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1.
Microsyst Nanoeng ; 10: 87, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38919163

RESUMO

The combination of flow elasticity and inertia has emerged as a viable tool for focusing and manipulating particles using microfluidics. Although there is considerable interest in the field of elasto-inertial microfluidics owing to its potential applications, research on particle focusing has been mostly limited to low Reynolds numbers (Re<1), and particle migration toward equilibrium positions has not been extensively examined. In this work, we thoroughly studied particle focusing on the dynamic range of flow rates and particle migration using straight microchannels with a single inlet high aspect ratio. We initially explored several parameters that had an impact on particle focusing, such as the particle size, channel dimensions, concentration of viscoelastic fluid, and flow rate. Our experimental work covered a wide range of dimensionless numbers (0.05 < Reynolds number < 85, 1.5 < Weissenberg number < 3800, 5 < Elasticity number < 470) using 3, 5, 7, and 10 µm particles. Our results showed that the particle size played a dominant role, and by tuning the parameters, particle focusing could be achieved at Reynolds numbers ranging from 0.2 (1 µL/min) to 85 (250 µL/min). Furthermore, we numerically and experimentally studied particle migration and reported differential particle migration for high-resolution separations of 5 µm, 7 µm and 10 µm particles in a sheathless flow at a throughput of 150 µL/min. Our work elucidates the complex particle transport in elasto-inertial flows and has great potential for the development of high-throughput and high-resolution particle separation for biomedical and environmental applications.

2.
Micromachines (Basel) ; 15(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38930678

RESUMO

Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.

3.
J Transl Med ; 22(1): 530, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38831317

RESUMO

BACKGROUND: Cancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression. METHODS: In this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors. RESULTS: We found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer. CONCLUSION: These results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.


Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas , Microambiente Tumoral , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Feminino , Transcrição Gênica , Perfilação da Expressão Gênica , Linhagem Celular Tumoral , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Fenótipo
4.
Anal Chem ; 96(23): 9493-9502, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38790145

RESUMO

The health hazards of micro- and nanoplastic contaminants in drinking water has recently emerged as an area of concern to policy makers and industry. Plastic contaminants range in size from micro- (5 mm to 1 µm) to nanoplastics (<1 µm). Microfluidics provides many tools for particle manipulation at the microscale, particularly in diagnostics and biomedicine, but has in general a limited capacity to process large volumes. Drinking water and environmental samples with low-level contamination of microplastics require processing of deciliter to liter sample volumes to achieve statistically relevant particle counts. Here, we introduce the EchoGrid, an acoustofluidics device for high throughput continuous flow particle enrichment into a robust array of particle clusters. The EchoGrid takes advantage of highly efficient particle capture through the integration of a micropatterned transducer for surface displacement-based acoustic trapping in a glass and polymer microchannel. Silica seed particles were used as anchor particles to improve capture performance at low particle concentrations and high flow rates. The device was able to maintain the silica grids at a flow rate of 50 mL/min. In terms of enrichment, the device is able to double the final pellet's microplastic concentration every 78 s for 23 µm particles and every 51 s for 10 µm particles at a flow rate of 5 mL/min. In conclusion, we demonstrate the usefulness of the EchoGrid by capturing microplastics in challenging conditions, such as large sample volumes with low microparticle concentrations, without sacrificing the potential of integration with downstream analysis for environmental monitoring.

5.
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
6.
BMJ Glob Health ; 8(12)2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38114235

RESUMO

Diagnostics are widely considered crucial in the fight against antimicrobial resistance (AMR), which is expected to kill 10 million people annually by 2030. Nevertheless, there remains a substantial gap between the need for AMR diagnostics versus their development and implementation. To help address this problem, target product profiles (TPP) have been developed to focus developers' attention on the key aspects of AMR diagnostic tests. However, during discussion between a multisectoral working group of 51 international experts from industry, academia and healthcare, it was noted that specific AMR-related TPPs could be extended by incorporating the interdependencies between the key characteristics associated with the development of such TPPs. Subsequently, the working group identified 46 characteristics associated with six main categories (ie, Intended Use, Diagnostic Question, Test Description, Assay Protocol, Performance and Commercial). The interdependencies of these characteristics were then identified and mapped against each other to generate new insights for use by stakeholders. Specifically, it may not be possible for diagnostics developers to achieve all of the recommendations in every category of a TPP and this publication indicates how prioritising specific TPP characteristics during diagnostics development may influence (or not) a range of other TPP characteristics associated with the diagnostic. The use of such guidance, in conjunction with specific TPPs, could lead to more efficient AMR diagnostics development.


