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1.
Artigo em Inglês | MEDLINE | ID: mdl-33405153

RESUMO

Mount of embodied carbon emissions flow along industrial chains and form a complex network. In order to reveal the structure and evolution characteristics of embodied carbon emission flow network among China's industrial sectors, this study applies a complex network theory to construct six embodied carbon emission flow networks with 30 sectors on the basis of China's input-output tables from 2002 to 2015. Through the analysis of complex network technology indicators, the overall structural characteristics of the network, the key sectors, and the key flow paths are analyzed. Main results show that six embodied carbon emission flow networks all have the small-world characteristics; there is an industrial cluster phenomenon in the network. During the study period, construction, manufacturing, and service-related industry community are the absorption sites for embodied carbon emissions. Coal- and petroleum-related industry communities are the divergent sites for embodied carbon emissions; moreover, electric and heat power and fuel processing are the important "suppliers" of embodied carbon emissions; construction and other service are the important "consumers" of embodied carbon emissions. Non-metallic products are the important "transmitters" of embodied carbon emissions. Metal smelting and chemical industry are at the core of the network because of their high weighted degree and betweenness centrality. The central effect of key sectors continues to increase over time; furthermore, the distribution of embodied carbon emission flows in the six networks all have long-tail characteristics, and this characteristic became more prominent over time. There are key edge-weights in the networks. About 11 to 15% of the edges carry 80% of the embodied carbon emissions. Further based on edge-weight analysis, this study identifies the key paths of embodied carbon emission flow in the six networks, and most key paths pass through construction. Thus, such key sectors and key flow paths should receive more attention when making carbon emission reduction policies.

2.
ChemRxiv ; 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32511284

RESUMO

The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training.

3.
Nucleic Acids Res ; 48(4): e19, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31828328

RESUMO

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.


Assuntos
Proteínas Argonauta/genética , Ácidos Nucleicos Livres/genética , Neoplasias/genética , Ácidos Nucleicos Peptídicos/genética , Alelos , Humanos , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/patologia , Ácidos Nucleicos Peptídicos/isolamento & purificação , Thermus thermophilus/genética
5.
Anal Chem ; 90(7): 4823-4831, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29542319

RESUMO

Rapid and quantitative molecular diagnostics in the field, at home, and at remote clinics is essential for evidence-based disease management, control, and prevention. Conventional molecular diagnostics requires extensive sample preparation, relatively sophisticated instruments, and trained personnel, restricting its use to centralized laboratories. To overcome these limitations, we designed a simple, inexpensive, hand-held, smartphone-based mobile detection platform, dubbed "smart-connected cup" (SCC), for rapid, connected, and quantitative molecular diagnostics. Our platform combines bioluminescent assay in real-time and loop-mediated isothermal amplification (BART-LAMP) technology with smartphone-based detection, eliminating the need for an excitation source and optical filters that are essential in fluorescent-based detection. The incubation heating for the isothermal amplification is provided, electricity-free, with an exothermic chemical reaction, and incubation temperature is regulated with a phase change material. A custom Android App was developed for bioluminescent signal monitoring and analysis, target quantification, data sharing, and spatiotemporal mapping of disease. SCC's utility is demonstrated by quantitative detection of Zika virus (ZIKV) in urine and saliva and HIV in blood within 45 min. We demonstrate SCC's connectivity for disease spatiotemporal mapping with a custom-designed website. Such a smart- and connected-diagnostic system does not require any lab facilities and is suitable for use at home, in the field, in the clinic, and particularly in resource-limited settings in the context of Internet of Medical Things (IoMT).


Assuntos
HIV/isolamento & purificação , Imagem Óptica , Patologia Molecular , Smartphone , Zika virus/isolamento & purificação , HIV/genética , Humanos , Internet , Medições Luminescentes , Técnicas de Amplificação de Ácido Nucleico , Zika virus/genética
6.
Biosens Bioelectron ; 109: 156-163, 2018 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-29550739

RESUMO

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.


