RESUMO
The ongoing coronavirus disease 2019 (COVID-19) pandemic demands rapid and straightforward diagnostic tools to prevent early-stage viral transmission. Although nasopharyngeal swabs are a widely used patient sample collection method for diagnosing COVID-19, using these samples for diagnosis without RNA extraction increases the risk of obtaining false-positive and -negative results. Thus, multiple purification steps are necessary, which are time-consuming, generate significant waste, and result in substantial sample loss. To address these issues, we developed surface-modified polymerase chain reaction (PCR) tubes using the tertiary aminated polymer poly(2-dimethylaminomethylstyrene) (pDMAMS) via initiated chemical vapor deposition. Introducing the clinical samples into the pDMAMS-coated tubes resulted in approximately 100% RNA capture efficiency within 25 min, which occurred through electrostatic interactions between the positively charged pDMAMS surface and the negatively charged RNA. The captured RNA is then detected via chamber digital PCR, enabling a sensitive, accurate, and rapid diagnosis. Our platform provides a simple and efficient RNA extraction and detection strategy that allows detection from 22 nasopharyngeal swabs and 21 saliva specimens with 0% false negatives. The proposed method can facilitate the diagnosis of COVID-19 and contribute to the prevention of early-stage transmission.
Assuntos
COVID-19 , Humanos , COVID-19/diagnóstico , Teste para COVID-19 , Reação em Cadeia da Polimerase , Polímeros , RNARESUMO
Quantifying the physical properties of individual exosomes containing amyloid-ß42 (Aß42) is crucial for a better understanding of an underpinning mechanism of Alzheimer's disease expression which is associated with the Aß42 transfer. Because of the lack of proper tools, however, there have been very few studies on how the amount of Aß42 affects the physical properties of exosomes. To answer the question, we investigated the physical properties of exosomes secreted by neuroblastoma by probing individual exosomes using electrostatic force microscopy. Interestingly, we observed that when the higher concentration of Aß42 oligomers was fed to cells, the higher surface charge of the exosomes appeared. This result indicates that the exosomes contain more Aß42 with the increase in Aß42 concentration in cell media, implying that they serve as transport vesicles for Aß42. Our approach could help to better understand how the neuronal exosomes are related to the propagation of neurodegenerative diseases and to seek how to make an early diagnosis of those diseases.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Exossomos/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Linhagem Celular , Exossomos/ultraestrutura , Camundongos , Microscopia de Força Atômica , Microscopia Eletroquímica de Varredura , Transporte Proteico , Eletricidade EstáticaRESUMO
Microscale emulsions are widely used in fundamental and applied sciences. To expand their utilization, various methods have been developed for manipulating and measuring the physical properties of fabricated emulsions inside microchannels. Herein, we present an electric emulsification platform that can produce emulsions and simultaneously detect their physical properties (size and production speed). The characterization of the emulsion properties during the fabrication process will broaden the application fields for microscale emulsions because it can avoid time-consuming post image processing and simplify the emulsification platform. To accomplish this, a "bottleneck" channel is implanted between two reservoirs of immiscible fluids (continuous and dispersion phases). This channel can not only confine one fluid within the other when the electric field is on, resulting in emulsification via electrohydrodynamically induced Rayleigh instability, but also act as a resistive pulse sensor (RPS). The fluctuation of the liquid/liquid interface during emulsification induces the fluctuation of the electric resistance in the bottleneck channel, as the two fluid phases have different electrical conductivities. With this simple but dual-functional channel, the emulsion size (radius of 5-10 µm) and production speed (7-12 Hz) can be controlled by adjusting the electric field and the channel-neck geometry. Additionally, the properties can be measured using the RPS; the data obtained through the RPS exhibit high correlations with the validated data obtained using a high-speed camera and microscopy (>95%). The proposed buffer-less electric emulsification with the embedded RPS is a simple and cost-effective emulsion production method that allows real-time emulsion characterization with a limited sample volume.
