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1.
Lab Chip ; 20(19): 3560-3568, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32844858

RESUMO

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s-1 and maximum cooling rate of 8 °C s-1. Target DNA concentrations in the range from 101 ± 1.4 copies per µL to 260 000 ± 20 000 copies per µL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 µm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 µm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per µL to 25 889 ± 737 copies per µL.


Assuntos
Reação em Cadeia da Polimerase/instrumentação , Algoritmos , Técnicas Biossensoriais , DNA/química , Fontes de Energia Elétrica , Humanos , Membranas Artificiais , Miniaturização , Polimetil Metacrilato
2.
PLoS One ; 15(8): e0237418, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790779

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2×105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Betacoronavirus/química , Infecções por Coronavirus/virologia , DNA Viral/genética , Confiabilidade dos Dados , Humanos , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/economia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Biotechniques ; 69(4): 317-325, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32815744

RESUMO

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.


Assuntos
Reação em Cadeia da Polimerase/métodos , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/instrumentação
5.
Biosens Bioelectron ; 165: 112349, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32510340

RESUMO

Timely detection and diagnosis are urgently needed to guide epidemiological measures, infection control, antiviral treatment, and vaccine research. In this review, biomarkers/indicators for diagnosis of coronavirus disease 2019 (COVID-19) or detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the environment are summarized and discussed. It is concluded that the detection methods targeting antibodies are not suitable for screening of early and asymptomatic cases since most patients had an antibody response at about 10 days after onset of symptoms. However, antibody detection methods can be combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) to significantly improve the sensitivity and specificity of diagnosis, and boost vaccine research. Fast, sensitive and accurate detection methods targeting antigens need to be developed urgently. Various specimens for diagnosis or detection are compared and analyzed. Among them, deep throat saliva and induced sputum are desired for RT-qPCR test or other early detection technologies. Chest computerized tomography (CT) scan, RT-qPCR, lateral flow immunochromatographic strip (LFICS) for diagnosis of COVID-19 are summarized and compared. Specially, potential electrochemical (EC) biosensor, surface enhanced Raman scattering (SERS)-based biosensor, field-effect transistor (FET)-based biosensor, surface plasmon resonance (SPR)-based biosensor and artificial intelligence (AI) assisted diagnosis of COVID-19 are emphasized. Finally, some commercialized portable detection device, current challenges and future directions are discussed.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Animais , Anticorpos Antivirais/análise , Antígenos Virais/análise , Técnicas Biossensoriais/métodos , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Desenho de Equipamento , Humanos , Pandemias , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Fitas Reagentes/análise , Tomografia Computadorizada por Raios X/instrumentação , Tomografia Computadorizada por Raios X/métodos
6.
Nat Commun ; 11(1): 2590, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444602

RESUMO

A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies.


Assuntos
Dieta Cetogênica , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mucosa Intestinal/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , DNA/isolamento & purificação , Fezes/microbiologia , Feminino , Dispositivos Lab-On-A-Chip , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase/instrumentação , RNA Ribossômico 16S , Verrucomicrobia/genética , Fluxo de Trabalho
7.
PLoS Biol ; 18(4): e3000667, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298256

RESUMO

As biodiversity loss continues to accelerate, there is a critical need for education and biomonitoring across the globe. Portable technologies allow for in situ molecular biodiversity monitoring that has been historically out of reach for many researchers in habitat nations. In the realm of education, portable tools such as DNA sequencers facilitate in situ hands-on training in real-time sequencing and interpretation techniques. Here, we provide step-by-step protocols as a blueprint for a terrestrial conservation genetics field training program that uses low-cost, portable devices to conduct genomics-based training directly in biodiverse habitat countries.