Assuntos
Técnicas Biossensoriais , Neisseria meningitidis/isolamento & purificação , Plasmodium falciparum/isolamento & purificação , Líquido Cefalorraquidiano/microbiologia , Colorimetria , Humanos , Limite de Detecção , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Infecções Meningocócicas/líquido cefalorraquidiano , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/patologia , Microfluídica , Neisseria meningitidis/patogenicidade , Ácidos Nucleicos/química , Ácidos Nucleicos/isolamento & purificação , Patologia Molecular , Plasmodium falciparum/patogenicidade , Sistemas Automatizados de Assistência Junto ao Leito , Impressão Tridimensional
7.
Biosensors (Basel) ; 8(1)2018 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-29495424

RESUMO

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges ('chips') that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Animais , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip/economia , Técnicas Analíticas Microfluídicas/economia , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito/economia , Smartphone/economia , Smartphone/instrumentação
8.
Anal Chem ; 90(2): 1209-1216, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29226671

RESUMO

To enable inexpensive molecular detection at the point-of-care and at home with minimal or no instrumentation, it is necessary to streamline unit operations and store reagents refrigeration-free. To address this need, a multifunctional enzymatic amplification reactor that combines solid-phase nucleic acid extraction, concentration, and purification; refrigeration-free storage of reagents with just-in-time release; and enzymatic amplification is designed, prototyped, and tested. A nucleic acid isolation membrane is placed at the reactor's inlet, and paraffin-encapsulated reagents are prestored within the reactor. When a sample mixed with chaotropic agents is filtered through the nucleic acid isolation membrane, the membrane binds nucleic acids from the sample. Importantly, the sample volume is decoupled from the reaction volume, enabling the use of relatively large sample volumes for high sensitivity. When the amplification reactor's temperature increases to its operating level, the paraffin encapsulating the reagents melts and moves out of the way. The reagents are hydrated, just-in-time, and the polymerase reaction proceeds. The amplification process can be monitored, in real-time. We demonstrate our reactors' ability to amplify both DNA and RNA targets using polymerase with both reverse-transcriptase and strand displacement activities to obtain sensitivities on-par with benchtop equipment and a shelf life exceeding 6 months.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Ácidos Nucleicos/análise , Reação em Cadeia da Polimerase/instrumentação , Extração em Fase Sólida/instrumentação , DNA Viral/análise , DNA Viral/genética , Desenho de Equipamento , Liofilização , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , Indicadores e Reagentes , Ácidos Nucleicos/genética , Infecções por Papillomavirus/virologia , Sistemas Automatizados de Assistência Junto ao Leito
10.
Clin Lab Int ; 41: 25-27, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28819345

RESUMO

The recent outbreak of Zika virus (ZIKV) infection has led to a serious threat to public health. Rapid and sensitive diagnostics for ZIKV infection are crucial because Zika infection is usually mild and often asymptomatic, but may have serious consequences to infants born to infected mothers. We report on a simple, sensitive, inexpensive, point-of-care (POC) diagnostic technology for rapid detection of ZIKV in saliva. We use a chemically heated cup for isothermal amplification without a need for electrical power. The detection results can be directly read out by eye.

11.
Methods Mol Biol ; 1572: 467-488, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28299706

RESUMO

Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.


Assuntos
Imunoensaio/instrumentação , Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos
12.
Clin Chem ; 63(3): 714-722, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28073898

RESUMO

BACKGROUND: The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS: To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP). RAMP's first-stage uses outer loop-mediated isothermal amplification (LAMP) primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent dye and a smartphone. RESULTS: In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed (<40 min); ease of use; and ability to cope with minimally processed samples. CONCLUSIONS: RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.


Assuntos
Técnicas Analíticas Microfluídicas , Reação em Cadeia da Polimerase Multiplex , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Temperatura , Humanos
13.
Lab Chip ; 17(3): 382-394, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28092381

RESUMO

The HIV pandemic affects 36.7 million people worldwide, predominantly in resource-poor settings. Nucleic acid-based molecular detection of HIV plays a significant role in antiretroviral treatment monitoring for HIV patients, as well as diagnosis of HIV infection in infants. Currently available molecular diagnostic methods are complex, time-consuming and relatively expensive, thus limiting their use in resource-poor settings. Recent advances in microfluidics technology have made possible low-cost integrated miniaturized devices for molecular detection and quantification of HIV at the point of care. We review recent technical advances in molecular testing of HIV using microfluidic technology, with a focus on assays based on isothermal nucleic acid amplification. Microfluidic components for sample preparation, isothermal amplification and result detection are discussed and compared. We also discuss the challenges and future directions for developing an integrated "sample-to-result" microfluidic platform for HIV molecular detection.


