Publications by authors named "Virginia Chu"

52 Publications

Accurate and rapid microfluidic ELISA to monitor Infliximab titers in patients with inflammatory bowel diseases.

Analyst 2022 Jan 13. Epub 2022 Jan 13.

Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN), Rua Alves Redol, 9, 1000-029 Lisbon, Portugal.

Inflammatory bowel disease (IBD) is a term used to describe disorders that involve chronic inflammation in the gastrointestinal tract, affecting more than 6.8 million people worldwide. Biological therapy is used in the most severe cases of IBD where anti-tumour necrosis factor-alpha (TNF-α) antibodies are the first choice for a biological treatment. When administrated to patients, these antibodies interact with TNF-α, usually overexpressed in these diseases, neutralizing its biological activity. Because of the chronic nature of these diseases, a recurring administration of the therapeutic antibodies is required, thus making therapy monitorization essential for the correct management of these diseases. The aim of this work is the development of an enzyme-linked immunosorbent assay (ELISA) microfluidic biosensor to quantify the therapeutic antibodies in IBD patient plasma samples, where the commercial monoclonal antibody Infliximab (IFX) is used as a model target. By providing a faster and more accurate measurement of IFX, the proposed method leads to improved therapy scheduling and a reduced risk of endogenous anti-drug antibodies (ADAs) reducing the efficacy of the treatment. The time needed between sample insertion and result output for the microfluidic ELISA (mELISA) is 24 minutes, drastically shorter than the time required by the conventional ELISA (cELISA). The mELISA presented in this work has a LoD of 0.026 μg mL, while commercially available solutions provide a LoD of 0.15 μg mL. Results acquired by the mELISA are highly correlated with the results obtained from the cELISA ( = 0.998; = 0.996; < 0.0001), demonstrating the validity of the microfluidic approach for the quantification of IFX from patient plasma and its potential for use at the point-of-care (POC).
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http://dx.doi.org/10.1039/d1an01810hDOI Listing
January 2022

Pre-miRNA-149 G-quadruplex as a molecular agent to capture nucleolin.

Eur J Pharm Sci 2022 Feb 16;169:106093. Epub 2021 Dec 16.

CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal. Electronic address:

One of the most significant challenges in capturing and detecting biomarkers is the choice of an appropriate biomolecular receptor. Recently, RNA G-quadruplexes emerged as plausible receptors due to their ability to recognize with high-affinity proteins. Herein, we have unveiled and characterized the capability of the precursor microRNA 149 to form a G-quadruplex structure and determined the role that some ligands may have in its folding and binding capacity to nucleolin. The G-quadruplex formation was induced by K ions and stabilized by ligands, as demonstrated by nuclear magnetic resonance and circular dichroism experiments. Surface plasmon resonance measurements showed a binding affinity of precursor microRNA 149 towards ligands in the micromolar range (10-10 M) and a strong binding affinity to nucleolin RNA-binding domains 1 and 2 (8.38 × 10 M). Even in the presence of the ligand PhenDC3, the binding remains almost identical and in the same order of magnitude (4.46 × 10 M). The molecular interactions of the RNA G-quadruplex motif found in precursor miRNA 149 (5'-GGGAGGGAGGGACGGG- 3') and nucleolin RNA-binding domains 1 and 2 were explored by means of molecular docking and molecular dynamics studies. The results showed that RNA G-quadruplex binds to a cavity between domains 1 and 2 of the protein. Then, complex formation was also evaluated through polyacrylamide gel electrophoresis. The results suggest that precursor microRNA 149/ligands and precursor microRNA 149/nucleolin RNA-binding domains 1 and 2 form stable molecular complexes. The in vitro co-localization of precursor microRNA 149 and nucleolin in PC3 cells was demonstrated using confocal microscopy. Finally, a rapid and straightforward microfluidic strategy was employed to check the ability of precursor microRNA 149 to capture nucleolin RNA-binding domains 1 and 2. The results revealed that precursor microRNA 149 can capture nucleolin RNA-binding domains 1 and 2 labeled with Fluorescein 5-isothiocyanate in a concentration-dependent manner, but PhenDC3 complexation seems to decrease the ability of precursor microRNA 149 to capture the protein. Overall, our results proved the formation of the G-quadruplex structure in the precursor microRNA 149 and the ability to recognize and detect nucleolin. This proof-of-concept study could open up a new framework for developing new strategies to design improved molecular receptors for capture and detection of nucleolin in complex biological samples.
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http://dx.doi.org/10.1016/j.ejps.2021.106093DOI Listing
February 2022