RESUMO
An excessive cholesterol level can lead to cardiovascular diseases, such as stroke, hypertension, and myocardial infarction. A non-invasive, painless method of determining the cholesterol level in blood would improve the user's convenience. To provide rapid and accurate determination of cholesterol, we have developed a simple, disposable, enzyme-based electrochemical biosensor that can detect salivary cholesterol. It is possible to detect low concentrations of cholesterol in saliva using the optimized vertical structure of the platinum nano-cluster (Pt-NC) and the immobilization of a proper volume of an enzyme. The biosensor exhibited a linear range from 2 to 486 µM, the limit of detection was about 2 µM, and the sensitivity of the sensor was calculated to be 132 µA mM-1 cm-2. It also showed good specificity for ascorbic acid, uric acid, dopamine, glucose, and lactate. In a test with an actual sample, the performance of the biosensor was confirmed by measuring total cholesterol in the saliva of a patient with hyperlipidemia. The cholesterol levels measured in the saliva of three patients with hyperlipidemia were 520, 460, and 290 µM. Therefore, the Pt-NC based enzyme sensor is a promising candidate for the detection of cholesterol in human saliva.
Assuntos
Técnicas Biossensoriais/métodos , Colesterol/análise , Nanoestruturas/química , Platina/química , Saliva/química , Ácido Ascórbico/química , Colesterol Oxidase/química , Colesterol Oxidase/metabolismo , Técnicas Eletroquímicas , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose/química , Humanos , Hiperlipidemias/diagnóstico , Limite de Detecção , Ácido Úrico/químicaRESUMO
Cancer-associated fibroblasts (CAFs) in the cancer microenvironment play an essential role in metastasis. Differentiation of endothelial cells into CAFs is induced by cancer cell-derived exosomes secreted from cancer cells that transfer molecular signals to surrounding cells. Differentiated CAFs facilitate migration of cancer cells to different regions through promoting extracellular matrix (ECM) modifications. However, in vitro models in which endothelial cells exposed to cancer cell-derived exosomes secreted from various cancer cell types differentiate into CAFs or a microenvironmentally controlled model for investigating cancer cell invasion by CAFs have not yet been studied. In this study, we propose a three-dimensional in vitro cancer cell invasion model for real-time monitoring of the process of forming a cancer invasion site through CAFs induced by exosomes isolated from three types of cancer cell lines. The invasiveness of cancer cells with CAFs induced by cancer cell-derived exosomes (eCAFs) was significantly higher than that of CAFs induced by cancer cells (cCAFs) through physiological and genetic manner. In addition, different genetic tendencies of the invasion process were observed in the process of invading cancer cells according to CAFs. Our 3D microfluidic platform helps to identify specific interactions among multiple factors within the cancer microenvironment and provides a model for cancer drug development.
Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/patologia , Diferenciação Celular , Células Endoteliais/citologia , Exossomos/patologia , Neoplasias/patologia , Microambiente Tumoral , Apoptose , Biomarcadores Tumorais/genética , Fibroblastos Associados a Câncer/metabolismo , Proliferação de Células , Células Endoteliais/metabolismo , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
EVs/exosomes are considered as the next generation of biomarkers, including for liquid biopsies. Consequently, the quantification of EVs/exosomes is crucial for facilitating EV/exosome research and applications. Paper-based enzyme-linked immunosorbent assay (p-ELISA) is a portable diagnostic system with low cost that is simple and easy to use; however, it shows low sensitivity and linearity. In this study, we develop p-ELISA for targeting EVs/exosomes by using streptavidin agarose resin-based immobilization (SARBI). This method reduces assay preparation times, provides strong binding, and retains good sensitivity and linearity. The time required for the total assay, including preparation steps and surface immobilization, was shortened to â¼2 h. We evaluated SARBI p-ELISA systems with/without CD63 capture Ab and then with fetal bovine serum (FBS) and EVs/exosome-depleted fetal bovine serum (dFBS). The results provide evidence supporting the selective capture ability of SARBI p-ELISA. We obtain semiquantitative p-ELISA results using an exosome standard (ES) and human serum (HS), with R2 values of 0.95 and 0.92, respectively.
