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1.
Nat Commun ; 12(1): 950, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574261

RESUMO

The advent of highly sensitive photodetectors and the development of photostabilization strategies made detecting the fluorescence of single molecules a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g., in point-of-care diagnostic settings, where costly equipment would be prohibitive. Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in NACHOS emit up to 461-fold (average of 89 ± 7-fold) brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia on the road.

2.
Plant J ; 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33506558

RESUMO

Volatile organic compounds (VOCs) released by plants serve as info- and defense chemicals in mutualistic and antagonistic interactions and mitigate effects of abiotic stress. Passive and dynamic sampling techniques combined with gas chromatography-mass spectrometry (GC-MS) analysis have become routine tools to measure emissions of VOCs and determine their various functions. More recently, knowledge of the roles of plant VOCs in the aboveground environment has led to the exploration of similar functions in the soil and rhizosphere. Moreover, VOC patterns have been recognized as sensitive and time-dependent markers of biotic and abiotic stress. This focused review addresses these developments by presenting recent progress in VOC sampling and analysis. We show advances in the use of small, inexpensive sampling devices and describe methods to monitor plant VOC emissions in the belowground environment. We further address latest trends in real-time measurements of volatilomes in plant phenotyping and most recent developments of small portable devices and VOC sensors for non-invasive VOC fingerprinting of plant disease. These technologies allow for innovative approaches to study plant VOC biology and application in agriculture.

3.
Sci Adv ; 7(2)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310733

RESUMO

Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 105 copies/µl) and exhibited a limit of detection (0.38 copies/µl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.


Assuntos
/diagnóstico , Testes Imediatos , Saliva/virologia , Smartphone , Animais , Sistemas CRISPR-Cas , Chlorocebus aethiops , Simulação por Computador , Feminino , Humanos , Limite de Detecção , Macaca mulatta , Masculino , Técnicas de Diagnóstico Molecular/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Células Vero , Carga Viral
4.
Front Chem ; 8: 837, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33024746

RESUMO

It is essential to develop effective analytical techniques for accurate and continuous monitoring of various biomanufacturing processes, such as the production of monoclonal antibodies and vaccines, through sensitive and quantitative detection of characteristic aqueous or gaseous metabolites and other analytes in the cell culture media. A comprehensive summary toward the use of mainstream techniques for bioprocess monitoring is critically reviewed here, which illustrates the instrumental and procedural advances and limitations of several major analytical tools in biomanufacturing applications. Despite those drawbacks present in modern detection systems such as mass spectrometry, gas chromatography or chemical/biological sensors, a considerable number of useful solutions and inspirations such as electronic or optoelectronic noses can be offered to greatly overcome the restrictions and facilitate the development of advanced analytical techniques that can target a more diverse range of key nutritious components, products or potential contaminants in different biomanufacturing processes.

5.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

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


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
6.
Curr Protoc Plant Biol ; 5(1): e20104, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32074406

RESUMO

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.


Assuntos
Lycopersicon esculentum , Phytophthora infestans/genética , DNA de Plantas , Folhas de Planta , Reação em Cadeia da Polimerase em Tempo Real
7.
Plant Dis ; 104(3): 708-716, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31967506

RESUMO

Phytophthora infestans is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for P. infestans and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of P. infestans. The LAMP assay was specific for P. infestans on potato and tomato and did not amplify other potato- or tomato-infecting Phytophthora species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of P. infestans and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.


Assuntos
Lycopersicon esculentum , Phytophthora infestans , Solanum tuberosum , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase
8.
Nat Plants ; 5(8): 856-866, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358961

