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1.
Biosens Bioelectron ; 258: 116337, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38703495

RESUMO

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.


Assuntos
Técnicas Biossensoriais , Adesão Celular , Neoplasias Cutâneas , Humanos , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Glicosilação , Neoplasias Cutâneas/patologia , Melanoma/patologia , Melanoma/diagnóstico , Queratinócitos/citologia , Pele/patologia , Pele/química , Lectinas/química , Lectinas/metabolismo , Linhagem Celular Tumoral , Melanócitos/citologia , Melanócitos/metabolismo , Microfluídica/métodos , Técnicas Analíticas Microfluídicas/instrumentação
2.
Biosens Bioelectron ; 258: 116350, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38705075

RESUMO

Early monitoring of cardiovascular disease (CVD) is crucial for its treatment and prognosis. Hence, highly specific and sensitive detection method is urgently needed. In this study, we propose a novel herringbone microfluid chip with aptamer functionalized core-shell photonic crystal (PhC) barcode integration for high throughput multiplex CVD detection. Based on the PhC derived from co-assembled carboxylated single-wall carbon nanotubes and silicon dioxide nanoparticles, we obtain core-shell PhC barcodes by hydrogel replicating and partially etching. These core-shell PhC barcodes not only retain the original structural colors coding element, but also fully expose a large number of carboxyl elements in the ore for the probe immobilization. We further combine the functionalized barcodes with herringbone groove microfluidic chip to elucidate its acceptability in testing clinical sample. It is demonstrated that the special design of microfluidic chip can significantly enhance fluid vortex resistance and contact frequency, improving the sample capture efficiency and detection sensitivity. These features indicate that our core-shell PhC barcodes-integrated herringbone microfluidic system possesses great potential for multiplex biomarker detection in clinical application.


Assuntos
Biomarcadores , Técnicas Biossensoriais , Dispositivos Lab-On-A-Chip , Nanotubos de Carbono , Nanotubos de Carbono/química , Humanos , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Aptâmeros de Nucleotídeos/química , Dióxido de Silício/química , Fótons , Nanopartículas/química , Técnicas Analíticas Microfluídicas/instrumentação
3.
Biosens Bioelectron ; 258: 116352, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38718635

RESUMO

The production of HbS - an abnormal hemoglobin (Hb) - in sickle cell disease (SCD) results in poorly deformable red blood cells (RBCs) that are prone to microcapillary occlusion, causing tissue ischemia and organ damage. Novel treatments, including gene therapy, may reduce SCD morbidity, but methods to functionally evaluate RBCs remain limited. Previously, we presented the microfluidic impedance red cell assay (MIRCA) for rapid assessment of RBC deformability, employing electrical impedance-based readout to measure RBC occlusion of progressively narrowing micropillar openings. We describe herein the design, development, validation, and clinical utility of the next-generation MIRCA assay, featuring enhanced portability, rapidity, and usability. It incorporates a miniaturized impedance analyzer and features a simplified wash-free operation that yields an occlusion index (OI) within 15 min as a new metric for RBC occlusion. We show a correlation between OI and percent fetal hemoglobin (%HbF), other laboratory biomarkers of RBC hemolysis, and SCD severity. To demonstrate the assay's versatility, we tested RBC samples from treatment-naïve SCD patients in Uganda that yielded OI levels similar to those from hydroxyurea (HU)-treated patients in the U.S., highlighting the role of %HbF in protecting against microcapillary occlusion independent of other pharmacological effects. The MIRCA assay could also identify a subset of HU-treated patients with high occlusion risks, suggesting that they may require treatment adjustments including a second-line therapy to improve their outcomes. This work demonstrates the potential of the MIRCA assay for accelerated evaluation of RBC health, function, and therapeutic effect in an ex vivo model of the microcapillary networks.


Assuntos
Anemia Falciforme , Técnicas Biossensoriais , Impedância Elétrica , Eritrócitos , Humanos , Anemia Falciforme/sangue , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Deformação Eritrocítica , Técnicas Analíticas Microfluídicas/instrumentação , Hemólise , Dispositivos Lab-On-A-Chip
4.
Methods Mol Biol ; 2804: 3-50, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753138

RESUMO

Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.


