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1.
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
2.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1283-1292, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748586

RESUMO

Point-of-care testing (POCT) is a test method performed on the sampling site or patient bedside. Accurate results can be achieved rapidly by the application of portable analytical instruments and compatible reagents. It has been widely used in the field of in vitro diagnosis (IVD). Paper-based microfluidics technology has great potential in developing POCT due to its advantages in low cost, simple operation, rapid detection, portable equipment, and unrestricted application conditions. In recent years, the development of paper-based microfluidic technology and its integration with various new technologies and methods have promoted the substantial development of POCT technology and methods. The classification and characteristic of the paper are summarized in this review. Paper-based microfluidic sample pretreatment methods, the flow control in the process of reaction and the signal detecting and analyzing methods for the testing results are introduced. The research progress of various kinds of microfluidic paper-based analytical devices (µPADs) toward POCT in recent years is reviewed. Finally, remaining problems and the future prospects in POCT application of paper-based microfluidics are discussed.


Assuntos
Testes Diagnósticos de Rotina , Técnicas Analíticas Microfluídicas , Papel , Testes Imediatos , Testes Diagnósticos de Rotina/métodos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação
3.
Nat Commun ; 11(1): 4244, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843650

RESUMO

Acoustical tweezers open major prospects in microbiology for cells and microorganisms contactless manipulation, organization and mechanical properties testing since they are biocompatible, label-free and have the potential to exert forces several orders of magnitude larger than their optical counterpart at equivalent power. Yet, these perspectives have so far been hindered by the absence of spatial selectivity of existing acoustical tweezers - i.e., the ability to select and move objects individually - and/or their limited resolution restricting their use to large particle manipulation only and/or finally the limited forces that they could apply. Here, we report precise selective manipulation and positioning of individual human cells in a standard microscopy environment with trapping forces up to ~200 pN without altering their viability. These results are obtained with miniaturized acoustical tweezers combining holography with active materials to synthesize specific wavefields called focused acoustical vortices designed to produce stiff localized traps with reduced acoustic power.


Assuntos
Acústica , Técnicas Citológicas/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Linhagem Celular Tumoral , Sobrevivência Celular , Desenho de Equipamento , Holografia , Humanos , Microscopia
5.
Proc Natl Acad Sci U S A ; 117(26): 14798-14804, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554496

RESUMO

Proper circulation of white blood cells (WBCs) in the pulmonary vascular bed is crucial for an effective immune response. In this branched vascular network, WBCs have to strongly deform to pass through the narrowest capillaries and bifurcations. Although it is known that this process depends on the cell mechanical properties, it is still poorly understood due to the lack of a comprehensive model of cell mechanics and of physiologically relevant experiments. Here, using an in-house microfluidic device mimicking the pulmonary capillary bed, we show that the dynamics of THP1 monocytes evolves along successive capillary-like channels, from a nonstationary slow motion with hops to a fast and smooth efficient one. We used actin cytoskeleton drugs to modify the traffic dynamics. This led us to propose a simple mechanical model that shows that a very finely tuned cortical tension combined with a high cell viscosity governs the fast transit through the network while preserving cell integrity. We finally highlight that the cortical tension controls the steady-state cell velocity via the viscous friction between the cell and the channel walls.


Assuntos
Capilares/fisiologia , Pulmão , Modelos Biológicos , Monócitos , Fenômenos Biomecânicos , Humanos , Pulmão/irrigação sanguínea , Pulmão/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Monócitos/citologia , Monócitos/fisiologia , Células THP-1
6.
Nat Commun ; 11(1): 2190, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366850

RESUMO

Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master mold using benchtop devices, design modifications are time consuming and require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to microfluidic devices made by soft lithography. Virtual channels are liquid-bound fluidic systems that can be created in glass cuvettes and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We demonstrate that the liquid-liquid interface imposes a hydrodynamic stress on confined samples, and the resulting strain can be used to calculate rheological parameters from simple linear models. In proof-of-principle experiments, we perform high-throughput rheology inside a flow cytometer cuvette and show the Young's modulus of isolated cells exceeds the one of the corresponding tissue by one order of magnitude.