Microchromatography integrated with impedance sensor for bioprocess optimization: Experimental and numerical study of column efficiency for evaluation of scalability.

J Chromatogr A 2022 Jan 8;1661:462678. Epub 2021 Nov 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:

In the last decade, there has been a growing interest in developing microfluidic systems as new scale-down models for accelerated and cost-effective biopharmaceutical process development. Nonetheless, the research in this field is still in its infancy and requires further investigation to simplify and accelerate the microfabrication process. In addition, integration of different label-free sensors into the microcolumn systems has utmost importance to minimize result discrepancies during the scale-up process. In this study, we developed a simple, low-cost integrated microcolumn (26 µl). Micromilling technology was employed to define the geometry and shape of microfluidic structures using poly(methylmethacrylate) (PMMA). The design of PMMA microstructure was transferred to polydimethylsiloxane (PDMS), and interdigitated planar microelectrodes (IDE) were integrated into the system. To evaluate the scalability of the developed microcolumn column, column performance was assessed and compared with a conventional 1-ml prepacked column. Computational Fluid Dynamics (CFD) studies were performed for both columns to understand the differences between theoretical and experimental results regarding retention time and peak broadening. Despite obtaining an acceptable asymmetric factor for the microcolumn (1.03 ± 0.02), the reduced plate height value was still higher than the recommended range with the value of 4.14 ± 0.18. Nevertheless, the consistency and significant improvement of microcolumn efficiency compared to previous studies provide the possibility of developing robust simulation tools for transferring acquired experimental data for larger-scale units.
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http://dx.doi.org/10.1016/j.chroma.2021.462678DOI Listing
January 2022

Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microfluidic Assays.

Methods Mol Biol 2021 ;2268:289-304

INESC Microsistemas e Nanotecnologias, Lisbon, Portugal.

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).
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http://dx.doi.org/10.1007/978-1-0716-1221-7_19DOI Listing
July 2021

Aptamer-based approaches to detect nucleolin in prostate cancer.

Talanta 2021 May 31;226:122037. Epub 2020 Dec 31.

CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, Covilhã, Portugal. Electronic address:

We have investigated the expression of nucleolin (NCL) in liquid biopsies of prostate cancer (PCa) patients and healthy controls to determine its correlation with tumor prognosis. To detect NCL we used a modified AS1411 aptamer designated by AS1411-N5. In presence of NCL, AS1411-N5 increases the fluorescence by assuming a G-quadruplex (G4) structure, while in the absence of NCL the fluorescence signal remains quenched. The structural characterization of AS1411-N5 was performed by biophysical studies, which demonstrated the formation of G4 parallel conformation in the presence of 100 mM K and the ability to recognize NCL with high affinity (K = 138.1 ± 5.5 nM). Furthermore, the clinical relevance of NCL in PCa liquid biopsies was assessed by using an NCL-based ELISA assay. The protein was measured in the peripheral blood mononuclear cells (PBMCs) cell lysate of 158 individuals, including PCa patients and healthy individuals. The results depicted a remarkable increase of NCL levels in the PBMC's lysate of PCa patients (mean of 626.1 pg/mL whole blood) when compared to healthy individuals (mean of 198.5 pg/mL whole blood). The ELISA results also provided evidence for the usefulness of determining NCL levels in advanced PCa stages. Furthermore, a microfluidic assay showed the ability of AS1411-N5 in recognizing NCL in spiked human plasma samples.
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http://dx.doi.org/10.1016/j.talanta.2020.122037DOI Listing
May 2021

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

Evidence for family-level variation of phenotypic traits in response to temperature of Brazilian Nyssorhynchus darlingi.