Assuntos
Testes Diagnósticos de Rotina , Resistência Microbiana a Medicamentos , Humanos , Testes Diagnósticos de Rotina/métodos
7.
Biomedicines ; 11(11)2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38002061

RESUMO

Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril-polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines-KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.

8.
Anal Chim Acta ; 1245: 340823, 2023 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-36737129

RESUMO

Cytokines play a very important role in our immune system by acting as mediators to put up a coordinated defense against foreign elements in our body. Elevated levels of cytokines in the body can signal to an ongoing response of the immune system to some abnormality. Thus, the quantification of a panel of cytokines can provide valuable information regarding the diagnosis of specific diseases and state of overall health of an individual. Conventional Enzyme Linked Immunosorbent Assay (ELISA) is the gold-standard for quantification of cytokines, however the need for trained personnel and expensive equipment limits its application to centralized laboratories only. In this context, there is a lack of simple, low-cost and portable devices which can allow for quantification of panels of cytokines at point-of-care and/or resource limited settings. Here, we report the development of a versatile, low-cost and portable bead-based centrifugal microfluidic platform allowing for multiplexed detection of cytokines with minimal hands-on time and an integrated colorimetric signal readout without the need for any external equipment. As a model, multiplexed colorimetric quantification of three target cytokines i.e., Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-2 (IL-2) was achieved in less than 30 min with limits of detection in ng/mL range. The developed platform was further evaluated using spiked-in plasma samples to test for matrix interference. The ease of use, low-cost and portability of the developed platform highlight its potential to serve as a sample-to-answer solution for detection of cytokine panels in resource limited settings.


Assuntos
Colorimetria , Microfluídica , Citocinas , Ensaio de Imunoadsorção Enzimática , Biomarcadores
9.
Bioengineering (Basel) ; 9(11)2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36354514

RESUMO

The liver exhibits complex geometrical morphologies of hepatic cells arranged in a hexagonal lobule with an extracellular matrix (ECM) organized in a specific pattern on a multi-scale level. Previous studies have utilized 3D bioprinting and microfluidic perfusion systems with various biomaterials to develop lobule-like constructs. However, they all lack anatomical relevance with weak control over the size and shape of the fabricated structures. Moreover, most biomaterials lack liver-specific ECM components partially or entirely, which might limit their biomimetic mechanical properties and biological functions. Here, we report 3D bioprinting of a sacrificial PVA framework to impart its trilobular hepatic structure to the decellularized liver extracellular matrix (dLM) hydrogel with polyethylene glycol-based crosslinker and tyrosinase to fabricate a robust multi-scale 3D liver construct. The 3D trilobular construct exhibits higher crosslinking, viscosity (182.7 ± 1.6 Pa·s), and storage modulus (2554 ± 82.1 Pa) than non-crosslinked dLM. The co-culture of HepG2 liver cells and NIH 3T3 fibroblast cells exhibited the influence of fibroblasts on liver-specific activity over time (7 days) to show higher viability (90-91.5%), albumin secretion, and increasing activity of four liver-specific genes as compared to the HepG2 monoculture. This technique offers high lumen patency for the perfusion of media to fabricate a densely populated scaled-up liver model, which can also be extended to other tissue types with different biomaterials and multiple cells to support the creation of a large functional complex tissue.

10.
Biosensors (Basel) ; 12(7)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35884324

RESUMO

Bioprinting is an acclaimed technique that allows the scaling of 3D architectures in an organized pattern but suffers from a scarcity of appropriate bioinks. Decellularized extracellular matrix (dECM) from xenogeneic species has garnered support as a biomaterial to promote tissue-specific regeneration and repair. The prospect of developing dECM-based 3D artificial tissue is impeded by its inherent low mechanical properties. In recent years, 3D bioprinting of dECM-based bioinks modified with additional scaffolds has advanced the development of load-bearing constructs. However, previous attempts using dECM were limited to low-temperature bioprinting, which is not favorable for a longer print duration with cells. Here, we report the development of a multi-material decellularized liver matrix (dLM) bioink reinforced with gelatin and polyethylene glycol to improve rheology, extrudability, and mechanical stability. This shear-thinning bioink facilitated extrusion-based bioprinting at 37 °C with HepG2 cells into a 3D grid structure with a further enhancement for long-term applications by enzymatic crosslinking with mushroom tyrosinase. The heavily crosslinked structure showed a 16-fold increase in viscosity (2.73 Pa s-1) and a 32-fold increase in storage modulus from the non-crosslinked dLM while retaining high cell viability (85-93%) and liver-specific functions. Our results show that the cytocompatible crosslinking of dLM bioink at physiological temperatures has promising applications for extended 3D-printing procedures.