Assuntos
Dimetilpolisiloxanos/química , Módulo de Elasticidade/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Polietilenoglicóis/química , Algoritmos , Desenho de Equipamento , Citometria de Fluxo , Células HEK293 , Células HL-60 , Humanos , Hidrodinâmica , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Modelos Teóricos , Reologia , Esferoides Celulares
7.
Nature ; 582(7811): 277-282, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-164175

RESUMO

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas Analíticas Microfluídicas/métodos , Viroses/diagnóstico , Viroses/virologia , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Farmacorresistência Viral/genética , Genoma Viral/genética , HIV/classificação , HIV/genética , HIV/isolamento & purificação , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , RNA Guia/genética , Sensibilidade e Especificidade
8.
Nat Biotechnol ; 38(6): 715-721, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32231335

RESUMO

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.


Assuntos
Anticorpos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , DNA/análise , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoglobulina G/genética , Camundongos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
9.
Transfusion ; 60(5): 1032-1041, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32237236

RESUMO

BACKGROUND: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices. STUDY DESIGN AND METHODS: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C. RESULTS: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average. CONCLUSIONS: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method.


Assuntos
Deformação Eritrocítica/fisiologia , Eritrócitos/citologia , Eritrócitos/fisiologia , Dispositivos Lab-On-A-Chip/normas , Técnicas Analíticas Microfluídicas , Bancos de Sangue/métodos , Bancos de Sangue/normas , Velocidade do Fluxo Sanguíneo/fisiologia , Preservação de Sangue/efeitos adversos , Preservação de Sangue/métodos , Preservação de Sangue/normas , Criopreservação , Contagem de Eritrócitos/instrumentação , Contagem de Eritrócitos/métodos , Contagem de Eritrócitos/normas , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Hemólise , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/normas , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Fatores de Tempo
10.
Nature ; 582(7811): 277-282, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349121

RESUMO

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas Analíticas Microfluídicas/métodos , Viroses/diagnóstico , Viroses/virologia , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Farmacorresistência Viral/genética , Genoma Viral/genética , HIV/classificação , HIV/genética , HIV/isolamento & purificação , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , RNA Guia/genética , Sensibilidade e Especificidade
11.
Food Chem ; 316: 126396, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32066068

RESUMO

This study employed the use of a microfluidic paper-based analytical device (µPAD) to determine the concentration of nitrite in pork and enhanced the limit of detection by analyzing the coffee-ring effect. The µPAD was fabricated by designing and embedding wax channels onto the cellulose-based filter paper through printing and subjecting the paper to heat treatment to allow wax penetration. Nitrite concentration was determined by monitoring the colorimetric reaction that occurred between nitrite and the added Griess reagent. The limit of detection of this device for nitrite in pork was determined to be 19.2 mg kg-1 by analyzing the inner-chamber reaction, while it could be as low as 1.1 mg kg-1 if the coffee-ring region was analyzed. The overall analysis could be completed within 15 min. This µPAD-based method has potential applications to routinely screen the nitrite concentration of meat products and ensure food safety and consumer health.


Assuntos
Nitritos/análise , Carne Vermelha/análise , Animais , Colorimetria , Etilenodiaminas/análise , Filtração , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Papel , Sulfanilamidas/análise , Suínos
12.
J Food Sci ; 85(3): 736-743, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32017096