Parasit Vectors 2020 Feb 10;13(1):55. Epub 2020 Feb 10.

Wadsworth Center, New York State Department of Health, New York State Route 5, Albany, NY, USA.

Background: Nyssorhynchus darlingi (also known as Anopheles darlingi) is the primary malaria vector in the Amazon River Basin. In Brazil, analysis of single nucleotide polymorphisms (SNPs) previously detected three major population clusters, and a common garden experiment in a laboratory setting revealed significant population variation in life history traits. Increasing temperatures and local level variation can affect life history traits, i.e. adult longevity, that alter vectorial capacity with implications for malaria transmission in Ny. darlingi.

Methods: We investigated the population structure of Ny. darlingi from 7 localities across Brazil utilizing SNPs and compared them to a comprehensive Ny. darlingi catalog. To test the effects of local level variation on life history traits, we reared F progeny from the 7 localities at three constant temperatures (20, 24 and 28 °C), measuring key life history traits (larval development, food-starved adult lifespan, adult size and daily survival).

Results: Using nextRAD genotyping-by-sequencing, 93 of the field-collected Ny. darlingi were genotyped at 33,759 loci. Results revealed three populations (K = 3), congruent with major biomes (Amazonia, Cerrado and Mata Atlântica), with greater F values between biomes than within. In the life history experiments, increasing temperature reduced larval development time, adult lifespan, and wing length in all localities. The variation of family responses for all traits within four localities of the Amazonia biome was significant (ANOVA, P < 0.05). Individual families within localities revealed a range of responses as temperature increased, for larval development, adult lifespan, wing length and survival time.

Conclusions: SNP analysis of several Brazilian localities provided results in support of a previous study wherein populations of Ny. darlingi were clustered by three major Brazilian biomes. Our laboratory results of temperature effects demonstrated that population variation in life history traits of Ny. darlingi exists at the local level, supporting previous research demonstrating the high plasticity of this species. Understanding this plasticity and inherent variation between families of Ny. darlingi at the local level should be considered when deploying intervention strategies and may improve the likelihood of successful malaria elimination in South America.
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http://dx.doi.org/10.1186/s13071-020-3924-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011564PMC
February 2020

Expression-Based Cell Lineage Analysis in Through a Course-Based Research Experience for Early Undergraduates.

G3 (Bethesda) 2019 11 5;9(11):3791-3800. Epub 2019 Nov 5.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095.

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The AL4 echnique for eal-time nd lonal xpression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.
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http://dx.doi.org/10.1534/g3.119.400541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829132PMC
November 2019

Microfluidic platform for rapid screening of bacterial cell lysis.

J Chromatogr A 2020 Jan 10;1610:460539. Epub 2019 Sep 10.

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:

Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency. This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.
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http://dx.doi.org/10.1016/j.chroma.2019.460539DOI Listing
January 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

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

Stepping responses to treadmill perturbations vary with severity of motor deficits in human SCI.

J Neurophysiol 2018 08 18;120(2):497-508. Epub 2018 Apr 18.

Sensory Motor Performance Program, Rehabilitation Institute of Chicago , Chicago, Illinois.