Assuntos
Bioimpressão , Bioimpressão/métodos , Matriz Extracelular/química , Hidrogéis/química , Fígado , Temperatura , Engenharia Tecidual/métodos , Alicerces Teciduais/química
11.
Macromol Biosci ; 22(10): e2200137, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35899862

RESUMO

Three-dimensional multicellular spheroids (MCSs) are complex structure of cellular aggregates and cell-to-matrix interaction that emulates the in-vivo microenvironment. This research field has grown to develop and improve spheroid generation techniques. Here, we present a new platform for spheroid generation using Layer-by-Layer (LbL) technology. Layer-by-Layer (LbL) containing cellulose nanofibrils (CNF) assemble on a standard 96 well plate. Various bi-layer numbers, multiple cell seeding concentration, and two tumor cell lines (HEK 293 T, HCT 116) are utilized to generate and characterize spheroids. The number and proliferation of generated spheroids, the viability, and the response to the anti-cancer drug are examined. The spheroids are formed and proliferated on the LbL-CNF coated wells with no significant difference in connection to the number of LbL-CNF bi-layers; however, the number of formed spheroids correlates positively with the cell seeding concentration (122 ± 17) and (42 ± 8) for HCT 116 and HEK 293T respectively at 700 cells ml-1 . The spheroids proliferate progressively up to (309, 663) µm of HCT 116 and HEK 293T respectively on 5 bi-layers coated wells with maintaining viability. The (HCT 116) spheroids react to the anti-cancer drug. We demonstrate a new (LbL-CNF) coating strategy for spheroids generation, with high performance and efficiency to test anti-cancer drugs.


Assuntos
Antineoplásicos , Celulose , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Celulose/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Esferoides Celulares
12.
Sci Rep ; 12(1): 3539, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35241725

RESUMO

Microfluidics has emerged rapidly over the past 20 years and has been investigated for a variety of applications from life sciences to environmental monitoring. Although continuous-flow microfluidics is ubiquitous, segmented-flow or droplet microfluidics offers several attractive features. Droplets can be independently manipulated and analyzed with very high throughput. Typically, microfluidics is carried out within planar networks of microchannels, namely, microfluidic chips. We propose that fibers offer an interesting alternative format with key advantages for enhanced optical coupling. Herein, we demonstrate the generation of monodisperse droplets within a uniaxial optofluidic Lab-in-a-Fiber scheme. We combine droplet microfluidics with laser-induced fluorescence (LIF) detection achieved through the development of an optical side-coupling fiber, which we term a periscope fiber. This arrangement provides stable and compact alignment. Laser-induced fluorescence offers high sensitivity and low detection limits with a rapid response time making it an attractive detection method for in situ real-time measurements. We use the well-established fluorophore, fluorescein, to characterize the Lab-in-a-Fiber device and determine the generation of [Formula: see text] 0.9 nL droplets. We present characterization data of a range of fluorescein concentrations, establishing a limit of detection (LOD) of 10 nM fluorescein. Finally, we show that the device operates within a realistic and relevant fluorescence regime by detecting reverse-transcription loop-mediated isothermal amplification (RT-LAMP) products in the context of COVID-19 diagnostics. The device represents a step towards the development of a point-of-care droplet digital RT-LAMP platform.


Assuntos
Dispositivos Lab-On-A-Chip , Vírus/isolamento & purificação , Fluorescência , Lasers
14.
Anal Chem ; 94(2): 787-792, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34931815

RESUMO

The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. Cell culture, nucleic acid amplification, or nanostructure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.