Assuntos
Conservação dos Recursos Naturais/métodos , Genética/educação , Genética/instrumentação , Biodiversidade , Código de Barras de DNA Taxonômico/instrumentação , Código de Barras de DNA Taxonômico/métodos , Ecossistema , Feminino , Genética/organização & administração , Humanos , Masculino , Peru , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos
10.
J Biosci Bioeng ; 130(1): 76-81, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32147250

RESUMO

The quantification of pathogens is important for assessing water safety and preventing disease outbreaks. Culture-independent approaches, such as quantitative PCR (qPCR) and digital PCR (dPCR), are useful techniques for quantifying pathogens in water samples. However, since pathogens are usually present at low concentrations in water, it is necessary to concentrate microbial cells before extracting their DNA. Many existing microbial concentration methods are inefficient or take a long time to perform. In this study, we applied a coagulation and foam separation method to concentrate environmental water samples of between 1000 and 5000 mL to 100 µL of DNA (i.e., a 1-5 × 104-fold concentration). The concentration process took <1 h. The DNA samples were then used to quantify various target pathogens using dPCR. One gene, the Shiga toxin gene (stx2) of Shiga toxin-producing Escherichia coli, was detected at 32 copies/100 mL in a river water sample. The coagulation and foam concentration method followed by dPCR reported herein is a fast, sensitive, and reliable method to quantify pathogen genes in environmental water samples.


Assuntos
Água Doce/microbiologia , Reação em Cadeia da Polimerase/métodos , Água Doce/química , Reação em Cadeia da Polimerase/instrumentação , Sensibilidade e Especificidade , Toxina Shiga/genética , Toxina Shiga/metabolismo , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/metabolismo
11.
Ginebra; World Health Organization; Mar. 19, 2020. 7 p.
Monografia em Inglês | BIGG - guias GRADE | ID: biblio-1053420

RESUMO

The purpose of this document is to provide interim guidance to laboratories and stakeholders involved in laboratory testing of patients who meet the definition of suspected case of pneumonia associated with a novel coronavirus identified in Wuhan, China.


Assuntos
Humanos , Pneumonia Viral/genética , Reação em Cadeia da Polimerase/instrumentação , Infecções por Coronavirus/genética , Betacoronavirus/genética , Ensaio de Imunoadsorção Enzimática/instrumentação , Vírus da SARS/genética , Serviços Laboratoriais de Saúde Pública
12.
ACS Appl Mater Interfaces ; 12(11): 12533-12540, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32101396

RESUMO

Emerging molecular diagnosis requires ultrafast polymerase chain reaction (PCR) on chip for rapid precise detection of infectious diseases in the point-of-care test. Here, we report nanoplasmonic on-chip PCR for rapid precision molecular diagnostics. The nanoplasmonic pillar arrays (NPA) comprise gold nanoislands on the top and sidewall of large-scale glass nanopillar arrays. The nanoplasmonic pillars enhance light absorption of a white light-emitting diode (LED) over the whole visible range due to strong electromagnetic hotspots between the nanoislands. As a result, they effectively induce photothermal heating for ultrafast PCR thermal cycling. The temperature profile of NPA exhibits 30 cycles between 98 and 60 °C for a total of 3 min and 30 s during the cyclic excitation of white LED light. The experimental results also demonstrate the rapid DNA amplification of both 0.1 ng µL-1 of λ-DNA in 20 thermal cycles and 0.1 ng µL-1 of complementary DNA of Middle East respiratory syndrome coronavirus in 30 thermal cycles using a conventional PCR volume of 15 µL. This nanoplasmonic PCR technique provides a new opportunity for rapid precision molecular diagnostics.