Assuntos
Técnicas Analíticas Microfluídicas , Ácidos Nucleicos , Sistemas Automatizados de Assistência Junto ao Leito , Proteínas , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Microfluídica/instrumentação , Ácidos Nucleicos/análise , Testes Imediatos , Proteínas/análise
5.
Biomed Microdevices ; 26(2): 24, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38709370

RESUMO

We report the fabrication and characterisation of magnetic liquid beads with a solid magnetic shell and liquid core using microfluidic techniques. The liquid beads consist of a fluorinated oil core and a polymer shell with magnetite particles. The beads are generated in a flow-focusing polydimethylsiloxane (PDMS) device and cured by photo polymerisation. We investigated the response of the liquid beads to an external magnetic field by characterising their motion towards a permanent magnet. Magnetic sorting of liquid beads in a channel was achieved with 90% efficiency. The results show that the liquid beads can be controlled magnetically and have potential applications in digital microfluidics including nucleic acid amplification, drug delivery, cell culture, sensing, and tissue engineering. The present paper also discusses the magnetophoretic behaviour of the liquid bead by varying its mass and magnetite concentration in the shell. We also demonstrated the two-dimensional self-assembly of magnetic liquid beads for potential use in digital polymerase chain reaction and digital loop mediated isothermal amplification.


Assuntos
Dimetilpolisiloxanos , Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/instrumentação , Campos Magnéticos , Microesferas
6.
Anal Chim Acta ; 1307: 342640, 2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719417

RESUMO

BACKGROUND: The analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. RESULTS: In this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. SIGNIFICANCE AND NOVELTY: By using the method of small flow perfusion (≤20 µL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.


Assuntos
Crioprotetores , Análise de Célula Única , Análise Espectral Raman , Análise Espectral Raman/métodos , Crioprotetores/química , Crioprotetores/farmacologia , Crioprotetores/isolamento & purificação , Dispositivos Lab-On-A-Chip , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Criopreservação/métodos , Animais
7.
Mikrochim Acta ; 191(5): 295, 2024 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-38700804

RESUMO

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.


Assuntos
Separação Celular , Doença Granulomatosa Crônica , Dispositivos Lab-On-A-Chip , Leucócitos , Técnicas Analíticas Microfluídicas , Humanos , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/sangue , Leucócitos/citologia , Separação Celular/instrumentação , Separação Celular/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
8.
PLoS One ; 19(5): e0295849, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38696491

RESUMO

INTRODUCTION: Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20. METHODS: An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined. RESULTS: Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 µs, which is lower than 56 µs for BSA and 16 µs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 µs, which confirms the reliability of MRPS measurements. CONCLUSION: The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.


Assuntos
Vesículas Extracelulares , Poloxâmero , Poloxâmero/química , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Humanos , Polissorbatos/química , Soroalbumina Bovina/química , Microfluídica/métodos , Molhabilidade , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Animais
9.
Lab Chip ; 24(10): 2791-2801, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38691394

RESUMO

Dilution is a standard fluid operation widely employed in the sample preparation process of many bio(chemical) assays. It serves multiple essential functions such as sample mixing with certain reagents at specific dilution ratios, reducing sample matrix effects, bringing target analytes within the linear assay detection range, among many others. Traditionally, sample processing is performed in laboratory settings through manual or automated pipetting. When working in resource-limited settings, however, neither trained personnel nor proper laboratory equipment are available limiting the accessibility to high-quality diagnostic tests. In this work, we present a novel standalone and fully automated microfluidic platform for the stepwise preparation of serial dilutions without the need for any active elements. Stepwise dilution is achieved using the coordinated burst action of hydrophobic burst valves to first isolate a precisely metered volume from an applied sample drop and subsequently merge it with a prefilled diluent liquid. Downstream, expansion chambers are used to mix both reagents into a homogeneous solution. The dilution module was characterized to generate accurate and reproducible (CV < 7%) dilutions for targeted dilution factors of 2, 5 and 10×, respectively. Three dilution modules were coupled in series to generate three-fold logarithmic (log5 or log10) dilutions, with excellent linearity (R2 > 0.99). Its compatibility with whole blood was furthermore illustrated, proving its applicability for automating and downscaling bioassays with complex biological matrices. Finally, autonomous on-chip serial dilution was demonstrated by incorporating the self-powered (i)SIMPLE technology as a passive driving source for liquid manipulation. We believe that the simplicity and modularity of the presented autonomous dilution platform are of interest to many point-of-care applications in which sample dilution and reagent mixing are of importance.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento
10.
Anal Methods ; 16(19): 3007-3019, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38695537

