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1.
Sci Rep ; 10(1): 443, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949259

RESUMO

Herein, we describe the synthesis of highly water-dispersible and biocompatible 3D adsorbents via a rapid two-step strategy employing a mesoporous magnetic nanomulberry-shaped Fe3O4 (MNM) on diatomaceous earth (DE) and cucurbituril (CB; MNM-DE-CB). Coating of CB on the surface of MNM-DE via hydrogen bonds not only enhanced the dispersibility of CB, but also improved the stability of MNM-DE. The ability of the adsorbent to remove dyes from water was investigated as a function of metal ions, solution pH, temperature, and concentration to determine optimum reaction conditions. Unlike MNM-DE, MNM-DE-CB exhibited highly efficient, rapid dye removal and recyclability in aqueous solution, and low cytotoxicity toward cancer cells in drug delivery tests. MNM-DE-CB is a promising green adsorbent with potential for diverse applications including water remediation, interface catalysis, bio-sample preparation, and drug delivery.

2.
Micromachines (Basel) ; 10(7)2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31252533

RESUMO

Blood plasma from patients is a powerful resource for diagnosing infectious disease due to it having many genetic materials as well as being relatively easy to obtain. Thus, various biosensors have been investigated for diagnosing diseases in blood plasma. However, there are no optimized and validated sensors for clinical use due to the low sensitivity, complexity, and difficulties of removing the inhibitors from plasma samples. In this study, we described a silicon microring resonator sensor used to detect Coxiella burnetii from the blood plasma of Q-fever patients in a label-free, real-time manner. Q-fever is an infectious disease caused by Coxiella burnetii via direct contact or inhalation aerosols. We validated this biosensor in the blood plasma of 35 clinical samples (including 16 Q fever samples infected with Coxiella burnetii and 19 samples infected with other febrile diseases. The biosensors are capable of rapid (10 min), highly sensitive (87.5%), and specific (89.5%) detection in plasma samples compared to the use of the conventional method.

3.
Lab Chip ; 19(13): 2256-2264, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31173022

RESUMO

The isolation of bio-molecules such as proteins and nucleic acids is a necessary step for both diagnostic and analytical processes in the broad fields of research and clinical applications. Although a myriad of isolation technologies have been developed, a method for simultaneous protein and nucleic acid isolation has not been explored for clinical use. Obtaining samples from certain cancers or rare diseases can be difficult. In addition, the heterogeneity of cancer tissues typically leads to inconsistent results when analyzing biomolecules. We here describe a homobifunctional imidoester (HI)-based microfluidic system for simultaneous DNA and protein isolation from either a solid or liquid single biopsy sample. An efficient and cost effective microfluidic design with less air bubbles was identified among several candidates using simulation and experimental results from the streamlining of isolation processing. HI groups were used as capture reagents for the simultaneous isolation of bio-molecules from a single specimen in a single microfluidic system. The clinical utility of this system for the simultaneous isolation of DNA and proteins within 40 min was validated in cancer cell lines and 23 tissue biopsies from colorectal cancer patients. The quantity of isolated protein and DNA was high using this system compared to the spin-column method. This HI-based microfluidic system shows good rapidity, affordability, and portability in the isolation of bio-molecules from limited samples for subsequent clinical analysis.

4.
Molecules ; 24(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987247

RESUMO

Infectious diseases, especially pathogenic infections, are a growing threat to public health worldwide. Since pathogenic bacteria usually exist in complex matrices at very low concentrations, the development of technology for rapid, convenient, and biocompatible sample enrichment is essential for sensitive diagnostics. In this study, a cucurbit[6]uril (CB) supermolecular decorated amine-functionalized diatom (DA) composite was fabricated to support efficient sample enrichment and in situ nucleic acid preparation from enriched pathogens and cells. CB was introduced to enhance the rate and effectiveness of pathogen absorption using the CB-DA composite. This novel CB-DA composite achieved a capture efficiency of approximately 90% at an Escherichia coli concentration of 106 CFU/mL within 3 min. Real-time PCR analyses of DNA samples recovered using the CB-DA enrichment system showed a four-fold increase in the early amplification signal strength, and this effective method for capturing nucleic acid might be useful for preparing samples for diagnostic systems.