In this study, we investigated the responses to tread perturbations during human stepping on a treadmill. Our approach was to test the effects of perturbations to a single leg using a split-belt treadmill in healthy participants and in participants with varying severity of spinal cord injury (SCI). We recruited 11 people with incomplete SCI and 5 noninjured participants. As participants walked on an instrumented treadmill, the belt on one side was stopped or accelerated briefly during midstance to late stance. A majority of participants initiated an unnecessary swing when the treadmill was stopped in midstance, although the likelihood of initiating a step was decreased in participants with more severe SCI. Accelerating or decelerating one belt of the treadmill during stance altered the characteristics of swing. We observed delayed swing initiation when the belt was decelerated (i.e., the hip was in a more flexed position at time of swing) and advanced swing initiation with acceleration (i.e., hip extended at swing initiation). Furthermore, the timing and leg posture of heel strike appeared to remain constant, reflected by a sagittal plane hip angle at heel strike that remained the same regardless of the perturbation. In summary, our results supported the current understanding of the role of sensory feedback and central drive in the control of stepping in participants with incomplete SCI and noninjured participants. In particular, the observation of unnecessary swing during a stop perturbation highlights the interdependence of central and sensory drive in walking control. NEW & NOTEWORTHY Using a novel approach with a split-belt treadmill, we tested the effects of hip angle perturbations to a single leg in healthy participants and participants with varying severity of spinal cord injury (SCI). A majority of participants initiated an unnecessary swing when the treadmill was stopped in midstance, although the likelihood of initiating a step decreased with the severity of SCI. Our results demonstrated interdependence of central and sensory drive in walking control.
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http://dx.doi.org/10.1152/jn.00486.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139440PMC
August 2018

Advances, challenges and opportunities for point-of-need screening of mycotoxins in foods and feeds.

Analyst 2018 Feb;143(5):1015-1035

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

The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains.
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http://dx.doi.org/10.1039/c7an01762fDOI Listing
February 2018

A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems.

J Chromatogr A 2017 Sep 5;1515:252-259. Epub 2017 Aug 5.

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

Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE.
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http://dx.doi.org/10.1016/j.chroma.2017.07.094DOI Listing
September 2017

Multiplexed capillary microfluidic immunoassay with smartphone data acquisition for parallel mycotoxin detection.

Biosens Bioelectron 2018 Jan 15;99:40-46. Epub 2017 Jul 15.

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

The field of microfluidics holds great promise for the development of simple and portable lab-on-a-chip systems. The use of capillarity as a means of fluidic manipulation in lab-on-a-chip systems can potentially reduce the complexity of the instrumentation and allow the development of user-friendly devices for point-of-need analyses. In this work, a PDMS microchannel-based, colorimetric, autonomous capillary chip provides a multiplexed and semi-quantitative immunodetection assay. Results are acquired using a standard smartphone camera and analyzed with a simple gray scale quantification procedure. The performance of this device was tested for the simultaneous detection of the mycotoxins ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON) which are strictly regulated food contaminants with severe detrimental effects on human and animal health. The multiplexed assay was performed approximately within 10min and the achieved sensitivities of<40, 0.1-0.2 and<10ng/mL for OTA, AFB1 and DON, respectively, fall within the majority of currently enforced regulatory and/or recommended limits. Furthermore, to assess the potential of the device to analyze real samples, the immunoassay was successfully validated for these 3 mycotoxins in a corn-based feed sample after a simple sample preparation procedure.
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http://dx.doi.org/10.1016/j.bios.2017.07.032DOI Listing
January 2018

A simple method for point-of-need extraction, concentration and rapid multi-mycotoxin immunodetection in feeds using aqueous two-phase systems.

J Chromatogr A 2017 Aug 5;1511:15-24. Epub 2017 Jul 5.

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:

The rapid detection of mycotoxins in feed samples is becoming an increasingly relevant challenge for the food production sector, in order to effectively enforce current regulations and assure food and feed safety. To achieve rapid mycotoxin detection, several biosensing strategies have been published, many reaching assay times of the order of a few minutes. However, the vast majority of these rely on sample preparation based on volatile organic solvents, often comprising complex multi-step procedures and devoid of clean-up and/or concentration effects. Here, a novel sample preparation methodology based on a green, non-toxic and inexpensive polyethylene glycol-sodium citrate aqueous two-phase system is reported, providing single-step extraction and concentration of three target mycotoxins within 20min: aflatoxin B1 (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON). With point-of-need applications in mind, the extraction procedure was optimized and validated using a rapid multi-toxin microfluidic competitive immunoassay. The assay was successfully tested with spiked complex solid matrices including corn, soy, chickpea and sunflower-based feeds and limits of detection of 4.6ngg±15.8%, 24.1ngg±8.1% and 129.7ngg±53.1% (±CV) were obtained in corn for AFB1, OTA and DON, respectively. These sensitivities are fit-for-purpose at the required regulatory and recommended limits for animal feed, providing an effective and safe semi-quantitative mycotoxin analysis that can be performed in the field.
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http://dx.doi.org/10.1016/j.chroma.2017.07.004DOI Listing
August 2017