RESUMO

We present a colorimetric probe based on polyvinylpyrrolidone-capped gold nanoparticles (PVP-AuNPs) that is sensitive and selective for cysteine (Cys). A microfluidic paper-based analytical device (µ-PAD) with embedded dried PVP-AuNPs at the polyethersulfone (PES) paper surface is used for Cys detection. When thiol molecules attach to PVP-AuNPs in the presence of Cys, they clump together, and this causes the solution's color to shift from red to blue within 5 minutes. The device is capable of detecting Cys levels between 1.0 µM and 50.0 µM with a limit of detection (LOD) of 0.2 µM under optimized conditions. The stability of the µ-PAD was tested for 100 days, demonstrating re-dispersibility to detect Cys levels in blood. Dried PVP-AuNP-µPADs were integrated with blood plasma separation modules for point-of-care (POC) Cys detection. Consequently, the device shows potential as a self-sustaining, quantification platform with a recovery percentage ranging from 98.44 to 111.9 in clinical samples.


Assuntos
Colorimetria , Cisteína , Ouro , Limite de Detecção , Nanopartículas Metálicas , Papel , Sistemas Automatizados de Assistência Junto ao Leito , Ouro/química , Cisteína/sangue , Cisteína/química , Nanopartículas Metálicas/química , Humanos , Colorimetria/métodos , Colorimetria/instrumentação , Povidona/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
11.
Lab Chip ; 24(10): 2811-2824, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38700452

RESUMO

The aging process has broad physiological impacts, including a significant decline in sensory function, which threatens both physical health and quality of life. One ideal model to study aging, neuronal function, and gene expression is the nematode Caenorhabditis elegans, which has a short lifespan and relatively simple, thoroughly mapped nervous system and genome. Previous works have identified that mechanosensory neuronal structure changes with age, but importantly, the actual age-related changes in the function and health of neurons, as well as the underlying genetic mechanisms responsible for these declines, are not fully understood. While advanced techniques such as single-cell RNA-sequencing have been developed to quantify gene expression, it is difficult to relate this information to functional changes in aging due to a lack of tools available. To address these limitations, we present a platform capable of measuring both physiological function and its associated gene expression throughout the aging process in individuals. Using our pipeline, we investigate the age-related changes in function of the mechanosensing ALM neuron in C. elegans, as well as some relevant gene expression patterns (mec-4 and mec-10). Using a series of devices for animals of different ages, we examined subtle changes in neuronal function and found that while the magnitude of neuronal response to a large stimulus declines with age, sensory capability does not significantly decline with age; further, gene expression is well maintained throughout aging. Additionally, we examine PVD, a harsh-touch mechanosensory neuron, and find that it exhibits a similar age-related decline in magnitude of neuronal response. Together, our data demonstrate that our strategy is useful for identifying genetic factors involved in the decline in neuronal health. We envision that this framework could be applied to other systems as a useful tool for discovering new biology.


Assuntos
Envelhecimento , Caenorhabditis elegans , Dispositivos Lab-On-A-Chip , Neurônios , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Envelhecimento/fisiologia , Neurônios/metabolismo , Neurônios/citologia , Mecanotransdução Celular , Técnicas Analíticas Microfluídicas/instrumentação
12.
Nat Commun ; 15(1): 4363, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38778087

RESUMO

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.


