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As the role of exosomes in physiological and pathological processes has been properly perceived, harvesting them and their internal components is critical for subsequent applications. This study is a debut of intermittent lysis, which has been integrated into a simple and easy-to-operate procedure on a single paper-based device to extract exosomal nucleic acid biomarkers for downstream analysis. Exosomes from biological samples were captured by anti-CD63-modified papers before being intermittently lysed by high-temperature, short-time treatment with double-distilled water to release their internal components. Exosomal nucleic acids were finally adsorbed by sol-gel silica for downstream analysis. Empirical trials not only revealed that sporadically dropping 95 °C ddH2O onto the anti-CD63-modified papers every 5 min for 6 times optimized the exosomal nucleic acids extracted by the anti-CD63 paper but also verified that the whole deployed procedure is applicable for point-of-care testing (POCT) in low-resource areas and for both in vitro (culture media) and in vivo (plasma and chronic lesion) samples. Importantly, downstream analysis of exosomal miR-21 extracted by the paper-based procedure integrated with this novel technique discovered that the content of exosomal miR-21 in chronic lesions related to their stages and the levels of exosomal carcinoembryonic antigen originated from colorectal cancer cells correlated to their exosomal miR-21.
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Exosomas , MicroARNs , Papel , Tetraspanina 30 , Exosomas/química , Humanos , Tetraspanina 30/metabolismo , MicroARNs/análisis , MicroARNs/sangre , Biomarcadores de Tumor/sangre , Pruebas en el Punto de AtenciónRESUMEN
Interleukin 6 (IL-6) has been regarded as a biomarker that can be applied as a predictor for the severity of COVID-19-infected patients. The IL-6 level also correlates well with respiratory dysfunction and mortality risk. In this work, three silanization approaches and two types of biorecognition elements were used on the silicon nanowire field-effect transistors (SiNW-FETs) to investigate and compare the sensing performance on the detection of IL-6. Experimental data revealed that the mixed-SAMs-modified silica surface could have superior surface morphology to APTES-modified and APS-modified silica surfaces. According to the data on detecting various concentrations of IL-6, the detection range of the aptamer-functionalized SiNW-FET was broader than that of the antibody-functionalized SiNW-FET. In addition, the lowest concentration of valid detection for the aptamer-functionalized SiNW-FET was 2.1 pg/mL, two orders of magnitude lower than the antibody-functionalized SiNW-FET. The detection range of the aptamer-functionalized SiNW-FET covered the concentration of IL-6, which could be used to predict fatal outcomes of COVID-19. The detection results in the buffer showed that the anti-IL-6 aptamer could produce better detection results on the SiNW-FETs, indicating its great opportunity in applications for sensing clinical samples.
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Técnicas Biosensibles , COVID-19 , Nanocables , Humanos , Silicio , Transistores Electrónicos , Interleucina-6 , Técnicas Biosensibles/métodos , COVID-19/diagnóstico , Dióxido de Silicio , AnticuerposRESUMEN
Emerging evidence has shown that microRNAs play pivotal roles in wound healing. MicroRNA-21 (miR-21) was previously found to upregulate in order to fulfill an anti-inflammation role for wounds. Exosomal miRNAs have been identified and explored as essential markers for diagnostic medicine. However, the role of exosomal miR-21 in wounds has yet to be well studied. In order to facilitate the early management of poorly healing wounds, we developed an easy-to-use, rapid, paper-based microfluidic-exosomal miR-21 extraction device to determine wound prognosis in a timely manner. We isolated and then quantitatively examined exosomal miR-21 in wound fluids from normal tissues and acute and chronic wounds. Eight improving wounds displayed lower levels of exosomal miR-21 expression after wound debridement. However, four instances of increased exosomal miR-21 expression levels were notably associated with patients with poor healing wounds despite aggressive wound debridement, indicating a predictive role of tissue exosomal miR-21 for wound outcome. Paper-based nucleic acid extraction device provides a rapid and user-friendly approach for evaluating exosomal miR-21 in wound fluids as a means of monitoring wounds. Our data suggest that tissue exosomal miR-21 is a reliable marker for determining current wound status.
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Exosomas , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Cicatrización de Heridas/genética , Proyectos de Investigación , Exosomas/genética , Exosomas/metabolismoRESUMEN
Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire field-effect transistors (SiNWFETs) is severely hindered due to the weakened signal, primarily caused by screening effects. In this study, aptamer as a signal amplifier, which has already been reported by our group, is integrated into SiNWFET immunosensors employing antigen-binding fragments (Fab) as the receptors to improve its detection limit for the first time. The Fab-SiNWFET immunosensors were developed by immobilizing Fab onto Si surfaces modified with either 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Fab/APTES-SiNWFETs), or mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) and GA (Fab/PEG-SiNWFETs), to detect the rabbit IgG at different concentrations in a high-ionic-strength environment (150 mM Bis-Tris Propane) followed by incubation with R18, an aptamer which can specifically target rabbit IgG, for signal enhancement. Empirical results revealed that the signal produced by the sensors with Fab probes was greatly enhanced compared to the ones with whole antibody (Wab) after detecting similar concentrations of rabbit IgG. The Fab/PEG-SiNWFET immunosensors exhibited an especially improved limit of detection to determine the IgG level down to 1 pg/mL, which has not been achieved by the Wab/PEG-SiNWFET immunosensors.