Assuntos
Exossomos , Papel , Sefarose/química , Estreptavidina/química , Anticorpos Imobilizados/imunologia , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Soro/química , Tetraspanina 29/imunologia , Tetraspanina 30/imunologiaRESUMO
Nanofluidic resistive pulse sensing (RPS) has been extensively used to measure the size, concentration, and surface charge of nanoparticles in electrically conducting solutions. Although various methods have been explored for improving detection performances, intrinsic problems including the extremely low particle-to-pore volume ratio (<0.01%) and fast nanoparticle translocation (10-1000 µs) still induce difficulties in detection, such as low signal magnitudes and short translocation times. Herein, we present an aqueous two-phase system (ATPS) in a nanofluidic RPS for amplifying translocation signals and decreasing translocation speeds simultaneously. Two immiscible aqueous liquids build a liquid-liquid interface inside nanopores. As particles translocate from a high-affinity liquid phase into a lower-affinity one, the high-affinity liquid forms a conformal coating on the particles, which increases the effective particle size and amplifies the current-blockage signal. The translocation time is also increased, as the ATPS interface impedes the particle translocation. For 20 nm particles, 7.92-fold and 5.82-fold enhancements of signal magnitude and translocation time can be achieved. To our knowledge, this is the first attempt to improve nanofluidic RPS by treating an interface of solution reservoirs for manipulating target particles rather than nanopores. This direct particle manipulation allows us to solve the two intrinsic problems all at once.
RESUMO
The ion concentration polarization (ICP) phenomenon at micronanofluidic interfaces has been extensively utilized to preconcentrate low-abundance biological samples. Although preconcentration by ICP is robust, its multiphysics phenomenon does not permit a clear prediction of the preconcentration conditions and sites. Here, we present a new method for spatiotemporally defining preconcentration, which can generate target-condensed plugs in a very specific region (<100 µm) regardless of the operating conditions (time, applied voltage, ionic strength, and pH). In contrast to previous devices that use only ion depletion, this device uses merged ICP zones with opposite polarity, i.e., ion depletion and ion enrichment. In this regard, ICP is initiated between two line-patterned cation exchange membranes. When voltage is applied across two membranes, an ion depletion (enrichment) zone occurs on the anodic (cathodic) side of the membranes. Two ICP zones are then merged and confined between the membranes. Consequently, the preconcentration action is also confined between the membranes. We demonstrate that fluorescent dyes are always preconcentrated at the designated location at all lengths of operating time and at broad voltage (0.5-100 V), ionic strength (1-100 mM KCl), and pH (3.7-10.3) ranges. This device successfully condenses proteins up to 10000-fold in a specific region of the channel (100 × 50 × 10 µm(3)) in 10 min. This work not only characterizes the unique scientific phenomenon of ICP overlapping but also opens the possibility of integrating ICP preconcentrators into commercial analysis equipment, which requires a known, stationary preconcentration site.
Assuntos
Corantes Fluorescentes/análise , Corantes Fluorescentes/isolamento & purificação , Técnicas Analíticas Microfluídicas , Proteínas/análise , Proteínas/isolamento & purificação , Análise Espaço-Temporal , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Íons/química , Íons/isolamento & purificação , Concentração Osmolar , Proteínas/química , Fatores de TempoRESUMO
BACKGROUND: This study aims to validate the performance and biocompatibility of an implantable inductive-type sensor for continuous monitoring of intraocular pressure (IOP) METHODS: The sensor is composed of a top layer integrated with an inductor and capacitor circuit, and a bottom layer integrated with ferrite. With IOP change, the sensor's bottom layer is mechanically deflected, which changes the distance between the bottom-layer ferrite and top-layer inductor, resulting in an alteration of inductance magnitude and the resonant frequency (RF). In-vitro measurement was conducted via air pressurization in a sealing jig (n = 3). Subsequently, the sensor was implanted into the anterior chamber of a rabbit eye. In-vivo measurement was performed while the IOP was elevated by infusion of balanced salt solution (BSS, 6 µL/min). Smaller-sized sensors later were implanted into two rabbit eyes, which were microscopically examined at 2, 4 and 8 weeks post-implantation. The eyes were then immediately enucleated for histological examination. RESULTS: The in-vitro measurement showed a significant RF shift as pressure in the jig was increased from 0 mmHg to 60 mmHg (average initial frequency: 10.86 MHz, average shift: 403 kHz). The in-vivo measurement also showed an RF decrease, from 12.80 MHz to 12.67 MHz, as the pressure was increased from 10 mmHg to 20 mmHg. Microscopic in-vivo evaluations and histological exams, performed at intervals up to 8 weeks post-implantation, showed no evidence of significant inflammation or deformity of the ocular-tissue structures. CONCLUSIONS: The implantable inductive-type IOP sensor demonstrated wireless pressure-sensing ability and favourable biocompatibility in the rabbit eye.
