b00990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644640PMC
August 2018

Microfluidic device for the point of need detection of a pathogen infection biomarker in grapes.

Analyst 2019 Aug;144(16):4871-4879

Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN) and IN - Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

Bacterial, fungal and viral infections in plant systems are on the rise, most of which tend to spread quickly amongst crops. These pathogens are also gaining resistance to known treatments, which makes their early detection a priority to avoid extensive loss of crops and the spreading of disease to animal systems. In this work, we propose a microfluidic platform coupled with integrated thin-film silicon photosensors for the detection of pathogen infections in grapes. This detection was achieved by monitoring the concentration of Azelaic Acid (AzA). This small organic acid plays a significant role in the defense mechanism in plant systems. In this platform, the enzyme tyrosinase was immobilized on microbeads inside a microfluidic system. By colorimetric monitoring of the inhibitory effect of AzA on the enzyme tyrosinase in real time, it was possible, in under 10 minutes, to detect different concentrations of AzA in both buffer and spiked solutions of grape juice, in both cases with limits of detection in the 5-10 nM range. In addition, with this microfluidic device, it was possible to clearly distinguish infected from healthy grape samples at three different grape maturation points. Healthy grape samples showed AzA concentrations in the range of 10-20 nM (post-dilution) while infected samples have an estimated increase of AzA of 10-30×, results which were confirmed using HPLC. In both juice and grape samples an integrated sample preparation stage that decreases the phenol content of the solutions was required to achieve fit-for-purpose sensitivities to AzA.
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http://dx.doi.org/10.1039/c9an01002eDOI Listing
August 2019

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

A microfluidic immunoassay platform for the detection of free prostate specific antigen: a systematic and quantitative approach.

Analyst 2015 Jul;140(13):4423-33

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

As a leading cause of cancer-related deaths in men globally, prostate cancer (PCa) demands immense attention for theranostic purposes. There is an increasing need for the development of rapid, sensitive, economical, miniaturized and multiplexable assays. Towards this goal, we present a systematic approach for the optimisation of a microfluidic sandwich immunoassay, which can be applied as a generic biosensor platform for PCa detection. Prostate specific antigen (PSA) was used as the model biomarker, and its free form was captured using commercially available antibodies and detected using chemiluminescence, both in spiked buffer and matrix solutions. Along with the optimisation of surface chemistry and microfluidic parameters, we report a bio-affinity amplification strategy based on biotin-streptavidin chemistry to bring the limits of detection for free-PSA from 21.4 ng mL(-1) down to 2.7 ng mL(-1), within the clinically relevant range. An estimate of the surface coverage and simulations of the interactions taking place in the microfluidic biosensor during the assay are also presented. This novel platform using a simple passive adsorption-based bio-affinity strategy, when coupled with multiplexing and integrated detection, can serve as a promising point-of-care diagnostic tool for PCa.
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http://dx.doi.org/10.1039/c5an00364dDOI Listing
July 2015

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

High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment.

Biomicrofluidics 2012 Mar 9;6(1):14109-141099. Epub 2012 Feb 9.

Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis.
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http://dx.doi.org/10.1063/1.3683161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365346PMC
March 2012

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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