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1.
J Vis Exp ; (167)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33491677

RESUMO

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, and chemoresistance. Although several in vitro models have been developed to study hypoxia-related cancer pathology, the complex interplay of the cancer microenvironment observed in vivo has not been reproduced yet owing to the lack of precise spatial control. Instead, 3D biofabrication approaches have been proposed to create microphysiological systems for better emulation of cancer ecology and accurate anticancer treatment evaluation. Herein, we propose a 3D cell-printing approach to fabricate a hypoxic cancer-on-a-chip. The hypoxia-inducing components in the chip were determined based on a computer simulation of the oxygen distribution. Cancer-stroma concentric rings were printed using bioinks containing glioblastoma cells and endothelial cells to recapitulate a type of solid cancer. The resulting chip realized central hypoxia and aggravated malignancy in cancer with the formation of representative pathophysiological markers. Overall, the proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models for cancer research.


Assuntos
Progressão da Doença , Dispositivos Lab-On-A-Chip , Neoplasias/patologia , Impressão Tridimensional , Hipóxia Tumoral , Linhagem Celular Tumoral , Sobrevivência Celular , Colágeno/farmacologia , Simulação por Computador , Criopreservação , Dimetilpolisiloxanos/química , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Células Estromais/patologia , Microambiente Tumoral
2.
Biosens Bioelectron ; 171: 112679, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33069957

RESUMO

The 2019 SARS CoV-2 (COVID-19) pandemic has illustrated the need for rapid and accurate diagnostic tests. In this work, a multiplexed grating-coupled fluorescent plasmonics (GC-FP) biosensor platform was used to rapidly and accurately measure antibodies against COVID-19 in human blood serum and dried blood spot samples. The GC-FP platform measures antibody-antigen binding interactions for multiple targets in a single sample, and has 100% selectivity and sensitivity (n = 23) when measuring serum IgG levels against three COVID-19 antigens (spike S1, spike S1S2, and the nucleocapsid protein). The GC-FP platform yielded a quantitative, linear response for serum samples diluted to as low as 1:1600 dilution. Test results were highly correlated with two commercial COVID-19 antibody tests, including an enzyme linked immunosorbent assay (ELISA) and a Luminex-based microsphere immunoassay. To demonstrate test efficacy with other sample matrices, dried blood spot samples (n = 63) were obtained and evaluated with GC-FP, yielding 100% selectivity and 86.7% sensitivity for diagnosing prior COVID-19 infection. The test was also evaluated for detection of multiple immunoglobulin isotypes, with successful detection of IgM, IgG and IgA antibody-antigen interactions. Last, a machine learning approach was developed to accurately score patient samples for prior COVID-19 infection, using antibody binding data for all three COVID-19 antigens used in the test.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Teste em Amostras de Sangue Seco , Desenho de Equipamento , Fluorescência , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Dispositivos Lab-On-A-Chip , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Sensibilidade e Especificidade
3.
Biosens Bioelectron ; 174: 112830, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33339696

RESUMO

To date, health organizations and countries around the world are struggling to completely control the spread of the coronavirus disease 2019 (COVID-19). Scientists and researchers are developing tests for the rapid detection of individuals who may carry the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), while striving to find a suitable vaccine to immunize healthy individuals. As there are clinically reported cases of asymptomatic carriers of SARS-CoV-2, fast and accurate diagnosis plays an important role in the control and further prevention of this disease. Herein, we present recent technologies and techniques that have been implemented for the diagnosis of COVID-19. We summarize the methods created by different research institutes as well as the commercial devices and kits developed by companies for the detection of SARS-CoV-2. The description of the existing methods is followed by highlighting their advantages and challenges. Finally, we propose some promising techniques that could potentially be applied to the detection of SARS-CoV-2, and tracing the asymptomatic carriers of COVID-19 rapidly and accurately in the early stages of infection, based on reviewing the research studies on the detection of similar infectious viruses, especially severe acute respiratory syndrome (SARS) coronavirus, and Middle East respiratory syndrome (MERS) coronavirus.


Assuntos
/métodos , /diagnóstico , Inteligência Artificial , Técnicas Biossensoriais , /instrumentação , Humanos , Dispositivos Lab-On-A-Chip , Tomografia Computadorizada por Raios X
4.
Anal Chem ; 92(21): 14297-14302, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33073982

RESUMO

With the global outbreak of the coronavirus disease 2019 (COVID-19), the highly infective, highly pathogenic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has attracted great attention. Currently, a method to simultaneously diagnose the seven known types human coronaviruses remains lacking and is urgently needed. In this work, we successfully developed a portable microfluidic system for the rapid, accurate, and simultaneous detection of SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, and four other human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. The disk-like microfluidic platform integrated with loop-mediated isothermal amplification provides highly accurate, sensitive, and specific results with a wide linear range within 40 min. The diagnostic tool achieved 100% consistency with the "gold standard" polymerase chain reaction in detecting 54 real clinical samples. The integrated system, with its simplicity, is urgently needed for the diagnosis of SARS-CoV-2 during the COVID-19 pandemic.