Systematic identification of anti-interferon function on hepatitis C virus genome reveals p7 as an immune evasion protein.

Proc Natl Acad Sci U S A 2017 02 3;114(8):2018-2023. Epub 2017 Feb 3.

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095;

Hepatitis C virus (HCV) encodes mechanisms to evade the multilayered antiviral actions of the host immune system. Great progress has been made in elucidating the strategies HCV employs to down-regulate interferon (IFN) production, impede IFN signaling transduction, and impair IFN-stimulated gene (ISG) expression. However, there is a limited understanding of the mechanisms governing how viral proteins counteract the antiviral functions of downstream IFN effectors due to the lack of an efficient approach to identify such interactions systematically. To study the mechanisms by which HCV antagonizes the IFN responses, we have developed a high-throughput profiling platform that enables mapping of HCV sequences critical for anti-IFN function at high resolution. Genome-wide profiling performed with a 15-nt insertion mutant library of HCV showed that mutations in the p7 region conferred high levels of IFN sensitivity, which could be alleviated by the expression of WT p7 protein. This finding suggests that p7 protein of HCV has an immune evasion function. By screening a liver-specific ISG library, we identified that IFI6-16 significantly inhibits the replication of p7 mutant viruses without affecting WT virus replication. In contrast, knockout of IFI6-16 reversed the IFN hypersensitivity of p7 mutant virus. In addition, p7 was found to be coimmunoprecipitated with IFI6-16 and to counteract the function of IFI6-16 by depolarizing the mitochondria potential. Our data suggest that p7 is a critical immune evasion protein that suppresses the antiviral IFN function by counteracting the function of IFI6-16.
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http://dx.doi.org/10.1073/pnas.1614623114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338388PMC
February 2017

Assessing Proprioception in Children: A Review.

J Mot Behav 2017 Jul-Aug;49(4):458-466. Epub 2016 Dec 9.

a Department of Occupational Therapy , Virginia Commonwealth University , Richmond , Virginia.

Proprioception is the subconscious and conscious awareness of the spatial and mechanical status of the musculoskeletal framework. When working with children with motor delays and sensory integrative dysfunction, occupational therapists routinely assess the client's proprioceptive system. However, currently available assessments for occupational therapists are primarily observer-based and concerns have been raised about the reliability of observer-based assessments of sensation. The author's purpose was to review measures of proprioception currently available to occupational therapists and explore direct measures of proprioception from neuroscience and rehabilitation that can be adapted for pediatric clinical use. Observer-based and direct measurements of proprioception assessments complement each other in meeting clinical needs. A better understanding of both types of evaluation will improve proprioceptive evaluation.
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http://dx.doi.org/10.1080/00222895.2016.1241744DOI Listing
September 2018

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies.

Front Neurosci 2016 15;10:511. Epub 2016 Nov 15.

Faculdade de Ciências Médicas, CEDOC - Chronic Diseases Research Center, Universidade Nova de LisboaLisbon, Portugal; Department of Neurodegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center GöttingenGöttingen, Germany.