Assuntos
Infecções por HIV/diagnóstico , Infecções por HIV/virologia , Técnicas Analíticas Microfluídicas , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Carga Viral
14.
Anal Chem ; 88(14): 7289-94, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27306491

RESUMO

The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.


Assuntos
Sistemas Automatizados de Assistência Junto ao Leito , Zika virus/isolamento & purificação , Primers do DNA/metabolismo , Violeta Genciana/química , Humanos , Microfluídica , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/química , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Saliva/virologia , Zika virus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia
15.
Sens Actuators B Chem ; 229: 232-238, 2016 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-26900258

RESUMO

Nucleic acid amplification-based diagnostics offer rapid, sensitive, and specific means for detecting and monitoring the progression of infectious diseases. However, this method typically requires extensive sample preparation, expensive instruments, and trained personnel. All of which hinder its use in resource-limited settings, where many infectious diseases are endemic. Here, we report on a simple, inexpensive, minimally-instrumented, smart cup platform for rapid, quantitative molecular diagnostics of pathogens at the point of care. Our smart cup takes advantage of water-triggered, exothermic chemical reaction to supply heat for the nucleic acid-based, isothermal amplification. The amplification temperature is regulated with a phase-change material (PCM). The PCM maintains the amplification reactor at a constant temperature, typically, 60-65°C, when ambient temperatures range from 12 to 35°C. To eliminate the need for an optical detector and minimize cost, we use the smartphone's flashlight to excite the fluorescent dye and the phone camera to record real-time fluorescence emission during the amplification process. The smartphone can concurrently monitor multiple amplification reactors and analyze the recorded data. Our smart cup's utility was demonstrated by amplifying and quantifying herpes simplex virus type 2 (HSV-2) with LAMP assay in our custom-made microfluidic diagnostic chip. We have consistently detected as few as 100 copies of HSV-2 viral DNA per sample. Our system does not require any lab facilities and is suitable for use at home, in the field, and in the clinic, as well as in resource-poor settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent.

16.
Lab Chip ; 16(3): 553-60, 2016 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-26732765

RESUMO

To meet stringent limit-of-detection specifications for low abundance target molecules, a relatively large volume of plasma is needed for many blood-based clinical diagnostics. Conventional centrifugation methods for plasma separation are not suitable for on-site testing or bedside diagnostics. Here, we report a simple, yet high-efficiency, clamshell-style, superhydrophobic plasma separator that is capable of separating a relatively large volume of plasma from several hundred microliters of whole blood (finger-prick blood volume). The plasma separator consists of a superhydrophobic top cover with a separation membrane and a superhydrophobic bottom substrate. Unlike previously reported membrane-based plasma separators, the separation membrane in our device is positioned at the top of the sandwiched whole blood film to increase the membrane separation capacity and plasma yield. In addition, the device's superhydrophobic characteristics (i) facilitates the formation of well-defined, contracted, thin blood film with a high contact angle; (ii) minimizes biomolecular adhesion to surfaces; (iii) increases blood clotting time; and (iv) reduces blood cell hemolysis. The device demonstrated a "blood in-plasma out" capability, consistently extracting 65 ± 21.5 µL of plasma from 200 µL of whole blood in less than 10 min without electrical power. The device was used to separate plasma from Schistosoma mansoni genomic DNA-spiked whole blood with a recovery efficiency of >84.5 ± 25.8%. The S. mansoni genomic DNA in the separated plasma was successfully tested on our custom-made microfluidic chip by using loop mediated isothermal amplification (LAMP) method.


Assuntos
Remoção de Componentes Sanguíneos/instrumentação , Remoção de Componentes Sanguíneos/métodos , DNA de Helmintos , Membranas Artificiais , Plasma/parasitologia , Schistosoma mansoni , Animais , DNA de Helmintos/sangue , DNA de Helmintos/isolamento & purificação , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino
17.
PLoS One ; 10(2): e0117852, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25675344

RESUMO

Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion.