Assuntos
Doenças Transmissíveis/diagnóstico , DNA/análise , Nanofibras/química , Reação em Cadeia da Polimerase/métodos , Doenças Transmissíveis/genética , DNA/metabolismo , Ouro/química , Humanos , Dispositivos Lab-On-A-Chip , Luz , Análise de Sequência com Séries de Oligonucleotídeos , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/instrumentação , Temperatura
13.
Talanta ; 206: 120200, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514845

RESUMO

Highly-sensitive and contamination-free droplet digital PCR (ddPCR) is an enabling technology and widely needed for accurate quantification of nucleic acid in clinical applications. In this paper, a novel droplet reader was developed by combining a "quasi" confocal laser-induced fluorescence (LIF) cytometry with a delicate microfluidic chip design. The droplets with a size of 90 µm was illuminated at an out-of-focus position by two aligned laser beams to generate maximum fluorescent signal. Additionally, the lateral offset position of the microfluidic chip should be precisely tuned so that the bandwidth of the FAM and VIC channels were configured at the matching sizes. Then, PMT gain voltages and pneumatic pressures were optimized for better droplet detection efficiencies. An aerosol adsorption experiment was performed to demonstrate that there was no aerosol contamination, and detected copy numbers of both mutants and wild types scaled linearly with the expected input copy numbers (r2>0.998) with a LoB of 0.0 copies and LoD of 3.0 copies. The results demonstrated that this droplet reader with the delicate chip is a convenient, highly-sensitive and contamination-free to detect fluorescence signals inside droplets after ddPCR, which is highly promising for broad applications of ddPCR in clinical diagnosis.


Assuntos
DNA/análise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Reação em Cadeia da Polimerase/métodos , Desenho de Equipamento , Receptores ErbB/genética , Antígeno HLA-B27/genética , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Mutação , Reação em Cadeia da Polimerase/instrumentação
14.
Biosci Biotechnol Biochem ; 84(1): 43-52, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31495297

RESUMO

To date, studies on the application of loop-mediated isothermal amplification (LAMP) in the detection of genetically modified organisms (GMOs) are stably increasing and demonstrates LAMP is a potential and promising method for on spot identification of GMOs. However, little information is known for detection of GM potato events by LAMP. In this report, we developed an optimized and visual LAMP assay with high specificity and sensitivity to rapidly amplify genomic DNA of potato EH92-527-1 within 45 min. The limit of detection of LAMP in our study is 10-fold higher than the conventional PCR. Furthermore, LAMP products can be directly observed via naked eyes by addition of SYBR Green I without gel electrophoresis analysis and PCR-based equipment. Therefore, the LAMP assay developed in this paper provides an efficient, convenient and cost-effective tool for the detection of GM potato EH92-527-1.


Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/genética , Sequência de Bases/genética , Percepção de Cores , Primers do DNA/genética , Enzimas de Restrição do DNA/genética , Eletroforese em Gel de Ágar , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Amplificação de Genes , Limite de Detecção , Compostos Orgânicos/química , Reação em Cadeia da Polimerase/economia , Sensibilidade e Especificidade , Temperatura , Tempo
15.
Talanta ; 207: 120303, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594577

RESUMO

Polymerase chain reaction (PCR) is commonly used for the analysis of nucleic acids in a variety of applications including clinical. There is, however, a need for a low cost portable PCR device that allows rapid identification of pathogenic bacteria. We report a shunting PCR microfluidic device comprising: polycarbonate microfluidic PCR chip; shunting thermal cycler and fluorescence detector. The microfluidic PCR chip - fabricated using micro-milling and thermal fusion bonding for sealing of the cover - was shunted between three double side temperature zones for thermal cycling. Rapid amplification was observed with heating and cooling rates of 1.8 °C/s and 2 °C/s respectively. Lock-in photodetector for fluorescence detection of the microfluidic PCR chip achieved at 95% confidence an LOD of 75pM FITC and 0.7 ng µl-1 of dsDNA using a QuantiFluor assay kit. The device was validated using universal primers - based on chromosomal DNA extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli) - for amplification of fragments of 250, 552 and 1500 bp. PCR amplification was demonstrated, with annealing temperatures ranging between 54 °C and 68 °C, and confirmed using gel electrophoresis. The developed shunting PCR microfluidic device will allow for low cost and portable nucleic acid amplification for the detection of infectious diseases.