RESUMO

By utilizing the coffee-ring effect and microfluidic paper-based analytical devices (µPADs), this study improved the sensitivity of the determination of norfloxacin in four different food matrices. Micro-PADs in this study were fabricated by designing and embedding wax channels onto cellulose-based filter paper through printing and subjecting the paper to heat to allow the wax to penetrate the paper. Determination of norfloxacin concentration in food samples was achieved by monitoring the colorimetric reaction that occurred between norfloxacin and the added iron (III) nitrate nonahydrate in 5 mM ammonia in each reaction chamber. A transition metal hydroxide was formed through this reaction that resulted in the formation of a solid precipitate to enable the antibiotic to bind to the iron molecule via coordination chemistry. This metal ion-antibiotic complex generated a visible color change. Following the colorimetric reaction, images were taken and subsequently analyzed via ImageJ to determine the relative pixel intensity that was used to infer norfloxacin concentration. The analytical sensitivity of this device was determined to be as low as 50 ppm when analyzing the inner-ring reaction, and as low as 5 ppm when analyzing the outer coffee ring thereby allowing for an alternative cheaper, faster, and more user-friendly method to detect norfloxacin than the conventional methods. PRACTICAL APPLICATION: This novel paper-based microfluidic device can achieve the detection of antibiotic residues in agrifoods in a faster, cheaper, and more user-friendly manner.


Assuntos
Antibacterianos/análise , Resíduos de Drogas/análise , Técnicas Analíticas Microfluídicas/métodos , Norfloxacino/análise , Colorimetria , Contaminação de Alimentos/análise , Técnicas Analíticas Microfluídicas/instrumentação , Smartphone
13.
Opt Lett ; 45(5): 1164-1167, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32108796

RESUMO

The generation and manipulation of small aqueous droplets is an important issue for nano- and biotechnology, particularly, when using microfluidic devices. The production of very small droplets has been frequently carried out by applying intense local electric fields to the fluid, which requires power supplies and metallic electrodes. This procedure complicates the device and reduces its versatility. In this work, we present a novel and flexible, to the best of our knowledge, electrodeless optoelectronic method for the production of tiny droplets of biologically friendly aqueous fluids. Our method takes advantage of the photoinduced electric fields generated by the bulk photovoltaic effect in iron-doped lithium niobate crystals. Two substrate configurations, presenting the polar ferroelectric axis either parallel or perpendicular to the active surface, have been successfully tested. In both crystal geometries, small droplets on the femtoliter scale have been obtained, although with a different spatial distributions correlated with the symmetry of the photovoltaic fields. The overall results demonstrate the effectiveness of the optoelectronic method to produce femtoliter droplets, both with pure water and with aqueous solutions containing biological material.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Fenômenos Ópticos , Água , Eletrodos , Hidrodinâmica
14.
Cardiovasc Eng Technol ; 11(3): 295-307, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32002815

RESUMO

OBJECTIVE: Arterial stiffness and endothelial function are two established surrogate markers of subclinical atherosclerosis and are quantified by three arterial parameters: elasticity, viscosity and radius of the arterial wall. Yet, the current methods for their assessment are unsuitable for routine use. Post-exercise response of the cardiovascular (CV) system serves as a more sensitive detection of subclinical arterial abnormalities that are not apparent at-rest. The objective of this study is to propose a novel method that can measure post-exercise response of arterial parameters and is also suitable for routine use. APPROACH: A microfluidic tactile sensor with a location-insensitive configuration was used for arterial pulse signal measurements on six asymptomatic male subjects, offering measurement reliability, ease use by a layperson, and affordability. By treating the arterial pulse signal as a vibration signal of the arterial wall, vibration-model-based analysis of only one measured pulse signal with no calibration was conducted for simultaneous estimation of three arterial parameters. Exercise-intensity-normalized percent changes in arterial parameters were utilized to remove the influence of variation in exercise intensity on post-exercise response, and then their measured values were compared for difference in post-exercise response between the subjects. MAIN RESULTS: One subject who was obese, on subject who had insomnia, and the oldest subject in the study demonstrated differences in post-exercise response at the radial artery (RA), as compared with the three subjects free of those three factors. Despite a lack of statistical significance, the observed difference at the RA between subjects was supported by (i) their consistency with the related findings in the literature, and (ii) their consistency with the measured values at the carotid artery (CA) and superficial temporal artery (STA) and the anatomical difference between the three arteries. SIGNIFICANCE: The proposed method has the potential of offering an affordable and convenient diagnosis tool for routine arterial health assessment.