Assuntos
Materiais Biocompatíveis , Nanocompostos , Manejo de Espécimes , Materiais Biocompatíveis/química , Linhagem Celular Tumoral , Diatomáceas/química , Humanos , Compostos Macrocíclicos/química , Técnicas Microbiológicas , Nanocompostos/química , Nanocompostos/ultraestrutura , Ácidos Nucleicos/química , Ácidos Nucleicos/isolamento & purificação , Manejo de Espécimes/métodos
5.
J Clin Microbiol ; 57(5)2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30814264

RESUMO

We evaluated the diagnostic performance of a simple and label-free pathogen enrichment method using homobifunctional imidoesters (HI) and a microfluidic system, called the SLIM assay, followed by real-time PCR from cerebrospinal fluid (CSF) in human immunodeficiency virus (HIV)-uninfected patients with suspected tuberculous meningitis (TBM). Patients with suspected TBM were prospectively enrolled in a tertiary hospital in an intermediate tuberculosis (TB)-burden country during a 30-month period. TBM was classified according to the uniform case definition. Definite and probable TBM were regarded as the reference standards for TBM, and possible TBM and not-TBM as the reference standards for not-TBM. Of 72 HIV-uninfected patients with suspected TBM, 10 were diagnosed with definite (n = 2) and probable (n = 8) TBM by the uniform case definition. The sensitivity of the SLIM assay was 100% (95% confidence interval [CI], 69 to 100%) compared with definite or probable TBM, and it was superior to those of mycobacterial culture (20% [95% CI, 3 to 56%]) and the Xpert MTB/RIF assay (0% [95% CI, 0 to 31%]). Of 21 possible TBM and 41 not-TBM patients by the uniform case definition, 5 possible TBM and no not-TBM patients gave positive results in the SLIM assay. The specificity of the SLIM assay was 92% (95% CI, 82 to 97%; 5/62). We demonstrated that the SLIM assay had a very high sensitivity and specificity with small samples of 10 cases of definite or probable TBM. Further studies are needed to confirm this finding and to compare the SLIM assay with mycobacterial culture, Xpert MTB/RIF, and Xpert MTB/RIF Ultra assays in a larger prospective cohort of patients with suspected TBM, including both HIV-infected and HIV-uninfected cases.

6.
Adv Sci (Weinh) ; 5(10): 1800614, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30356899

RESUMO

Cell-free nucleic acids (cfNAs) are emerging diagnostic biomarkers for monitoring the treatment and recurrence of cancers. In particular, the biological role and clinical usefulness of cfNAs obtained from the plasma of patients with various cancers are popular and still intensely explored, yet most studies are limited by technical problems during cfNA isolation. A dimethyl dithiobispropionimidate (DTBP)-based microchannel platform that enables spontaneous cfNA capture in 15 min with minimal cellular background and no requirements for use of bulky instruments is reported first. This platform identified KRAS and BRAF hot-spot mutations following cfDNA isolation from the blood plasma and tissues obtained from 30 colorectal cancer patients. The correlation of mutations between the primary tissues and plasma from the patients was high using this platform with whole genome sequencing compared to the spin-column method. This platform can also be combined with various detection approaches (biooptical sensor, Sanger sequencing, and polymerase chain reaction (PCR)) for rapid, simple, low-cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies has applications in cancer treatment and precision medicine.

7.
Biosens Bioelectron ; 119: 79-85, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30103157

RESUMO

Rapid identification of emerging infectious pathogens is crucial for preventing public health threats. Various pathogen detection techniques have been introduced; however, most techniques are time-consuming and lack multiple-target detection specificity. Although multiple-target detection techniques can distinguish emerging infectious pathogens from related pathogens, direct amplification methods have not been widely examined. Here, we present a novel arch-shaped multiple-target sensor capable of rapid pathogen identification using direct amplification in clinical samples. In this study, an arch-shaped amplification containing primer sequences was designed to rapidly amplify multiple targets. Further, the sensing platform allowed for sensitive and specific detection of human coronavirus, Middle East respiratory syndrome, Zika virus, and Ebola virus down to several copies. This platform also simultaneously distinguished between Middle East respiratory syndrome and human coronavirus in clinical specimens within 20 min. This arch-shaped multiple-target sensing assay can provide rapid, sensitive, and accurate diagnoses of emerging infectious diseases in clinical applications.


Assuntos
Técnicas Biossensoriais/métodos , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/virologia , Doenças Transmissíveis Emergentes/virologia , Humanos , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Vírus/genética
8.
Biosens Bioelectron ; 111: 66-73, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29653418

RESUMO

Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.


Assuntos
Técnicas Biossensoriais/instrumentação , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , Imidoésteres/química , Dispositivos Lab-On-A-Chip , Doenças Transmissíveis/sangue , Doenças Transmissíveis/diagnóstico , Desenho de Equipamento , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/diagnóstico , Herpes Zoster/sangue , Herpes Zoster/diagnóstico , Herpesvirus Humano 3/isolamento & purificação , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Saliva/microbiologia , Saliva/virologia
9.
Anal Chem ; 90(8): 5108-5115, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29561136

RESUMO

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.