Assuntos
Pressão Intraocular/fisiologia , Monitorização Fisiológica/instrumentação , Próteses e Implantes , Tonometria Ocular/instrumentação , Animais , Desenho de Equipamento , Masculino , Teste de Materiais , Coelhos , Telemetria/instrumentaçãoRESUMO
Preventing unexpected explosive attacks and tracing explosion-related molecules require the development of highly sensitive gas-vapor detection systems. For that purpose, a micromechanical cantilever-based olfactory sensing system including a sample preconcentrator was developed to detect 2,4-dinitrotoluene (2,4-DNT), which is a well-known by-product of the explosive molecule trinitrotoluene (TNT) and exists in concentrations on the order of parts per billion in the atmosphere at room temperature. A peptide receptor (His-Pro-Asn-Phe-Ser-Lys-Tyr-Ile-Leu-His-Gln-Arg) that has high binding affinity for 2,4-DNT was immobilized on the surface of the cantilever sensors to detect 2,4-DNT vapor for highly selective detection. A micro-preconcentrator (µPC) was developed using Tenax-TA adsorbent to produce higher concentrations of 2,4-DNT molecules. The preconcentration was achieved via adsorption and thermal desorption phenomena occurring between target molecules and the adsorbent. The µPC directly integrated with a cantilever sensor and enhanced the sensitivity of the cantilever sensor as a pretreatment tool for the target vapor. The response was rapidly saturated within 5 min and sustained for more than 10 min when the concentrated vapor was introduced. By calculating preconcentration factor values, we verified that the cantilever sensor provides up to an eightfold improvement in sensing performance.
Assuntos
Técnicas Biossensoriais/instrumentação , Dinitrobenzenos/análise , Gases/análise , Fenômenos Mecânicos , Microtecnologia/instrumentação , Olfato , Modelos Teóricos , Peptídeos/análise , Processamento de Sinais Assistido por Computador , VolatilizaçãoRESUMO
Alzheimer's Disease (AD) is one of the most common neurodegenerative disorders in elderly people. It is diagnosed by detecting amyloid beta (Aß) protein in cerebrospinal fluid (CSF) obtained by lumbar puncture or through expensive positron emission tomography (PET) imaging. Although blood-based diagnosis of AD offers a less invasive and cost-effective alternative, the quantification of Aß is technically challenging due to its low abundance in peripheral blood. To address this, we developed a compact yet highly sensitive microwell-based electrochemical sensor with a densely packed microelectrode array (20 by 20) for enhancing sensitivity. Employing microwells on the working and counter electrodes minimized the leakage current from the metallic conductors into the assay medium, refining the signal fidelity. We achieved a detection limit <10 fg/mL for Aß by elevating the signal-to-noise ratio, thus capable of AD biomarker quantification. Moreover, the microwell structure maintained the performance irrespective of variations in bead number, indicative of the sensor's robustness. The sensor's efficacy was validated through the analysis of Aß concentrations in plasma samples from 96 subjects, revealing a significant distinction between AD patients and healthy controls with an area under the receiver operating characteristic curve (AUC) of 0.85. Consequently, our novel microwell-based electrochemical biosensor represents a highly sensitive platform for detecting scant blood-based biomarkers, including Aß, offering substantial potential for advancing AD diagnostics.
Assuntos
Doença de Alzheimer , Técnicas Biossensoriais , Humanos , Idoso , Peptídeos beta-Amiloides , Tomografia por Emissão de Pósitrons/métodos , Biomarcadores/líquido cefalorraquidiano , Microeletrodos , Proteínas tau , Fragmentos de PeptídeosRESUMO
Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.