Although, the precise molecular mechanisms underlying Parkinson's disease (PD) are still elusive, it is now known that spreading of alpha-synuclein (aSyn) pathology and neuroinflammation are important players in disease progression. Here, we developed a novel microfluidic cell-culture platform for studying the communication between two different cell populations, a process of critical importance not only in PD but also in many biological processes. The integration of micro-valves in the device enabled us to control fluid routing, cellular microenvironments, and to simulate paracrine signaling. As proof of concept, two sets of experiments were designed to show how this platform can be used to investigate specific molecular mechanisms associated with PD. In one experiment, naïve H4 neuroglioma cells were co-cultured with cells expressing aSyn tagged with GFP (aSyn-GFP), to study the release and spreading of the protein. In our experimental set up, we induced the release of the contents of aSyn-GFP producing cells to the medium and monitored the protein's diffusion. In another experiment, H4 cells were co-cultured with N9 microglial cells to assess the interplay between two cell lines in response to environmental stimuli. Here, we observed an increase in the levels of reactive oxygen species in H4 cells cultured in the presence of activated N9 cells, confirming the cross talk between different cell populations. In summary, the platform developed in this study affords novel opportunities for the study of the molecular mechanisms involved in PD and other neurodegenerative diseases.
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http://dx.doi.org/10.3389/fnins.2016.00511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108800PMC
November 2016

Miniaturization of aqueous two-phase extraction for biological applications: From micro-tubes to microchannels.

Biotechnol J 2016 Dec 14;11(12):1498-1512. Epub 2016 Sep 14.

IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.

Aqueous two-phase extraction (ATPE) is a biocompatible liquid-liquid (L-L) separation technique that has been under research for several decades towards the purification of biomolecules, ranging from small metabolites to large animal cells. More recently, with the emergence of rapid-prototyping techniques for fabrication of microfluidic structures with intricate designs, ATPE gained an expanded range of applications utilizing physical phenomena occurring exclusively at the microscale. Today, research is being carried simultaneously in two different volume ranges, mL-scale (microtubes) and nL-scale (microchannels). The objective of this review is to give insight into the state of the art at both microtube and microchannel-scale and to analyze whether miniaturization is currently a competing or divergent technology in a field of applications including bioseparation, bioanalytics, enhanced fermentation processes, catalysis, high-throughput screening and physical/chemical compartmentalization. From our perspective, both approaches are worthy of investigation and, depending on the application, it is likely that either (i) one of the approaches will eventually become obsolete in particular research areas such as purification at the preparative scale or high-throughput screening applications; or (ii) both approaches will function as complementing techniques within the bioanalytics field.
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http://dx.doi.org/10.1002/biot.201600356DOI Listing
December 2016

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

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

Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen.

Stem Cell Res 2016 05 29;16(3):640-50. Epub 2016 Mar 29.

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD 20892, USA. Electronic address:

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.
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http://dx.doi.org/10.1016/j.scr.2016.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4903911PMC
May 2016

Children With Dystonia Can Learn a Novel Motor Skill: Strategies That are Tolerant to High Variability.

IEEE Trans Neural Syst Rehabil Eng 2016 Aug 25;24(8):847-858. Epub 2016 Jan 25.

Children with dystonia are characterized by highly variable and seemingly uncontrolled movements. An important question for any rehabilitative effort is whether these children can learn and improve their performance. This study compared children with dystonia due to cerebral palsy, typically developing children, and healthy adults in their ability to acquire a novel sensorimotor skill. Using a virtual setup, subjects threw a virtual ball tethered to a post to hit a virtual target. Multiple combinations of release angle and velocity of the arm at ball release could achieve a target hit-the task was redundant and afforded solutions with different sensitivity to variability. Subjects performed 200 trials for two target locations that presented different types of redundancy. We hypothesized that children with dystonia develop strategies that are tolerant to their high variability. Estimating this variability highlighted the insufficiency of traditional outcome measures. Therefore, additional analyses of data distributions and of ball release timing were applied. Results showed that: 1) children with dystonia reduced their performance error despite their high variability; 2) this improvement was brought about by finding error-tolerant solutions; and 3) they generated arm trajectories that created time windows for ball release that were tolerant to timing variability. While reduced in magnitude, the performance improvements in children with dystonia paralleled those in healthy children and adults. These findings demonstrate that children with dystonia are able to adapt their behavior to their high variability, an important basis for any rehabilitative intervention.
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http://dx.doi.org/10.1109/TNSRE.2016.2521404DOI Listing
August 2016

DNA aptamer-based sandwich microfluidic assays for dual quantification and multi-glycan profiling of cancer biomarkers.