Assuntos
Neoplasias da Mama , Microfluídica , Medicina de Precisão , Ensaios Antitumorais Modelo de Xenoenxerto , Medicina de Precisão/métodos , Humanos , Animais , Camundongos , Feminino , Linhagem Celular Tumoral , Microfluídica/métodos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
13.
Anal Chim Acta ; 1308: 342575, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38740448

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a prevalent neurodegenerative disease with no effective treatment. Efficient and rapid detection plays a crucial role in mitigating and managing AD progression. Deep learning-assisted smartphone-based microfluidic paper analysis devices (µPADs) offer the advantages of low cost, good sensitivity, and rapid detection, providing a strategic pathway to address large-scale disease screening in resource-limited areas. However, existing smartphone-based detection platforms usually rely on large devices or cloud servers for data transfer and processing. Additionally, the implementation of automated colorimetric enzyme-linked immunoassay (c-ELISA) on µPADs can further facilitate the realization of smartphone µPADs platforms for efficient disease detection. RESULTS: This paper introduces a new deep learning-assisted offline smartphone platform for early AD screening, offering rapid disease detection in low-resource areas. The proposed platform features a simple mechanical rotating structure controlled by a smartphone, enabling fully automated c-ELISA on µPADs. Our platform successfully applied sandwich c-ELISA for detecting the ß-amyloid peptide 1-42 (Aß 1-42, a crucial AD biomarker) and demonstrated its efficacy in 38 artificial plasma samples (healthy: 19, unhealthy: 19, N = 6). Moreover, we employed the YOLOv5 deep learning model and achieved an impressive 97 % accuracy on a dataset of 1824 images, which is 10.16 % higher than the traditional method of curve-fitting results. The trained YOLOv5 model was seamlessly integrated into the smartphone using the NCNN (Tencent's Neural Network Inference Framework), enabling deep learning-assisted offline detection. A user-friendly smartphone application was developed to control the entire process, realizing a streamlined "samples in, answers out" approach. SIGNIFICANCE: This deep learning-assisted, low-cost, user-friendly, highly stable, and rapid-response automated offline smartphone-based detection platform represents a good advancement in point-of-care testing (POCT). Moreover, our platform provides a feasible approach for efficient AD detection by examining the level of Aß 1-42, particularly in areas with low resources and limited communication infrastructure.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Biomarcadores , Ensaio de Imunoadsorção Enzimática , Papel , Smartphone , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/sangue , Humanos , Biomarcadores/sangue , Biomarcadores/análise , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/sangue , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/análise , Dispositivos Lab-On-A-Chip , Aprendizado Profundo , Automação , Técnicas Analíticas Microfluídicas/instrumentação
14.
Anal Chim Acta ; 1308: 342639, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38740452

RESUMO

BACKGROUND: Calcium and magnesium ions are highly abundant and important cations in human body. At the same time, both dyscalcemia and dysmagnesemia are frequently encountered in the clinical practice. As deficiency or excess of Ca(II) or Mg(II) can cause severe symptoms, determining these ions in serum is of great importance. Concentration of these ions in biological samples is typically assayed in clinical laboratories with the use of expensive and specialized equipment. Since those methods cannot be easily adapted for self-diagnosis purposes, there is a great need to develop a convenient tool for reliable determination of calcium and magnesium in serum at the point-of-care. RESULTS: The colorimetric methods employed for calcium and magnesium analysis were o-cresophtalein complexone assay and xylidyl blue assay, respectively. Analytical signal acquisition was accomplished using an ordinary flatbed scanner or smartphone and free software. For increased user-friendliness the device was optimized to perform simultaneous determination of calcium and magnesium ions in only 10 min. In the optimized conditions, the limit of detection for calcium ions was 0.09 mmol L-1, while for magnesium it was 0.04 mmol L-1. Determination of both ions requires only 4 µL of serum sample. The developed paper-based sensors were validated with control human serum samples and the obtained relative errors for majority of samples were below 20 %. SIGNIFICANCE: In this paper, a microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum is reported for the first time. Additionally, this is also the first report on colorimetric determination in serum of any of these ions in paper-based format. Simultaneous detection of both ions allows for fast and user-friendly screening of disturbance in calcium and magnesium homeostasis.