RESUMO

Plant pathogen detection conventionally relies on molecular technology that is complicated, time-consuming and constrained to centralized laboratories. We developed a cost-effective smartphone-based volatile organic compound (VOC) fingerprinting platform that allows non-invasive diagnosis of late blight caused by Phytophthora infestans by monitoring characteristic leaf volatile emissions in the field. This handheld device integrates a disposable colourimetric sensor array consisting of plasmonic nanocolorants and chemo-responsive organic dyes to detect key plant volatiles at the ppm level within 1 min of reaction. We demonstrate the multiplexed detection and classification of ten individual plant volatiles with this field-portable VOC-sensing platform, which allows for early detection of tomato late blight 2 d after inoculation, and differentiation from other pathogens of tomato that lead to similar symptoms on tomato foliage. Furthermore, we demonstrate a detection accuracy of ≥95% in diagnosis of P. infestans in both laboratory-inoculated and field-collected tomato leaves in blind pilot tests. Finally, the sensor platform has been beta-tested for detection of P. infestans in symptomless tomato plants in the greenhouse setting.


Assuntos
Aplicativos Móveis , Doenças das Plantas , Smartphone , Solanum tuberosum/microbiologia , Phytophthora infestans/fisiologia , Doenças das Plantas/microbiologia , Folhas de Planta/química , Compostos Orgânicos Voláteis/análise
9.
ACS Nano ; 13(6): 6540-6549, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31179687

RESUMO

In-field molecular diagnosis of plant diseases via nucleic acid amplification is currently limited by cumbersome protocols for extracting and isolating pathogenic DNA from plant tissues. To address this challenge, a rapid plant DNA extraction method was developed using a disposable polymeric microneedle (MN) patch. By applying MN patches on plant leaves, amplification-assay-ready DNA can be extracted within a minute from different plant species. MN-extracted DNA was used for direct polymerase chain reaction amplification of plant plastid DNA without purification. Furthermore, using this patch device, extraction of plant pathogen DNA ( Phytophthora infestans) from both laboratory-inoculated and field-infected leaf samples was performed for detection of late blight disease in tomato. MN extraction achieved 100% detection rate of late blight infections for samples after 3 days of inoculation when compared to the conventional gold standard cetyltrimethylammonium bromide (CTAB)-based DNA extraction method and 100% detection rate for all blind field samples tested. This simple, cell-lysis-free, and purification-free DNA extraction method could be a transformative approach to facilitate rapid sample preparation for molecular diagnosis of various plant diseases directly in the field.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA Fúngico/química , Phytophthora/genética , Doenças das Plantas/microbiologia , Código de Barras de DNA Taxonômico/instrumentação , DNA Fúngico/genética , Lycopersicon esculentum/microbiologia , Metagenômica/instrumentação , Metagenômica/métodos , Agulhas , Phytophthora/patogenicidade , Folhas de Planta/química , Folhas de Planta/microbiologia
10.
Anal Chem ; 91(16): 10448-10457, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31192585

RESUMO

Developing easy-to-use and miniaturized detectors is essential for in-field monitoring of environmentally hazardous substances, such as the cyanotoxins. We demonstrated a differential fluorescent sensor array made of aptamers and single-stranded DNA (ssDNA) dyes for multiplexed detection and discrimination of four common cyanotoxins with an ordinary smartphone within 5 min of reaction. The assay reagents were preloaded and dried in a microfluidic chip with a long shelf life over 60 days. Upon the addition of analyte solutions, competitive binding of cyanotoxin to the specific aptamer-dye conjugate occurred. A zone-specific and concentration-dependent reduction in the green fluorescence was observed as a result of the aptamer conformation change. The aptasensors are fully optimized by quantification of their dissociation constants, tuning the stoichiometric ratios of reaction mixtures, and implementation of an internal intensity correction step. The fluorescent sensor array allowed for accurate identification and measurement of four important cyanotoxins, including anatoxin-a (ATX), cylindrospermopsin (CYN), nodularin (NOD), and microcystin-LR (MC-LR), in parallel, with the limit of detection (LOD) down to a few nanomolar (<3 nM), which is close to the World Health Organization's guideline for the maximum concentration allowed in drinking water. The smartphone-based sensor platform also showed remarkable chemical specificity against potential interfering agents in water. The performance of the system was tested and validated with real lake water samples that were contaminated with trace levels of individual cyanotoxins as well as binary, ternary, and quaternary mixtures. Finally, a smartphone app interface has been developed for rapid on-site data processing and result display.