Assuntos
Infecções por HIV/virologia , HIV/classificação , HIV/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Biologia Computacional/métodos , Genes Virais , Genótipo , HIV-1/classificação , HIV-1/genética , Humanos , Reprodutibilidade dos Testes
18.
PLoS Negl Trop Dis ; 9(12): e0004318, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26720725

RESUMO

Parasitic helminths such as schistosomes, as well as filarial and soil-transmitted nematodes, are estimated to infect at least a billion people worldwide, with devastating impacts on human health and economic development. Diagnosis and monitoring of infection dynamics and efficacy of treatment depend almost entirely on methods that are inaccurate, labor-intensive, and unreliable. These shortcomings are amplified and take on added significance in mass drug administration programs, where measures of effectiveness depend on accurate monitoring of treatment success (or failure), changes in disease transmission rates, and emergence of possible drug resistance. Here, we adapt isothermal molecular assays such as loop-mediated isothermal amplification (LAMP) to a simple, hand-held, custom-made field-ready microfluidic device that allows sensitive and specific detection of schistosome cell-free nucleic acids in serum and plasma (separated with a point-of-care plasma separator) from Schistosoma mansoni-infected mice. Cell-free S. mansoni DNA was detected with our device without prior extraction from blood. Our chip exhibits high sensitivity (~2 x 10(-17) g/µL), with a positive signal for S. mansoni DNA detectable as early as one week post infection, several weeks before parasite egg production commences. These results indicate that incorporation of isothermal amplification strategies with our chips could represent a strategy for rapid, simple, low-cost diagnosis of both pre-patent and chronic schistosome infections as well as potential monitoring of treatment efficacy.


Assuntos
Microfluídica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/diagnóstico , Animais , DNA de Helmintos/química , DNA de Helmintos/genética , Feminino , Humanos , Camundongos , Schistosoma mansoni/genética , Esquistossomose mansoni/parasitologia , Fatores de Tempo
19.
Microarrays (Basel) ; 4(4): 474-89, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27600235

RESUMO

Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of "lab on a chip" NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase ("membrane") to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 10³ virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

20.
Acc Chem Res ; 47(10): 2941-50, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25111636

RESUMO

Circulating tumor cells (CTCs) are cancer cells that break away from either a primary tumor or a metastatic site and circulate in the peripheral blood as the cellular origin of metastasis. With their role as a "tumor liquid biopsy", CTCs provide convenient access to all disease sites, including that of the primary tumor and the site of fatal metastases. It is conceivable that detecting and analyzing CTCs will provide insightful information in assessing the disease status without the flaws and limitations encountered in performing conventional tumor biopsies. However, identifying CTCs in patient blood samples is technically challenging due to the extremely low abundance of CTCs among a large number of hematologic cells. To address this unmet need, there have been significant research endeavors, especially in the fields of chemistry, materials science, and bioengineering, devoted to developing CTC detection, isolation, and characterization technologies. Inspired by the nanoscale interactions observed in the tissue microenvironment, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with high efficiency. The working mechanism of NanoVelcro cell-affinity substrates mimics that of Velcro: when the two fabric strips of a Velcro fastener are pressed together, tangling between the hairy surfaces on two strips leads to strong binding. Through continuous evolution, three generations (gens) of NanoVelcro CTC chips have been established to achieve different clinical utilities. The first-gen NanoVelcro chip, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, was created for CTC enumeration. Side-by-side analytical validation studies using clinical blood samples suggested that the sensitivity of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch. In conjunction with the use of the laser microdissection (LMD) technique, second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation. The individually isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger sequencing and next-generation sequencing, NGS) to verify the CTC's role as tumor liquid biopsy. Created by grafting of thermoresponsive polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive NanoVelcro) have demonstrated the capture and release of CTCs at 37 and 4 °C, respectively. The temperature-dependent conformational changes of polymer brushes can effectively alter the accessibility of the capture agent on SiNS, allowing for rapid CTC purification with desired viability and molecular integrity. This Account summarizes the continuous evolution of NanoVelcro CTC assays from the emergence of the original idea all the way to their applications in cancer research. We envision that NanoVelcro CTC assays will lead the way for powerful and cost-efficient diagnostic platforms for researchers to better understand underlying disease mechanisms and for physicians to monitor real-time disease progression.


Assuntos
Separação Celular/métodos , Técnicas Analíticas Microfluídicas/métodos , Nanoestruturas/química , Células Neoplásicas Circulantes/patologia , Separação Celular/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentação
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