Assuntos
Escherichia coli K12/genética , Escherichia coli K12/isolamento & purificação , Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/instrumentação , Espectrometria de Fluorescência , Fatores de Tempo
16.
Clin Microbiol Infect ; 26(4): 411-420, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31574340

RESUMO

BACKGROUND: Mobile microbiology is an evolving concept that has the potential to reduce morbidity and mortality associated with infectious diseases on a global level. Molecular methods used in the context of mobile microbiology ensure rapid and accurate aetiological diagnostics and allow timely initiation of clinical care. The great majority of published data regarding molecular diagnostics in mobile laboratories have focused on emerging viral infections and using laboratory-developed assays. Use of clinically validated and commercially available molecular diagnostic instruments in routine diagnostics of infectious diseases in mobile laboratories has received only limited attention in the field. OBJECTIVES: This review summarizes the suitability of a range of portable diagnostic molecular instruments for application in mobile laboratories by taking into account the instruments' analytical concepts, technical features and environmental requirements, as well as results of major validation studies. SOURCES: Data on technical features of selected portable instruments were mainly extracted from manufacturers' websites. Information on validation studies of various molecular assays developed for the selected instruments was extracted from peer-reviewed publications searched for through PubMed. CONTENT: Eight portable diagnostic molecular instruments (Alere q, GeneXpert Edge, GeneXpert Omni, Genedrive, PanNAT, Revogene, cobas Liat and ID Now) that are commercially available or in the launching stage are presented and evaluated in the context of the mobile microbiology concept, with particular emphasis on technical features and environmental requirements. Both the cobas Liat and the Alere i assays have been extensively validated in a variety of studies carried out in both adult and paediatric patients from various settings (ranging from primary care to emergency care departments in tertiary centres). Most studies showed comparable performance of cobas Liat and Alere i molecular assays with the standard-of-care in vitro diagnostics molecular assays routinely performed in dedicated/centralized molecular diagnostics laboratories. In addition, acceptable performance of Alere q and Genedrive instruments has been shown in implementations studies for early infant diagnosis of children born to human immunodeficiency virus-positive mothers and detection of hepatitis C virus RNA, respectively. Additional validation studies on existing (GeneXpert Edge, PanNAT, Revogene) and emerging (GeneXpert Omni) technologies are warranted. IMPLICATIONS: Several portable molecular diagnostic platforms reviewed are suitable for mobile microbiology applications. Further development in this field should be directed toward providing a broader range of assays per instrument, multiplexing, reducing the frequency of invalid results, and price cutting.


Assuntos
Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Humanos , Laboratórios , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/instrumentação , Sensibilidade e Especificidade
17.
Biomed Microdevices ; 22(1): 5, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31823015

RESUMO

The reasons for restricting continuous flow polymerase chain reaction (CF-PCR) microfluidic chip from lab to application are that it is not portable and requires costly external precision pumps for sample injection. Herein, we employed water as the substitute for PCR solution, and investigated the effect of the cross-section, width-to-depth ratio, and the length ratio for three temperature zones of the micro channel on the thermal and flow distribution of fluid in micro tube by finite element analysis. Results show that the central velocity is uniform and stable velocity occupies the most if the cross-section is rectangular. The deviation between predefined temperature and theoretical temperature is slight and the fluid flux is the most if width-to-depth ratio is 1:1. It is suitable for the short DNA replication if the high temperature zone Wh is larger than the low temperature zone Wl, and vice versa. Then a portable CF-PCR microfluidic chip was fabricated and an automatic sample injection system was developed. As an application, we have successfully amplified the DNA of Treponema denticola in the chip within 8 min. Such a study may offer new insight into the design of CF-PCR microfluidic chip and promote it from lab-scale research to full-scale application.