Assuntos
Aptâmeros de Nucleotídeos/química , Toxinas Bacterianas/análise , Técnicas Biossensoriais/métodos , Microcistinas/análise , Peptídeos Cíclicos/análise , Tropanos/análise , Uracila/análogos & derivados , Poluentes Químicos da Água/análise , Alcaloides , Técnicas Biossensoriais/instrumentação , DNA de Cadeia Simples/química , Fluorescência , Água Doce/química , Humanos , Dispositivos Lab-On-A-Chip , Lagos/química , Limite de Detecção , Toxinas Marinhas , Análise em Microsséries , Smartphone , Uracila/análise
11.
ACS Omega ; 4(1): 637-642, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30775643

RESUMO

Smartphone-based fluorescence microscopy has been rapidly developing over the last few years, enabling point-of-need detection of cells, bacteria, viruses, and biomarkers. These mobile microscopy devices are cost-effective, field-portable, and easy to use, and benefit from economies of scale. Recent developments in smartphone camera technology have improved their performance, getting closer to that of lab microscopes. Here, we report the use of DNA origami nanobeads with predefined numbers of fluorophores to quantify the sensitivity of a smartphone-based fluorescence microscope in terms of the minimum number of detectable molecules per diffraction-limited spot. With the brightness of a single dye molecule as a reference, we compare the performance of color and monochrome sensors embedded in state-of-the-art smartphones. Our results show that the monochrome sensor of a smartphone can achieve better sensitivity, with a detection limit of ∼10 fluorophores per spot. The use of DNA origami nanobeads to quantify the minimum number of detectable molecules of a sensor is broadly applicable to evaluate the sensitivity of various optical instruments.

12.
Anal Chem ; 90(15): 8881-8888, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30004217

RESUMO

We present an approach to estimate the concentration of a biomolecule in a solution by sampling several nanoliter-scale volumes and determining if the volumes contain any biomolecules. In this method, varying volume fractions (nanoliter-scale) of a sample of nucleic acids are introduced to an array of uniform volume reaction wells (100 µL), which are then fluorescently imaged to determine if signal is above a threshold after nucleic acid amplification, all without complex instrumentation. The nanoliter volumes are generated and introduced using the simple positioning of a permanent magnet, and imaging is performed with a cellphone-based fluorescence detection scheme, both methods suitable for limited-resource settings. We use the length of time a magnetic field is applied to generate a calibrated number of nanoliter ferrodrops of sample mixed with ferrofluid at a step emulsification microfluidic junction. Each dose of ferrodrops is then transferred into larger microliter scale reaction wells on chip through a simple shift of the external magnet. Nucleic acid amplification is achieved using loop-mediated isothermal amplification (LAMP). By repeating each nanoliter dosage a number of times to calculate the probability of a positive signal at each dosage, we can use a binomial probability distribution to estimate the sample nucleic acid concentration. Using this approach we demonstrate detection of lambda DNA molecules down to 25 copies per microliter. The ability to dose separate nanoliter-scale volumes of a low-volume sample across wells in this platform is suited for multiplexed assays. This platform has the potential to be applied to a range of diseases by mixing a sample with magnetic nanoparticles.


Assuntos
DNA/análise , Nanopartículas de Magnetita/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Emulsões/química , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Tamanho da Amostra
13.
Nano Res ; 11(10): 5439-5473, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-32218913

RESUMO

Plasmonics-based biosensing assays have been extensively employed for biomedical applications. Significant advancements in use of plasmonic assays for the construction of point-of-care (POC) diagnostic methods have been made to provide effective and urgent health care of patients, especially in resourcelimited settings. This rapidly progressive research area, centered on the unique surface plasmon resonance (SPR) properties of metallic nanostructures with exceptional absorption and scattering abilities, has greatly facilitated the development of cost-effective, sensitive, and rapid strategies for disease diagnostics and improving patient healthcare in both developed and developing worlds. This review highlights the recent advances and applications of plasmonic technologies for highly sensitive protein and nucleic acid biomarker detection. In particular, we focus on the implementation and penetration of various plasmonic technologies in conventional molecular diagnostic assays, and discuss how such modification has resulted in simpler, faster, and more sensitive alternatives that are suited for point-of-use. Finally, integration of plasmonic molecular assays with various portable POC platforms for mobile health applications are highlighted.