Assuntos
Bactérias , Doenças Transmissíveis , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos
15.
Electrophoresis ; 42(23): 2538-2551, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34510466

RESUMO

Improved sample preparation has the potential to address unmet needs for fast turnaround sepsis tests. In this work, we report elasto-inertial based rapid bacteria separation from diluted blood at high separation efficiency. In viscoelastic flows, we demonstrate novel findings where blood cells prepositioned at the outer wall entering a spiral device remain fully focused throughout the channel length while smaller bacteria migrate to the opposite wall. Initially, using microparticles, we show that particles above a certain size cut-off remain fully focused at the outer wall while smaller particles differentially migrate toward the inner wall. We demonstrate particle separation at 1 µm resolution at a total throughput of 1 mL/min. For blood-based experiments, a minimum of 1:2 dilution was necessary to fully focus blood cells at the outer wall. Finally, Escherichia coli spiked in diluted blood were continuously separated at a total flow rate of 1 mL/min, with efficiencies between 82 and 90% depending on the blood dilution. Using a single spiral, it takes 40 min to process 1 mL of blood at a separation efficiency of 82%. The label-free, passive, and rapid bacteria isolation method has a great potential for speeding up downstream phenotypic and genotypic analysis.


Assuntos
Bactérias , Técnicas Analíticas Microfluídicas , Sepse , Bactérias/isolamento & purificação , Células Sanguíneas/microbiologia , Humanos , Microfluídica , Sepse/sangue , Sepse/microbiologia
16.
ACS Sens ; 6(9): 3357-3366, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34410700

RESUMO

Sepsis is a serious bloodstream infection where the immunity of the host body is compromised, leading to organ failure and death of the patient. In early sepsis, the concentration of bacteria is very low and the time of diagnosis is very critical since mortality increases exponentially with every hour after infection. Common culture-based methods fail in fast bacteria determination, while recent rapid diagnostic methods are expensive and prone to false positives. In this work, we present a sepsis kit for fast detection of bacteria in whole blood, here achieved by combining selective cell lysis and a sensitive colorimetric approach detecting as low as 103 CFU/mL bacteria in less than 5 h. Homemade selective cell lysis buffer (combination of saponin and sodium cholate) allows fast processing of whole blood in 5 min while maintaining bacteria alive (100% viability). After filtration, retained bacteria on filter paper are incubated under constant illumination with the electrochromic precursors, i.e., ferricyanide and ferric ammonium citrate. Viable bacteria metabolically reduce iron(III) complexes, initiating a photocatalytic cascade toward Prussian blue formation. As a proof of concept, we combine this method with antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC) using two antibiotics (ampicillin and gentamicin). Although this kit is used to demonstrate its applicability to sepsis, this approach is expected to impact other key sectors such as hygiene evaluation, microbial contaminated food/beverage, or UTI, among others.


Assuntos
Compostos Férricos , Sepse , Bactérias , Humanos , Sepse/diagnóstico
18.
Lab Chip ; 21(15): 2932-2944, 2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34114589

RESUMO

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


Assuntos
COVID-19 , Teste para COVID-19 , Humanos , Microfluídica , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade , Smartphone
19.
Sci Rep ; 11(1): 8467, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875755

RESUMO

Passive particle manipulation using inertial and elasto-inertial microfluidics have received substantial interest in recent years and have found various applications in high throughput particle sorting and separation. For separation applications, elasto-inertial microfluidics has thus far been applied at substantial lower flow rates as compared to inertial microfluidics. In this work, we explore viscoelastic particle focusing and separation in spiral channels at two orders of magnitude higher Reynolds numbers than previously reported. We show that the balance between dominant inertial lift force, dean drag force and elastic force enables stable 3D particle focusing at dynamically high Reynolds numbers. Using a two-turn spiral, we show that particles, initially pinched towards the inner wall using an elasticity enhancer, PEO (polyethylene oxide), as sheath migrate towards the outer wall strictly based on size and can be effectively separated with high precision. As a proof of principle for high resolution particle separation, 15 µm particles were effectively separated from 10 µm particles. A separation efficiency of 98% for the 10 µm and 97% for the 15 µm particles was achieved. Furthermore, we demonstrate sheath-less, high throughput, separation using a novel integrated two-spiral device and achieved a separation efficiency of 89% for the 10 µm and 99% for the 15 µm particles at a sample flow rate of 1 mL/min-a throughput previously only reported for inertial microfluidics. We anticipate the ability to precisely control particles in 3D at extremely high flow rates will open up several applications, including the development of ultra-high throughput microflow cytometers and high-resolution separation of rare cells for point of care diagnostics.

20.
ACS Sens ; 6(3): 842-851, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33724791

RESUMO

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


Assuntos
Produtos Biológicos , Microfluídica , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Humanos
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