Assuntos
Replicação do DNA , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/instrumentação , DNA Bacteriano/genética , Temperatura , Treponema denticola/genética
18.
Methods Enzymol ; 629: 1-15, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31727235

RESUMO

Circulating tumor DNA (ctDNA) analyses are minimally invasive and accessible for risk stratification or treatment response monitoring of cancer patients. Compared to tumor biopsy analysis, they are not only less invasive, but also provide a more representative picture, allowing capturing both tumor heterogeneity and multiple tumor sites. Development of new technologies such as droplet-based digital PCR (ddPCR) has greatly improved the sensitivity, specificity and precision of the detection of rare ctDNA sequences in the pool of total circulating free DNA. In this chapter, we discuss the application of ddPCR in the analysis of ctDNA, and present the protocols for obtaining variant allele frequency or copy number variation analysis.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , DNA Tumoral Circulante/isolamento & purificação , Neoplasias/diagnóstico , Reação em Cadeia da Polimerase/métodos , Alelos , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Variações do Número de Cópias de DNA , Frequência do Gene , Humanos , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Neoplasias/sangue , Neoplasias/genética , Neoplasias/mortalidade , Reação em Cadeia da Polimerase/instrumentação , Prognóstico , Medição de Risco
19.
Lab Chip ; 19(24): 4104-4116, 2019 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-31720646

RESUMO

Digital polymerase chain reaction (dPCR) is a powerful tool for genetic analysis, providing superior sensitivity and accuracy. In many applications that demand minuscule reaction volumes, such as single cell analysis, efficient and reproducible sample handling and digitization is pivotal for accurate absolute quantification of targets, but remains a significant technical challenge. In this paper, we described a robust and flexible microfluidic alternating-pull-push active digitization (µAPPAD) strategy that confers close to 100% sample digitization efficiency for microwell-based dPCR. Our strategy employs pneumatic valve control to periodically manipulate air pressure inside the chip to greatly facilitate the vacuum-driven partition of solution into microwells, enabling efficient digitization of a small-volume solution with significantly reduced volume variability. The µAPPAD method was evaluated on both tandem-channel and parallel-channel chips, which achieved a digitization efficiency of 99.5 ± 0.3% and 94.6 ± 0.9% within 10.5 min and 2 min, respectively. To assess the analytical performance of the µAPPAD chip, we calibrated it for absolution dPCR quantitation of λDNA across a range of concentrations. The results obtained with our chip matched well with the theoretical curve computed from Poisson statistics. Compared to the existing methods for highly efficient sample digitization, not only does our technology greatly reduce the constraints on microwell geometries and channel design, but also benefits from the intrinsic amenability of the pneumatic valve technique with device integration and automation. Thus we envision that the µAPPAD technology will provide a scalable and widely adaptable platform to promote the development of advanced lab-on-a-chip systems integrating microscale sample processing with dPCR for a broad scope of applications, such as single cell analysis of tumor heterogeneity and genetic profiling of circulating exosomes directly in clinical samples.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Reação em Cadeia da Polimerase , Bacteriófago lambda/genética , DNA Viral/química , DNA Viral/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
20.
Bioanalysis ; 11(23): 2175-2188, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31724446

RESUMO

Aim: Nowadays, IFN-α is considered a promising therapeutic target for systemic lupus erythematosus. An immuno-PCR (iPCR) was developed to quantify low amounts of IFN-α in human plasma followed by a deep analysis of the methodologic robustness throughout quality by design approach. Results: An accurate, sensitive, selective and versatile iPCR was validated. The critical iPCR procedural steps were identified, applying a Plackett-Burman design. Also, this assay demonstrated an outstanding LOD of 0.3 pg/ml. A significant aspect relies on its high versatility to detect and quantify other cytokines in human plasma as the appropriate biotinylated antibody is employed. Conclusion: This reliable iPCR assay can be clinically used as an alternative method for quantitating and detecting low IFN-α2b concentrations in human plasma samples.


Assuntos
Imunoensaio/métodos , Imunoensaio/normas , Interferon-alfa/sangue , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Adulto , Idoso , Desenho de Equipamento , Feminino , Humanos , Imunoensaio/instrumentação , Interferon-alfa/genética , Interferon-alfa/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/instrumentação , Voluntários , Adulto Jovem
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