Assuntos
Nanoestruturas/química , Ácidos Nucleicos/análise , Reação em Cadeia da Polimerase/métodos , Bactérias/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Detergentes/química , Dimetil Suberimidato/química , Endopeptidase K/metabolismo , Células HCT116 , Humanos , Ácidos Nucleicos/isolamento & purificação , Tamanho da Partícula , Testes Imediatos , Temperatura Ambiente , Óxido de Zinco/química
10.
Anal Biochem ; 544: 87-92, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29289485

RESUMO

Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.


Assuntos
Doenças Transmissíveis/diagnóstico , DNA Bacteriano/genética , Técnicas Analíticas Microfluídicas , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real , Doenças Transmissíveis/genética
11.
J Biophotonics ; 11(4): e201700167, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29024445

RESUMO

Recent zoonotic outbreaks, such as Zika, Middle East respiratory syndrome and Ebola, have highlighted the need for rapid and accurate diagnostic assays that can be used to aid pathogen control. Q fever is a zoonotic disease caused by the transmission of Coxiella burnetii that can cause serious illness in humans through aerosols and is considered a potential bioterrorism agent. However, the existing assays are not suitable for the detection of this pathogen due to its low levels in real samples. We here describe a rapid bio-optical sensor for the accurate detection of Q fever and validate its clinical utility. By combining a bio-optical sensor, that transduces the presence of the target DNA based on binding-induced changes in the refractive index on the waveguide surface in a label-free and real-time manner, with isothermal DNA amplification, this new diagnostic tool offers a rapid (<20 min), 1-step DNA amplification/detection method. We confirmed the clinical sensitivity (>90%) of the bio-optical sensor by detecting C. burnetii in 11 formalin-fixed, paraffin-embedded liver biopsy samples from acute Q fever hepatitis patients and in 16 blood plasma samples from patients in which Q fever is the cause of fever of unknown origin.


Assuntos
Técnicas Biossensoriais/instrumentação , Dispositivos Ópticos , Febre Q/diagnóstico , Coxiella burnetii/genética , Coxiella burnetii/isolamento & purificação , Coxiella burnetii/fisiologia , Febre de Causa Desconhecida/sangue , Hepatite/diagnóstico , Humanos , Reação em Cadeia da Polimerase , Febre Q/microbiologia , Fatores de Tempo
12.
Biosens Bioelectron ; 99: 443-449, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-28818785

RESUMO

Versatile, simple and efficient sample preparation is desirable for point-of-care testing of emerging diseases such as zoonoses, but current sample preparation assays are insensitive, labour-intensive and time-consuming and require multiple instruments. We developed a single-tube sample preparation approach involving direct pathogen enrichment and extraction from human specimens using diatomaceous earth (DE). Amine-modified DE was used to directly enrich a zoonotic pathogen, Brucella, in a large sample volume. Next, a complex of amine-modified DE and dimethyl suberimidate was used for nucleic acid extraction from the enriched pathogen. Using our single-tube approach, the pathogen can be enriched and extracted within 60min at a level of 1 colony formation unit (CFU) from a 1ml sample volume in the same tube. The performance of this approach is 10-100 times better than that of a commercial kit (102 to 103CFU/ml) but does not require a large centrifuge. Finally, we combined the single-tube approach with a bio-optical sensor for rapid and accurate zoonotic pathogen detection in human urine samples. Using the combination system, Brucella in human urine can be efficiently enriched (~ 8-fold) and the detection limit is enhanced by up to 100 times (1CFU/ml bacteria in urine) compared with the commercial kit. This combined system is fast and highly sensitive and thus represents a promising approach for disease diagnosis in the clinical setting.


Assuntos
Técnicas Biossensoriais , Brucella/isolamento & purificação , Terra de Diatomáceas/química , Brucella/patogenicidade , Humanos , Limite de Detecção , Testes Imediatos
13.
Clin Chem ; 64(3): 556-565, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29208659

RESUMO

BACKGROUND: Scrub typhus and severe fever with thrombocytopenia syndrome (SFTS) are the most common tick-borne illnesses in South Korea. Early differentiation of SFTS from scrub typhus in emergency departments is essential but difficult because of their overlapping epidemiology, shared risk factors, and similar clinical manifestations. METHODS: We compared the diagnostic performance of one-step isothermal nucleic acid amplification with bio-optical sensor detection (iNAD) under isothermal conditions, which is rapid (20-30 min), with that of real-time PCR, in patients with a confirmed tick-borne illness. Fifteen patients with confirmed SFTS who provided a total of 15 initial blood samples and 5 follow-up blood samples, and 21 patients with confirmed scrub typhus, were evaluated. RESULTS: The clinical sensitivity of iNAD (100%; 95% CI, 83-100) for SFTS was significantly higher than that of real-time PCR (75%; 95% CI, 51-91; P = 0.047), while its clinical specificity (86%; 95% CI, 65-97) was similar to that of real-time PCR (95%; 95% CI, 77-99; P = 0.61). The clinical sensitivity of iNAD for scrub typhus (100%; 95% CI, 81-100) was significantly higher than that of real-time PCR for scrub typhus (67%; 95% CI, 43-85; P = 0.009), while its clinical specificity (90%; 95% CI, 67-98) was similar to that of real-time PCR (95%; 95% CI, 73-100; P > 0.99). CONCLUSIONS: iNAD is a valuable, rapid method of detecting SFTS virus and Orientia tsutsugamushi with high clinical sensitivity and specificity.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Febre por Flebótomos/diagnóstico , Tifo por Ácaros/diagnóstico , Doenças Transmitidas por Carrapatos/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos , Reação em Cadeia da Polimerase em Tempo Real , República da Coreia , Estudos Retrospectivos , Sensibilidade e Especificidade
14.
Oncotarget ; 8(48): 83860-83871, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-29137388

