Publications by authors named "Inês F Pinto"

9 Publications

  • Page 1 of 1

Microfluidics as a high-throughput solution for chromatographic process development - The complexity of multimodal chromatography used as a proof of concept.

J Chromatogr A 2021 Nov 8;1658:462618. Epub 2021 Oct 8.

iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

High-throughput technologies are fundamental to expedite the implementation of novel purification platforms. The possibility of performing process development within short periods of time while saving consumables and biological material are prime features for any high-throughput screening device. In this work, a microfluidic device is evaluated as high-throughput solution for a complete study of chromatographic operation conditions on ten different multimodal resins. The potential of this class of purification solutions is generally hindered by its complexity. Taking this into consideration, the microfluidic platform was herein applied and assessed as a tool for high-throughput applications. The commercially available multimodal ligands were studied for the binding of three antibody-based biomolecules (polyclonal mixture of whole antibodies, Fab and Fc fragments) at different pH and salt conditions, in a total of 450 experiments. The results obtained with the microfluidic device were comparable to a standard 96-well filtering microplate high-throughput tool. Additionally, five of the ten multimodal ligands tested were packed into a bench-scale column to perform a final validation of the microfluidic results obtained. All the data acquired in this work using different screening protocols corroborate each other, showing that microfluidic chromatography is a valuable tool for the fast implementation of a new purification step, particularly, if the goal is to narrow the downstream possibilities by being a first point of decision.
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http://dx.doi.org/10.1016/j.chroma.2021.462618DOI Listing
November 2021

Knowing more from less: miniaturization of ligand-binding assays and electrophoresis as new paradigms for at-line monitoring and control of mammalian cell bioprocesses.

Curr Opin Biotechnol 2021 10 7;71:55-64. Epub 2021 Jul 7.

KTH Royal Institute of Technology, Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Stockholm, Sweden; AdBIOPRO, Competence Centre for Advanced BioProduction by Continuous Processing, KTH, Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.copbio.2021.06.018DOI Listing
October 2021

Sample-to-answer COVID-19 nucleic acid testing using a low-cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out.

Lab Chip 2021 08 11;21(15):2932-2944. Epub 2021 Jun 11.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, Solna, Sweden. and AIMES - Center for the Advancement of Integrated Medical and Engineering Sciences at Karolinska Institutet and, KTH Royal Institute of Technology, Stockholm, Sweden.

With its origin estimated around December 2019 in Wuhan, China, the ongoing SARS-CoV-2 pandemic is a major global health challenge. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used to selectively remove primer dimers, inactivate the reaction post-amplification and allowing enhanced fluorescence detection via a smartphone camera. Sample-to-answer analysis within 1 hour from sample collection and a detection limit of approximately 100 RNA copies in 10 μL reaction volume were achieved. The platform was validated with a panel of 162 nasopharyngeal swab samples collected from patients with COVID-19 symptoms, providing a sensitivity of 96.6% (82.2-99.9%, 95% CI) for samples with Ct values below 26 and a specificity of 100% (90-100%, 95% CI), thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics to resource-limited settings.
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http://dx.doi.org/10.1039/d1lc00266jDOI Listing
August 2021

Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production.

ACS Sens 2021 03 16;6(3):842-851. Epub 2021 Mar 16.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, 171 21 Solna, Sweden.

The biopharmaceutical market has been rapidly growing in recent years, creating a highly competitive arena where R&D is critical to strike a balance between clinical safety and profitability. Toward process optimization, the recent development and adoption of new process analytical technologies (PAT) highlight the dynamic complexity of mammalian/human cell culture processes, as well as the importance of fine-tuning and modeling key metabolites and proteins. In this context, simple, rapid, and cost-effective devices allowing routine at-line monitoring of specific proteins during process development and production are currently lacking. Here, we report the development of a versatile microfluidic protein analysis cartridge allowing the multiplexed bead-based immunodetection of specific proteins directly from complex mixtures with minimal hands-on time. Colorimetric quantification of Chinese hamster ovary (CHO) host cell proteins as key impurities, monoclonal antibodies as target biopharmaceuticals, and lactate dehydrogenase as a marker of cell viability was achieved with limits of detection in the 1-10 ng/mL range and analysis times as short as 30 min. The device was further demonstrated for the monitoring of a Rituximab-producing CHO cell bioreactor over the course of 8 days, providing comparable recoveries to standard enzyme-linked immunosorbent assay (ELISA) kits. The high sensitivity combined with robustness to matrix interference highlights the potential of the device to perform at-line measurements spanning from the bioreactor to the downstream processing.
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http://dx.doi.org/10.1021/acssensors.0c01884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034812PMC
March 2021

Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography.

Biosens Bioelectron 2020 Oct 26;166:112442. Epub 2020 Jul 26.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden. Electronic address:

Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of ~1 μm-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.
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http://dx.doi.org/10.1016/j.bios.2020.112442DOI Listing
October 2020

Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules.

Biotechnol J 2019 Oct 23;14(10):e1800593. Epub 2019 Jul 23.

IBB - Institute for Bioengineering and Biosciences Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais 1, 1049-001, Lisbon, Portugal.

The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiol-reactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions.
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http://dx.doi.org/10.1002/biot.201800593DOI Listing
October 2019

Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range.

Biosens Bioelectron 2019 Mar 18;128:68-75. Epub 2018 Dec 18.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, SE-171 65 Solna, Sweden. Electronic address:

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200 × 200 µm area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 μL of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.
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http://dx.doi.org/10.1016/j.bios.2018.12.004DOI Listing
March 2019

Multiplexed microfluidic fluorescence immunoassay with photodiode array signal acquisition for sub-minute and point-of-need detection of mycotoxins.

Lab Chip 2018 05;18(11):1569-1580

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

Portable, rapid, cost effective and simple analytical tools are in increasing demand to facilitate the routine monitoring of target chemical/biological compounds at the point-of-need. Such devices are highly relevant within the context of food safety, particularly concerning the screening of highly toxic and strictly regulated mycotoxins. To achieve ultrarapid detection of mycotoxins, namely aflatoxin B1, ochratoxin A and deoxynivalenol, at the point-of-need, a novel multiplexed bead-based microfluidic competitive immunosensor, coupled with an array of a-Si:H thin-film photodiodes for integrated fluorescence signal acquisition, is reported. Simultaneously measuring the initial binding rate for each analyte of the sample under analysis against an internal reference, this device provided limits of detection below 1 ng mL-1 for all mycotoxins in a single-step assay and within 1 minute after mixing the sample under analysis with a fluorescent conjugate. The compatibility of the device with the analysis of mycotoxins spiked in corn samples was further demonstrated after performing a sample preparation procedure based on aqueous two-phase extraction. The short times of analysis and sensitivities in the low ng mL-1 range make these devices potentially competitive with the lateral flow devices that are currently the standard for this application. Furthermore, this device architecture and concept is amenable of being expanded to other analytes in food safety, biomedical and other applications.
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http://dx.doi.org/10.1039/c8lc00259bDOI Listing
May 2018

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies.

Anal Chem 2016 08 28;88(16):7959-67. Epub 2016 Jul 28.

Instituto de Engenharia de Sistemas e Computadores-Microsistemas e Nanotecnologias, and Institute of Nanoscience and Nanotechnology, 1000-029 Lisbon, Portugal.

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.
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http://dx.doi.org/10.1021/acs.analchem.6b00781DOI Listing
August 2016
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