Biosens Bioelectron 2016 May 19;79:313-9. Epub 2015 Dec 19.

Department of Electronic & Electrical Engineering, University of Bath, Bath BA2 7AY, United Kingdom. Electronic address:

Two novel sandwich-based immunoassays for prostate cancer (PCa) diagnosis are reported, in which the primary antibody for capture is replaced by a DNA aptamer. The assays, which can be performed in parallel, were developed in a microfluidic device and tested for the detection of free Prostate Specific Antigen (fPSA). A secondary antibody (Aptamer-Antibody Assay) or a lectin (Aptamer-Lectin Assay) is used to quantify, by chemiluminescence, both the amount of fPSA and its glycosylation levels. The use of aptamers enables a more reliable, selective and controlled sensing of the analyte. The dual approach provides sensitive detection of fPSA along with selective fPSA glycoprofiling, which is of significant importance in the diagnosis and prognosis of PCa, as tumor progression is associated with changes in fPSA glycosylation. With these approaches, we can potentially detect 0.5 ng/mL of fPSA and 3 ng/mL of glycosylated fPSA using Sambucus nigra (SNA) lectin, both within the relevant clinical range. The approach can be applied to a wide range of biomarkers, thus providing a good alternative to standard antibody-based immunoassays with significant impact in medical diagnosis and prognosis.
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http://dx.doi.org/10.1016/j.bios.2015.12.058DOI Listing
May 2016

Evidence for temporal population replacement and the signature of ecological adaptation in a major Neotropical malaria vector in Amazonian Peru.

Malar J 2015 Sep 29;14:375. Epub 2015 Sep 29.

Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY, USA.

Background: The major Neotropical malaria vector, Anopheles darlingi, was reintroduced into the Iquitos, Loreto, Peru area during the early 1990s, where it displaced other anophelines and caused a major malaria epidemic. Since then, case numbers in Loreto have fluctuated, but annual increases have been reported since 2012.

Methods: The population genetic structure of An. darlingi sampled before and after the introduction of long-lasting insecticidal nets (LLINs) was investigated to test the hypothesis of temporal population change (2006 vs. 2012). Current samples of An. darlingi were used to test the hypothesis of ecological adaptation to human modified (highway) compared with wild (riverine) habitat, linked to forest cover. In total, 693 An. darlingi from nine localities in Loreto, Peru area were genotyped using 13 microsatellite loci. To test the hypothesis of habitat differentiation in An. darlingi biting time patterns, HBR and EIR, four collections of An. darlingi from five localities (two riverine and three highway) were analysed.

Results: Analyses of microsatellite loci from seven (2006) and nine settlements (2012-2014) in the Iquitos area detected two distinctive populations with little overlap, although it is unclear whether this population replacement event is associated with LLIN distribution or climate. Within the 2012-2014 population two admixed subpopulations, A and B, were differentiated by habitat, with B significantly overrepresented in highway, and both in near-equal proportions in riverine. Both subpopulations had a signature of expansion and there was moderate genetic differentiation between them. Habitat and forest cover level had significant effects on HBR, such that Plasmodium transmission risk, as measured by EIR, in peridomestic riverine settlements was threefold higher than in peridomestic highway settlements. HBR was directly associated with available host biomass rather than forest cover.

Conclusions: A population replacement event occurred between 2006 and 2012-2014, concurrently with LLIN distribution and a moderate El Niño event, and prior to an increase in malaria incidence. The likely drivers of this replacement cannot be determined with current data. The present-day An. darlingi population is composed of two highly admixed subpopulations, which appear to be in an early stage of differentiation, triggered by anthropogenic alterations to local habitat.
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http://dx.doi.org/10.1186/s12936-015-0863-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587789PMC
September 2015
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