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Técnicas Biosensibles , Nanocables , Animales , Inmunoensayo , Límite de Detección , Proteínas/análisis , Conejos , SilicioRESUMEN
Aptamers, in sensing technology, are famous for their role as receptors in versatile applications due to their high specificity and selectivity to a wide range of targets including proteins, small molecules, oligonucleotides, metal ions, viruses, and cells. The outburst of field-effect transistors provides a label-free detection and ultra-sensitive technique with significantly improved results in terms of detection of substances. However, their combination in this field is challenged by several factors. Recent advances in the discovery of aptamers and studies of Field-Effect Transistor (FET) aptasensors overcome these limitations and potentially expand the dominance of aptamers in the biosensor market.
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Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodosRESUMEN
During recent years, field-effect transistor biosensors (Bio-FET) for biomedical applications have experienced a robust development with evolutions in FET characteristics as well as modification of bio-receptor structures. This review initially provides contemplation on this progress by briefly summarizing remarkable studies on two aforementioned aspects. The former includes fabricating unprecedented nanostructures and employing novel materials for FET transducers whereas the latter primarily synthesizes compact molecules as bio-probes (antibody fragments and aptamers). Afterwards, a future perspective on research of FET-biosensors is also predicted depending on current situations as well as its great demand in clinical trials of disease diagnosis. From these points of view, FET-biosensors with infinite advantages are expected to continuously advance as one of the most promising tools for biomedical applications.
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Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Nanoestructuras/química , Transistores Electrónicos , Anticuerpos/química , Aptámeros de Nucleótidos/química , Diseño de Equipo , Humanos , Sondas Moleculares/químicaRESUMEN
Field-effect transistors (FETs) have been developed as pH sensors by using various device structures, fabrication technologies, and sensing film materials. Different transistor structures, like extended-gate (EG) FETs, floating-gate FET sensors, and dual-gate (DG) FETs, can enhance the sensor performance. In this article, we report the effects of using solution-gate and bottom-gate FET configurations on pH sensing and investigate the influence of different ionic concentrations of buffers in the measured signals. The surface charge of hafnium dioxide (HfO2) affected by the buffer pH, with/without the modification of polyethylene glycol (PEG) terminated with hydroxyl groups, and the location of applied gate voltage are vital factors to the sensor performance in pH sensing. Based on the results, the solution-gate FET exhibits good pH sensitivity even in the high ionic strength solutions of bis-tris propane (BTP), and these values of pH sensitivity are close to the Nernst limit (59.2 mV/pH). In general, silane-PEG-OH modification can reduce the deviations of measured signals in pH sensing. The performance of bottom-gate FET is inferior in the BTP buffers with high ionic solutions but suitable to be operated in low ionic concentrations, such as 0.1, 1, and 10 mM BTP buffers. The size of the ions was also studied and discussed. The solution-gate FET demonstrates excellent performance under high ionic strengths, meaning a more significant potential for detecting biological molecules under physiological conditions.
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This study proposes a paper/PMMA hybrid device designed to isolate exosomes and extract exosomal miRNA, followed by quantitative analysis. It aims to provide simplified and convenient sample preparation for potential point-of-care testing (POCT) processes. In contrast to previous work conducted by our research team, which focused on isolating exosomes and exosomal nucleic acids, this study introduces a novel approach by integrating paper and a PMMA mold with a microvalve controlled design. This innovative method enables the entire process to be performed on paper. The pressure on the paper could be adjusted by turning the screw upon the valve to change the pore size and permeability of the paper, which achieved the effect of controlling the flow rate of fluids. The paper was designed to have an immunoaffinity area for capturing exosomes and a sol-gel silica coating area for extracting miRNA. The paper-based ELISA (p-ELISA) exhibited a limit of detection and a limit of quantitation of 6 × 107 and 5.4 × 108 particles/mL, respectively, for exosome measurement. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that the Ct (threshold cycle) value for quantifying the miR-21 in the miRNAs extracted by the proposed paper/PMMA hybrid device was comparable to the Ct value of the commercial extraction kit. The developed paper/PMMA hybrid device with a microvalve-controlled design should be incorporated into the POCT system to extract exosomal miRNAs.