RESUMO

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs.

15.
PLoS One ; 12(7): e0180237, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28672014

RESUMO

BACKGROUND: Serologic diagnosis is one of the most widely used diagnostic methods for Q fever, but the window period in antibody response of 2 to 3 weeks after symptom onset results in significant diagnostic delay. We investigated the diagnostic utility of Q fever PCR from formalin-fixed liver tissues in Q fever patients with acute hepatitis. METHODS: We reviewed the clinical and laboratory data in patients with Q fever hepatitis who underwent liver biopsy during a 17-year period, and whose biopsied tissues were available. We also selected patients who revealed granuloma in liver biopsy and with no Q fever diagnosis within the last 3 years as control. Acute Q fever hepatitis was diagnosed if two or more of the following clinical, serologic, or histopathologic criteria were met: (1) an infectious hepatitis-like clinical feature such as fever (≥ 38°C) with elevated hepatic transaminase levels; (2) exhibition of a phase II immunoglobulin G (IgG) antibodies titer by IFA of ≥ 1:128 in single determination, or a four-fold or greater rise between two separate samples obtained two or more weeks apart; (3) histologic finding of biopsy tissue showing characteristic fibrin ring granuloma. RESULTS: A total of 11 patients with acute Q fever hepatitis were selected and analyzed. Of the 11 patients, 3 (27%) had exposure to zoonotic risk factors and 7 (63%) met the serologic criteria. Granulomas with either circumferential or radiating fibrin deposition were observed in 10 cases on liver biopsy and in 1 case on bone marrow biopsy. 8 (73%) revealed positive Coxiella burnetii PCR from their formalin-fixed liver tissues. In contrast, none of 10 patients with alternative diagnosis who had hepatic granuloma revealed positive C. burnetii PCR from their formalin-fixed liver tissues. CONCLUSIONS: Q fever PCR from formalin-fixed liver tissues appears to be a useful adjunct for diagnosing Q fever hepatitis.


Assuntos
Coxiella burnetii/isolamento & purificação , Formaldeído , Hepatite/complicações , Febre Q/patologia , Fixação de Tecidos , Doença Aguda , Adulto , Idoso , Coxiella burnetii/genética , Humanos , Masculino , Pessoa de Meia-Idade , Febre Q/complicações , Estudos Retrospectivos
16.
Anal Chem ; 89(14): 7502-7510, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28633525

RESUMO

The isolation of nucleic acids in the lab on a chip is crucial to achieve the maximal effectiveness of point-of-care testing for detection in clinical applications. Here, we report on the use of a simple and versatile single-channel microfluidic platform that combines dimethyl pimelimidate (DMP) for nucleic acids (both RNA and DNA) extraction without electricity using a thin-film system. The system is based on the adaption of DMP into nonchaotropic-based nucleic acids and the capture of reagents into a low-cost thin-film platform for use as a microfluidic total analysis system, which can be utilized for sample processing in clinical diagnostics. Moreover, we assessed the use of the DMP system for the extraction of nucleic acids from various samples, including mammalian cells, bacterial cells, and viruses from human disease, and we also confirmed that the quality and quantity of the nucleic acids extracted were sufficient to allow for the robust detection of biomarkers and/or pathogens in downstream analysis. Furthermore, this DMP system does not require any instruments and electricity, and has improved time efficiency, portability, and affordability. Thus, we believe that the DMP system may change the paradigm of sample processing in clinical diagnostics.

17.
Biosens Bioelectron ; 90: 187-194, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27894035

RESUMO

Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Viroses/diagnóstico , Coronavirus/genética , Coronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/genética , Humanos , Influenza Humana/diagnóstico , Influenza Humana/genética , Influenzavirus A/genética , Influenzavirus A/isolamento & purificação , Influenzavirus B/genética , Influenzavirus B/isolamento & purificação , Limite de Detecção , RNA Viral/genética , Viroses/genética
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