Assuntos
Infecções por Coronavirus/diagnóstico por imagem , DNA Viral/análise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Pneumonia Viral/diagnóstico por imagem , Betacoronavirus , Técnicas de Laboratório Clínico , Coronavirus Humano 229E , Infecções por Coronavirus/diagnóstico , Coronavirus Humano NL63 , Diagnóstico Diferencial , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias
5.
Nat Commun ; 11(1): 5328, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087704

RESUMO

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 105 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.


Assuntos
Testes de Sensibilidade Microbiana/métodos , Bactérias/química , Bactérias/efeitos dos fármacos , Fenômenos Biofísicos , Farmacorresistência Bacteriana , Impedância Elétrica , Desenho de Equipamento , Humanos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Meropeném/administração & dosagem , Testes de Sensibilidade Microbiana/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
6.
PLoS One ; 15(10): e0225020, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031388

RESUMO

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (µPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed µPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed µPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the µPAD design for channel width (3 mm), agar volume (780 µL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed µPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the µPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and µPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, µPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Técnicas Analíticas Microfluídicas/instrumentação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Desenho de Equipamento , Dispositivos Lab-On-A-Chip/economia , Viabilidade Microbiana , Modelos Biológicos , Papel , Reprodutibilidade dos Testes , Ceras
7.
Lab Chip ; 20(22): 4255-4261, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33064114

RESUMO

COVID-19 is a widespread and highly contagious disease in the human population. COVID-19 is caused by SARS-CoV-2 infection. There is still a great demand for point-of-care tests for detection, epidemic prevention and epidemiological investigation, both now and after the epidemic. We present a lateral flow immunoassay kit based on a selenium nanoparticle-modified SARS-CoV-2 nucleoprotein, which detects anti-SARS-CoV-2 IgM and anti-SARS-CoV-2 IgG in human serum, and the results can be read by the naked eye in 10 minutes. We expressed and purified the SARS-CoV-2 nucleoprotein in HEK293 cells, with a purity of 98.14% and a concentration of 5 mg mL-1. Selenium nanoparticles were synthesized by l-ascorbic acid reduction of seleninic acid at room temperature. After conjugation with the nucleoprotein, a lateral flow kit was successfully prepared. The IgM and IgG detection limits of the lateral flow kit reached 20 ng mL-1 and 5 ng mL-1, respectively, in human serum. A clinical study sample comprising 90 COVID-19-diagnosed patients and 263 non-infected controls was used to demonstrate a sensitivity and specificity of 93.33% and 97.34%, respectively, based on RT-PCR and clinical results. No cross-reactions with rheumatoid factor and positive serum for anti-nuclear antibodies, influenza A, and influenza B were observed. Moreover, the lateral flow kit remained stable after storage for 30 days at 37 °C. Our results demonstrate that the selenium nanoparticle lateral flow kit can conveniently, rapidly, and sensitively detect anti-SARS-CoV-2 IgM and IgG in human serum and blood; it can also be suitable for the epidemiological investigation of COVID-19.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pneumonia Viral/sangue , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Desenho de Equipamento , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Dispositivos Lab-On-A-Chip , Limite de Detecção , Nanopartículas/química , Pandemias , Pneumonia Viral/imunologia , Testes Imediatos , Fitas Reagentes/análise , Selênio/química
8.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2227-2230, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018450

RESUMO

Organ-on-a-chip has the potential to replace preclinical trials which have been problematic for decades due to unaffordable cost and time. The performance of in vitro tumor-on-a-chip depends on how accurately the system represents analogous tumor-microenvironment (TME) and TME associated phenomena. In this study, we have focused on angiogenesis, one of the most significant features of TME for tumor growth and metastasis. Angiogenesis in TME is triggered through cascaded interactions among TME associated neighboring cells including immune cells, tumor cells, and fibroblast cells [1]. Therefore, temporally-controlled TME-on-a-chip is desired for an accurate representation of angiogenesis. However, conventional microfluidic devices cannot temporarily manipulate the condition of interacting cells and secreted signal molecules. Here, we proposed a hydrogel-based variable TME-on-a-chip with diffusion switch channels. The channels between hydrogel walls enable temporal diffusion control by controlling inflow. The diffusion control was observed in diffusion experiment with a fluorescent dye. Furthermore, experiment of HUVEC's migration toward diffused VEGF also confirmed that TME-on-a-chip is capable of reproducing an angiogenic switch triggering through temporal diffusion control. Due to a simple fabrication procedure, the design of the microfluidic device can be easily modified to represent more complex variable TME models.