Assuntos
Artérias/fisiopatologia , Doenças Cardiovasculares/diagnóstico , Teste de Esforço , Exercício Físico , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Fluxo Pulsátil , Transdutores de Pressão , Rigidez Vascular , Adulto , Doenças Cardiovasculares/fisiopatologia , Elasticidade , Nível de Saúde , Humanos , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Modelos Cardiovasculares , Valor Preditivo dos Testes , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Processamento de Sinais Assistido por Computador , Fatores de Tempo , Vibração , Viscosidade , Adulto Jovem
15.
Talanta ; 209: 120571, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892085

RESUMO

Single-cell detection methods are already of great significance for many bioanalysis applications, and droplet microfluidics technology is understood as particularly a powerful tool. Salmonella infection is a major hygienic problem worldwide that causes major public health and economic damage, and preventing Salmonella outbreaks requires detection food-borne detection methods that are rapid, portable, and reliable, ideally without the need for complicated pre-treatment protocol steps. Herein, we present a single-cell-level analysis method based on droplet microfluidics that can sensitively and rapidly detect Salmonella directly from food samples. Specifically, this method achieves single-cell encapsulation of Salmonella in droplets of a growth medium with resazurin that enables fluorescence-based detection of pathogens within 5 h. The ratio of positive droplets in a Poisson Distribution is used for quantitation, and the detection limit of our system determined to be 50 CFU/mL, a value lower than conventional analytical methods for assessing Salmonella contamination. Our experimental results demonstrate the precise and highly sensitive performance of a single-cell-precision, droplet-based microfluidic chip analytical method for monitoring pathogenic bacteria in food. Beyond our example case of Salmonella detection from milk samples, our work lays the foundation for a new generation of microfluidics-based analytical technologies for both public health and food safety applications which can undoubtedly benefit from increases in the sensitivity and rapidity of food-borne pathogen detection.


Assuntos
Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/microbiologia , Técnicas Analíticas Microfluídicas/instrumentação , Infecções por Salmonella/microbiologia , Salmonella/isolamento & purificação , Animais , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Análise de Alimentos/instrumentação , Microbiologia de Alimentos , Humanos , Limite de Detecção , Leite/microbiologia , Análise de Célula Única/instrumentação
16.
PLoS One ; 15(1): e0227294, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940389

RESUMO

BACKGROUND: Cell-free DNA detection is becoming a surrogate assay for tumor genotyping. Biological fluids often content a very low amount of cell-free tumor DNA and assays able to detect very low allele frequency mutant with a few quantities of DNA are required. We evaluated the ability of the fully-automated molecular diagnostics platform Idylla for the detection of KRAS, NRAS and BRAF hotspot mutations in plasma from patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: First, we evaluated the limit of detection of the system using two set of laboratory made samples that mimic mCRC patient plasma, then plasma samples from patients with mCRC were assessed using Idylla system and BEAMing digital PCR technology. RESULTS: Limits of detection of 0.1%, 0.4% and 0.01% for KRAS, NRAS and BRAF respectively have been reached. With our laboratory made samples, sensitivity up to 0.008% has been reached. Among 15 patients' samples tested for KRAS mutation, 2 discrepant results were found between Idylla and BEAMing dPCR. A 100% concordance between the two assays has been found for the detection of NRAS and BRAF mutations in plasma samples. CONCLUSIONS: The Idylla system does not reach as high sensitivity as assays like ddPCR but has an equivalent sensitivity to modified NGS technics with a lower cost and a lower time to results. These data allowed to consider the Idylla system in a routine laboratory workflow for KRAS, NRAS and BRAF mutations detection in plasma.


Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/isolamento & purificação , Neoplasias Colorretais/diagnóstico , Análise Mutacional de DNA/instrumentação , Técnicas de Genotipagem/instrumentação , Linhagem Celular Tumoral , DNA Tumoral Circulante/genética , Ensaios Clínicos como Assunto , Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , GTP Fosfo-Hidrolases/genética , Frequência do Gene , Técnicas de Genotipagem/métodos , Humanos , Limite de Detecção , Proteínas de Membrana/genética , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Sensibilidade e Especificidade
17.
Proc Natl Acad Sci U S A ; 117(6): 3301-3306, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31974311

RESUMO

Genome-scale technologies have enabled mapping of the complex molecular networks that govern cellular behavior. An emerging theme in the analyses of these networks is that cells use many layers of regulatory feedback to constantly assess and precisely react to their environment. The importance of complex feedback in controlling the real-time response to external stimuli has led to a need for the next generation of cell-based technologies that enable both the collection and analysis of high-throughput temporal data. Toward this end, we have developed a microfluidic platform capable of monitoring temporal gene expression from over 2,000 promoters. By coupling the "Dynomics" platform with deep neural network (DNN) and associated explainable artificial intelligence (XAI) algorithms, we show how machine learning can be harnessed to assess patterns in transcriptional data on a genome scale and identify which genes contribute to these patterns. Furthermore, we demonstrate the utility of the Dynomics platform as a field-deployable real-time biosensor through prediction of the presence of heavy metals in urban water and mine spill samples, based on the the dynamic transcription profiles of 1,807 unique Escherichia coli promoters.


Assuntos
Técnicas Biossensoriais/instrumentação , Monitoramento Ambiental , Perfilação da Expressão Gênica , Aprendizado de Máquina , Regiões Promotoras Genéticas/genética , Bases de Dados Genéticas , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Desenho de Equipamento , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Genes Bacterianos/genética , Genômica/instrumentação , Genômica/métodos , Ensaios de Triagem em Larga Escala , Metais Pesados/toxicidade , Técnicas Analíticas Microfluídicas/instrumentação , Transcriptoma/genética
18.
Chem Asian J ; 15(1): 79-84, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31778028

RESUMO

ß-Amino acid N-carboxy anhydrides (ß-NCAs) are rarely used in the synthesis of ß-peptides, which is due mainly to the poor availability of these potentially useful substrates. Herein, we describe the heretofore challenging synthesis of ß-NCAs via a single-step, rapid, and mild formation using pH flash switching and flash dilution, which are aspects of micro-flow technology. We synthesized 15 ß-NCAs in good to excellent yields that included acid-labile ß-NCAs that cannot be readily synthesized using the conventional Leuchs approach. Scaled-up synthesis using this process can be readily achieved via continuous operation.


Assuntos
Aminoácidos/síntese química , Anidridos/síntese química , Técnicas Analíticas Microfluídicas , Aminoácidos/química , Anidridos/química , Técnicas Analíticas Microfluídicas/instrumentação , Estrutura Molecular
19.
J Chromatogr A ; 1610: 460537, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31537305

RESUMO

A device with four parallel channels was designed and manufactured by 3D printing in titanium. A simple experimental setup allowed splitting of the mobile phase in four parallel streams, such that a single sample could be analysed four times simultaneously. The four capillary channels were filled with a monolithic stationary phase, prepared using a zwitterionic functional monomer in combination with various dimethacrylate cross-linkers. The resulting stationary phases were applicable in both reversed-phase and hydrophilic-interaction retention mechanisms. The mobile-phase composition was optimized by means of a window diagram so as to obtain the highest possible resolution of dopamine precursors and metabolites on all columns. Miniaturized electrochemical detectors with carbon fibres as working electrodes and silver micro-wires as reference electrodes were integrated in the device at the end of each column. Experimental separations were successfully compared with those predicted by a three-parameter retention model. Finally, dopamine was determined in human urine to further confirm applicability of the developed device.


Assuntos
Cromatografia Líquida/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Dopamina/urina , Desenho de Equipamento , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microeletrodos , Titânio
20.
J Chromatogr A ; 1610: 460539, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31543341

RESUMO

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


Assuntos
Bactérias , Técnicas Analíticas Microfluídicas , Proteínas Recombinantes , Bactérias/química , Bactérias/citologia , Bactérias/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo
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