Assuntos
Cálcio , Magnésio , Papel , Magnésio/sangue , Humanos , Cálcio/sangue , Colorimetria , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Limite de Detecção
15.
J Sep Sci ; 47(9-10): e2400120, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38772720

RESUMO

Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in developing an accurate and label-free tool for herbal medicine authentication. Here, a high-resolution microfluidic-based method is developed for identifying herbal species by protoplast subpopulations. Moso bamboo and henon bamboo are used as a model to be differentiated based on protoplast. Their biophysical properties factors are characterized to be 7.09 (± 0.39) × 108 V/m2 and 6.54 (± 0.26) × 108 V/m2, respectively. Their biophysical distributions could be distinguished by the Cramér-von Mises criterion with a 94.60% confidence level. The subpopulations of each were compared with conventional flow cytometry indicating the existence of subpopulations and the differences between the two species. The subsets divided by a biophysical factor of 8.05(± 0.51) × 108 V/m2 suggest good consistency with flow cytometry. The work demonstrated the possibility of microfluidics manipulation on protoplast for medication safety use taking advantage of dielectrophoresis. The device is promising in developing a reliable and accurate way of identifying herbal species with difficulties in authentication.


Assuntos
Folhas de Planta , Protoplastos , Análise de Célula Única , Protoplastos/citologia , Folhas de Planta/química , Citometria de Fluxo , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação
16.
Methods Mol Biol ; 2804: 77-89, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753141

RESUMO

Extracellular vesicles (EVs) are secreted by cells and found in biological fluids such as blood, with concentration correlated with oncogenic signals, making them attractive biomarkers for liquid biopsy. The current gold-standard method for EVs isolation requires an ultracentrifugation (UC) step among others. The cost and complexity of this technique are forbiddingly high for many researchers, as well as for routine use in biological laboratories and hospitals. This chapter reports on a simple microfluidic method for EVs isolation, based on a microfluidic size sorting technique named Deterministic Lateral Displacement (DLD). With the design of micrometric DLD array, we demonstrated the potential of our DLD devices for the isolation of nano-biological objects such as EVs, with main population size distribution consistent with UC technique.


Assuntos
Vesículas Extracelulares , Dispositivos Lab-On-A-Chip , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Cultura de Células/métodos , Ultracentrifugação/métodos
17.
Methods Mol Biol ; 2804: 91-100, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753142

RESUMO

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.


Assuntos
Separação Imunomagnética , Dispositivos Lab-On-A-Chip , Células Neoplásicas Circulantes , Células Neoplásicas Circulantes/patologia , Separação Imunomagnética/métodos , Humanos , Separação Celular/métodos , Separação Celular/instrumentação , Neoplasias/patologia , Neoplasias/sangue , Linhagem Celular Tumoral , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
18.
Methods Mol Biol ; 2804: 127-138, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753145

RESUMO

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.


Assuntos
Cricetulus , Células CHO , Animais , Anticorpos Monoclonais/imunologia , Reatores Biológicos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Imunoensaio/métodos , Imunoensaio/instrumentação , Microfluídica/métodos , Microfluídica/instrumentação , Cricetinae
19.
Methods Mol Biol ; 2804: 65-75, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753140

RESUMO

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).


Assuntos
Ácidos Nucleicos Livres , Dispositivos Lab-On-A-Chip , Humanos , Ácidos Nucleicos Livres/isolamento & purificação , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Reação em Cadeia da Polimerase/métodos , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Magnetismo/métodos , Neoplasias/sangue , Neoplasias/genética , Neoplasias/diagnóstico
20.
Methods Mol Biol ; 2804: 141-162, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753146

RESUMO

Protein secretion is a key cellular functionality, particularly in immunology, where cells can display large heterogeneity in this crucial activity in addition to binary secretion behavior. However, few methods enable quantitative secretion rate measurements at the single-cell level, and these methods are mostly based on microfluidics systems. Here, we describe such a microfluidic single-cell method for precisely measuring protein secretion rates in detail, building on the published droplet-based microfluidic platform DropMap. We give an updated, detailed guide toward quantifying protein secretion rates, discussing its setup and limitations. We illustrate the protocol on two key immunological analytes, immunoglobulin G, and interferon-γ.


Assuntos
Interferon gama , Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Interferon gama/metabolismo , Imunoglobulina G/metabolismo , Proteínas/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/métodos , Microfluídica/instrumentação
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