Assuntos
Doenças Hematológicas , Neoplasias , Difusão , Humanos , Dispositivos Lab-On-A-Chip , Microambiente Tumoral
9.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2249-2252, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018455

RESUMO

We report on a unique microfluidic device that can enrich nanoparticles in a continuous flow by railing them along activated tracks (electrodes). This was achieved based on dielectrophoretic force and electrohydrodynamic drag (electrothermal rolls and AC electroosmosis) in both low and high conductive media. The results have implication for the isolation of high quality and pure nanoparticles such as exosomes from biofluids for applications in cancer diagnosis and prognosis.


Assuntos
Técnicas Analíticas Microfluídicas , Nanopartículas , Condutividade Elétrica , Eletro-Osmose , Dispositivos Lab-On-A-Chip
10.
J Vis Exp ; (162)2020 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-32865534

RESUMO

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (µmBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model µmBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.


Assuntos
Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Dispositivos Lab-On-A-Chip , Aprendizado de Máquina , Tomografia , Microambiente Tumoral , Animais , Humanos , Camundongos
11.
Anal Bioanal Chem ; 412(28): 7685-7699, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32870351

RESUMO

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.


Assuntos
Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/instrumentação , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana/instrumentação , Animais , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/imunologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Citocinas/imunologia , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/métodos , Humanos , Testes de Sensibilidade Microbiana/métodos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia , Vírion/efeitos dos fármacos , Vírion/imunologia , Viroses/tratamento farmacológico , Viroses/imunologia
12.
Nat Commun ; 11(1): 4489, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895384

RESUMO

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Carcinoma Hepatocelular/diagnóstico , Detecção Precoce de Câncer/métodos , Vesículas Extracelulares/genética , Neoplasias Hepáticas/diagnóstico , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Química Click/instrumentação , Química Click/métodos , Química Computacional , Simulação por Computador , Diagnóstico Diferencial , Dimetilpolisiloxanos/química , Progressão da Doença , Detecção Precoce de Câncer/instrumentação , Feminino , Células Hep G2 , Humanos , Dispositivos Lab-On-A-Chip , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Nanoestruturas/química , Nanofios/química , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
13.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
14.
Mycorrhiza ; 30(6): 789-796, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32918101

RESUMO

Arbuscular mycorrhizal fungi are beneficial components often included in biofertilizers. Studies of the biology and utilization of these fungi are key to their successful use in the biofertilizer industry. The acquisition of isolated spores is a required step in these studies; however, spore quality control and spore separation are bottlenecks. Filtered and centrifuged spores have to be hand-picked under a microscope. The conventional procedure is skill-demanding, labor-intensive, and time-consuming. Here, we developed a microfluidic device to aid manual separation of spores from a filtered and centrifuged suspension. The device is a single spore streamer equipped with a manual temporary flow diversion (MTFD) mechanism to select single spores. Users can press a switch to generate MTFD when the spore arrives at the selection site. The targeted spore flows in a stream to the collection chamber via temporary cross flow. Using the device, spore purity, the percentage of spore numbers against the total number of particles counted in the collecting chamber reached 96.62% (median, n = 10) which is greater than the spore purity obtained from the conventional method (88.89% (median, n = 10)).


Assuntos
Micorrizas , Dispositivos Lab-On-A-Chip , Esporos Fúngicos
15.
PLoS One ; 15(9): e0238581, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881948

RESUMO

Electrowetting-on-dielectric is a decent technique to manipulate discrete volumes of liquid in form of droplets. In the last decade, electrowetting-on-dielectric systems, also called digital microfluidic systems, became more frequently used for a variety of applications because of their high flexibility and reconfigurability. Thus, one design can be adapted to different assays by only reprogramming. However, this flexibility can only be useful if the entire system is portable and easy to use. This paper presents the development of a portable, stand-alone digital microfluidic system based on a Linux-based operating system running on a Raspberry Pi, which is unique. We present "PortaDrop" exhibiting the following key features: (1) an "all-in-one box" approach, (2) a user-friendly, self-explaining graphical user interface and easy handling, (3) the ability of integrated electrochemical measurements, (4) the ease to implement additional lab equipment via Universal Serial Bus and the General Purpose Interface Bus as well as (5) a standardized experiment documentation. We propose that PortaDrop can be used to carry out experiments in different applications, where small sample volumes in the nanoliter to picoliter range need to be handled an analyzed automatically. As a first application, we present a protocol, where a droplet is consequently exchanged by droplets of another medium using passive dispensing. The exchange is monitored by electrical impedance spectroscopy. It is the first time, the media exchange caused by passive dispensing is characterized by electrochemical impedance spectroscopy. Summarizing, PortaDrop allows easy combination of fluid handling by means of electrowetting and additional sensing.