14.
Anal Chem ; 90(1): 690-695, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29136461

RESUMO

Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.


Assuntos
Bioensaio/métodos , Telefone Celular , MicroRNAs/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Orthomyxoviridae/genética , Sistemas Automatizados de Assistência Junto ao Leito
15.
Proc Natl Acad Sci U S A ; 114(34): E7054-E7062, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28784765

RESUMO

The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the "D4 assay") that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are "on-chip," and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.


Assuntos
Análise Química do Sangue/métodos , Imunoensaio/métodos , Polímeros/química , Biomarcadores/sangue , Análise Química do Sangue/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Leptina/sangue , Sistemas Automatizados de Assistência Junto ao Leito , Impressão
16.
Sci Rep ; 7(1): 2124, 2017 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-28522808

RESUMO

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.


Assuntos
Aumento da Imagem/métodos , Pontos Quânticos/química , Smartphone , Vidro/química , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Testes Imediatos , Prata/química
17.
ACS Nano ; 11(3): 2934-2943, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28234452

RESUMO

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.


Assuntos
Telefone Celular , DNA/análise , Substâncias Intercalantes/química , Naftalenossulfonatos/química , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Bacteriófago lambda/química , Telefone Celular/economia , Fluorescência , Estrutura Molecular , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito/economia , Espectrometria de Fluorescência/economia , Espectrometria de Fluorescência/instrumentação
18.
Nat Commun ; 8: 13913, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28094784

RESUMO

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.


Assuntos
Telefone Celular/estatística & dados numéricos , DNA/genética , Neoplasias/diagnóstico , Patologia Molecular/métodos , Análise de Sequência de DNA/métodos , Humanos , Microscopia , Mutação , Neoplasias/genética , Patologia Molecular/instrumentação , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA/instrumentação
19.
ACS Nano ; 9(8): 7857-66, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26159546

RESUMO

Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.


Assuntos
Anticorpos Antivirais/sangue , Computadores de Mão/economia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Sistemas Automatizados de Assistência Junto ao Leito/economia , Telefone Celular/instrumentação , Colorimetria/economia , Colorimetria/instrumentação , Colorimetria/métodos , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/instrumentação , Herpes Genital/sangue , Herpes Genital/diagnóstico , Herpes Genital/imunologia , Herpes Simples/sangue , Herpes Simples/diagnóstico , Herpes Simples/imunologia , Humanos , Aprendizado de Máquina , Sarampo/sangue , Sarampo/diagnóstico , Sarampo/imunologia , Aplicativos Móveis , Caxumba/sangue , Caxumba/diagnóstico , Caxumba/imunologia , Fibras Ópticas , Testes Imediatos , Sensibilidade e Especificidade
20.
Anal Chem ; 87(13): 6434-45, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26068279

RESUMO

Providing means for researchers and citizen scientists in the developing world to perform advanced measurements with nanoscale precision can help to accelerate the rate of discovery and invention as well as improve higher education and the training of the next generation of scientists and engineers worldwide. Here, we review some of the recent progress toward making optical nanoscale measurement tools more cost-effective, field-portable, and accessible to a significantly larger group of researchers and educators. We divide our review into two main sections: label-based nanoscale imaging and sensing tools, which primarily involve fluorescent approaches, and label-free nanoscale measurement tools, which include light scattering sensors, interferometric methods, photonic crystal sensors, and plasmonic sensors. For each of these areas, we have primarily focused on approaches that have either demonstrated operation outside of a traditional laboratory setting, including for example integration with mobile phones, or exhibited the potential for such operation in the near future.


Assuntos
Nanotecnologia , Óptica e Fotônica , Microscopia de Fluorescência
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