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Exosomas , MicroARNs , Polimetil Metacrilato , Exosomas/química , MicroARNs/análisisRESUMEN
MicroRNA (miRNA) sensing plays an essential role in the diagnosis of several diseases, especially cancers, for appropriate intervention and treatment. However, quantifying miRNA demands highly sensitive and selective assays which can distinguish analogous sequences with low abundance in bio-samples and determine wide range of concentrations. In this report, we present a novel technique satisfying all those requirements by modifying silicon nanowire field-effect transistors (SiNWFETs) with 2-component mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) at different ratios (silane-PEG-NH2:silane-PEG-OH = 1:1, 1:3, and 1:5) and glutaraldehyde to immobilize DNA probes for miRNA-21 detection, a biomarker in several types of cancers. Empirical results reveal that all the fabricated PEG-SiNWFET DNA biosensors could quantify miRNA-21 within 1 fM - 10 pM. Especially, the ones modified with silane-PEG-NH2:silane-PEG-OH = 1:3 exhibited an outstanding performance to recognize miRNA-21 at an ultra-low concentration of 10 aM in the dynamic range up to 6 orders of magnitude (10 aM - 10 pM). This approach is more convenient, analytical competitive, and cost-effective in comparison with currently used methods for nucleic acid testing because of label- and amplification-free characteristics. It is therefore not only feasible for miRNA detection by SiNWFET-based biosensors but also potential for clinical applications of disease diagnosis with oligonucleotide biomarkers.
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Técnicas Biosensibles , MicroARNs , Nanocables , MicroARNs/genética , Polietilenglicoles , Silicio , Transistores ElectrónicosRESUMEN
This chapter introduces neutralized DNA (nDNA) as a novel design for the primers of PCR and RT-PCR by methylating phosphate groups of some oligonucleotides in their structures. It starts with an introduction of the nDNA which possesses an electrically chimeric neutral backbone as well as the proposed standards in designing nDNA as a novel primer for PCR and RT-PCR , concluded from various experimental results presented afterward. The primary content comprises empirical data from PCR to compare nDNA and unmodified DNA as primers in terms of ability to distinguish and amplify mismatch templates, activities of polymerase enzymes, melting temperature of double-stranded sequences, and the trials and discussions on various modified positions of the nDNA primers. In summary, nDNA exhibited outstanding performance as a primer for PCR and RT-PCR , compared to unmodified DNA, and is expected to be expanded in diverse applications which require enhanced specificity.
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Reacción en Cadena de la Polimerasa , ADN/genética , Oligonucleótidos/genética , Fosfatos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Exosomes, nanovesicles derived from cells, contain a variety of biomolecules that can be considered biomarkers for disease diagnosis, including microRNAs (miRNAs). Given knowledge and demand, inexpensive, robust, and easy-to-use tools that are compatible with downstream nucleic acid detection should be developed to replace traditional methodologies for point-of-care testing (POCT) applications. This study deploys a paper-based extraction kit for exosome and exosomal miRNA analytical system with some quantifying methods to serve as an easy sample preparation for a possible POCT process. Exosomes concentrated from HCT116 cell cultures were arrested on paper-based immunoaffinity devices, which were produced by immobilizing anti-CD63 antibodies on Whatman filter paper, before being subjected to paper-based silica devices for nucleic acids to be trapped by silica nanoparticles adsorbed onto Whatman filter paper. Concentrations of captured exosomes were quantified by enzyme-linked immunosorbent assay (ELISA), demonstrating that paper-based immunoaffinity devices succeeded in capturing and determining exosome levels from cells cultured in both neutral and acidic microenvironments, whereas microRNA 21 (miR-21), a biomarker for various types of cancers and among the nucleic acids absorbed onto the silica devices, was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to prove that paper-based silica devices were capable of trapping exosomal nucleic acids. The developed paper-based kit and the devised procedure was successfully exploited to isolate exosomes and exosomal nucleic acids from different biological samples (platelet-poor plasma and lesion fluid) as clinical applications.
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Silicon nanowire field-effect transistors (SiNW-FETs) have been demonstrated as a highly sensitive platform for label-free detection of a variety of biological and chemical entities. However, detecting signal from immunoassays by nano-FETs is severely hindered by the distribution of different charged groups of targeted entities, their binding orientation, and distances to the surface of the FET. Aptamers have been widely applied as a recognition element for plentiful biosensors because of small molecular sizes and moderate to high specific binding affinity with different types of molecules. In this study, we propose an effective approach to enhance the electrical responses of both direct (6×-histidine) and sandwich (amyloid ß 1-42) immunoassays in SiNW-FETs with R18, a highly negative charged RNA aptamer against rabbit immunoglobulin G (IgG). Empirical results presented that the immunosensors targeted with R18 expressed a significantly stabilized and amplified signal compared to the ones without this aptamer. The research outcome provides applicability of the highly negative charged aptamer as a bioamplifier for immunoassays by FETs.