Assuntos
Eletroumectação/instrumentação , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Tampões (Química) , Técnicas Eletroquímicas/instrumentação , Software
16.
Biosens Bioelectron ; 169: 112578, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32911317

RESUMO

The ongoing global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to active research in its associated diagnostics and medical treatments. While quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most reliable method to detect viral genes of SARS-CoV-2, serological tests for specific antiviral antibodies are also important as they identify false negative qRT-PCR responses, track how effectively the patient's immune system is fighting the infection, and are potentially helpful for plasma transfusion therapies. In this work, based on the principle of localized surface plasmon resonance (LSPR), we develop an opto-microfluidic sensing platform with gold nanospikes, fabricated by electrodeposition, to detect the presence and amount of antibodies specific to the SARS-CoV-2 spike protein in 1µL of human plasma diluted in 1mL of buffer solution, within ∼30min. The target antibody concentration can be correlated with the LSPR wavelength peak shift of gold nanospikes caused by the local refractive index change due to the antigen-antibody binding. This label-free microfluidic platform achieves a limit of detection of ∼0.08ng/mL (∼0.5pM), falling under the clinical relevant concentration range. We demonstrate that our opto-microfluidic platform offers a promising point-of-care testing tool to complement standard serological assays and make SARS-CoV-2 quantitative diagnostics easier, cheaper, and faster.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/sangue , Nanoestruturas/química , Pneumonia Viral/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Ressonância de Plasmônio de Superfície/instrumentação , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Desenho de Equipamento , Ouro/química , Humanos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Nanoestruturas/ultraestrutura , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia
17.
J Oleo Sci ; 69(10): 1331-1337, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-32908098

RESUMO

The development of actuators for power sources is essential for the efficient manipulation of fluids in microfluidics systems. In this work, a capacitor-type three-layer paper actuator was fabricated by sandwiching a polyelectrolyte layer between two films of poly(3,4-ethylenedioxythiophene) doped with poly(4-styrenesulfonate) (PEDOT/PSS). The paper actuator exhibited stable large electromechanical deformations in bilateral symmetry under alternating square-wave electric field. The actuation properties were examined in a function of voltage (±0.5, ±1, ±1.5, ±2, and ±2.5 V) and frequency (1, 0.5, 0.2, and 0.05 Hz). In addition, the PEDOT/PSS electrode films with different thicknesses were prepared, and the effects of actuator thickness on actuation properties were examined. As a result, it was found that the actuator displacement increased considerably with reducing actuator thickness. In addition, the actuator with a thickness of 48 µm demonstrated a maximum displacement of 5.8 mm at a voltage of 1.5 V and a frequency of 0.05 Hz. The proposed actuator can be potentially used in the development of power sources for micropumps and check valves of microfluidic devices.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Eletrodos , Dispositivos Lab-On-A-Chip , Microfluídica , Papel , Polímeros/química , Poliestirenos/química , Tiofenos/química , Compostos Bicíclicos Heterocíclicos com Pontes/classificação , Eletricidade , Polímeros/classificação
18.
Nat Commun ; 11(1): 4511, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908128

RESUMO

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.


Assuntos
Cristalografia/instrumentação , Elétrons , Dispositivos Lab-On-A-Chip , Lasers , Aldeído Liases/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Hidrodinâmica
19.
Nat Commun ; 11(1): 4149, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811832

RESUMO

Many bacteria can form wall-deficient variants, or L-forms, that divide by a simple mechanism that does not require the FtsZ-based cell division machinery. Here, we use microfluidic systems to probe the growth, chromosome cycle and division mechanism of Bacillus subtilis L-forms. We find that forcing cells into a narrow linear configuration greatly improves the efficiency of cell growth and chromosome segregation. This reinforces the view that L-form division is driven by an excess accumulation of surface area over volume. Cell geometry also plays a dominant role in controlling the relative positions and movement of segregating chromosomes. Furthermore, the presence of the nucleoid appears to influence division both via a cell volume effect and by nucleoid occlusion, even in the absence of FtsZ. Our results emphasise the importance of geometric effects for a range of crucial cell functions, and are of relevance for efforts to develop artificial or minimal cell systems.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Divisão Celular/fisiologia , Segregação de Cromossomos/fisiologia , Formas L/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip/microbiologia , Bacillus subtilis/citologia , Bacillus subtilis/fisiologia , Parede Celular/fisiologia , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/fisiologia , Formas L/citologia , Formas L/fisiologia , Modelos Biológicos
20.
Nat Protoc ; 15(9): 2920-2955, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788719

RESUMO

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.


Assuntos
Sistema Imunitário/citologia , Dispositivos Lab-On-A-Chip , Fenótipo , Análise de Célula Única/instrumentação , Animais , Linfócitos B/citologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência
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