Assuntos
Técnicas Biossensoriais , COVID-19 , Animais , Camundongos , SARS-CoV-2 , COVID-19/diagnóstico , Saliva , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos , Sensibilidade e Especificidade , Anticorpos AntiviraisRESUMO
Multiple target detection using a cantilever is essential for biosensor, chemical sensor, and electronic nose systems. We report a novel microcantilever array chip that includes four microreaction chambers in a chip, which consequently contains four different functionalized surfaces for multitarget detection. For model tests, we designed microcantilever chips and demonstrated the ability of binding of 2,4-dinitrotoluene (DNT) targets onto four different surfaces. We used peptide receptors that are known to have highly selective binding. By simply using four microreaction chambers, we immobilized DNT specific peptide (HPNFSKYILHQRC; SP), DNT nonspecific peptide (TSMLLMSPKHQAC; NSP), and self-assembled monolayer (SAM) as well as a bare cantilever. After flowing DNT gases through the cantilever chip, we could monitor the four different binding signals simultaneously. The shifts in NSP provided information as a negative control because it contained information of temperature fluctuations and mechanical vibration from gas flow. By utilizing the differential signal of the SP and NSP, we acquired 7.5 Hz in resonant responses that corresponds with 160 part per billion (ppb) DNT concentration, showing the exact binding response by eliminating the inevitable thermal noise, vibration noise, as well as humidity effects on the peptide surface.
Assuntos
Técnicas Biossensoriais , Dinitrobenzenos/análise , Eletrônica , Nariz , Técnicas Biossensoriais/instrumentação , Eletrônica/instrumentação , Umidade , Oligopeptídeos/química , Propriedades de Superfície , Temperatura , VibraçãoRESUMO
Owing to their excellent durability, tunable physical properties, and biofunctionality, block copolymer-based membranes provide a platform for various biotechnological applications. However, conventional approaches for fabricating block copolymer membranes produce only planar or suspended polymersome structures, which limits their utilization. This study is the first to demonstrate that an electric-field-assisted self-assembly technique can allow controllable and scalable fabrication of 3-dimensional block copolymer artificial cell membranes (3DBCPMs) immobilized on predefined locations. Topographically and chemically structured microwell array templates facilitate uniform patterning of block copolymers and serve as reactors for the effective growth of 3DBCPMs. Modulating the concentration of the block copolymer and the amplitude/frequency of the electric field generates 3DBCPMs with diverse shapes, controlled sizes, and high stability (100% survival over 50 days). In vitro protein-membrane assays and mimicking of human intestinal organs highlight the potential of 3DBCPMs for a variety of biological applications such as artificial cells, cell-mimetic biosensors, and bioreactors.
Assuntos
Células Artificiais , Técnicas Biossensoriais , Humanos , Membranas Artificiais , Polímeros/química , Propriedades de SuperfícieRESUMO
This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Peptídeos beta-Amiloides/análise , Fluorescência , Peroxidase do Rábano Silvestre/química , Humanos , Oxazinas/análise , Oxazinas/química , OxirreduçãoRESUMO
Speciated hourly measurements of fine aerosols were made for more than two years at an urban, an industrial and a port site in Busan, Korea. A Generalized Additive Model (GAM) was designed to deconvolve factors contributing to the pollutant concentrations at multiple scales. The model yields estimates of source contributions to pollution by separately identifying the signals in the time series due to meteorology, vertical mixing, horizontal wind transport and temporal variations such as diurnal, weekly, seasonal and annual trends. The GAM model was expanded to include FLEXPART back trajectory clusters generated using fuzzy c-means clustering. This made it possible to quantify the impact of long-range transport using the Trajectory Cluster Contribution Function (TCCF). TCCF provides a development of methods such as Concentration Field Analysis and Potential Source Contribution Function by providing numerical estimates of concentration changes associated with different air mass transport patterns while accounting for possible confounding factors from meteorology. The GAM simulations identified the importance of local transport for primary pollutants and long-range transport from China for secondary pollutants. Local factors accounted for up to 72% of the variance in concentrations of NO2 and elemental carbon whereas large-scale/seasonal factors accounted for up to 56% of PM2.5 and 80% of inorganic species. The algorithm further identified the importance of the weekend effect and the holiday effect at the different sites in Busan. The residual from the analysis was used to estimate the impact of the COVID-19 pandemic. The signature of the pandemic was different between the pollutants as well as from site to site. The model was able to distinguish small impacts from local pollutants at the residential site; short-lived acute impacts from industrial changes; and longer-term changes due to the early pandemic response in China.
RESUMO
Although urinary exosomal microRNAs (miRNAs) have recently emerged as potential biomarkers, clinical applications are still limited due to their low concentration in small volumes of clinical samples. Therefore, the development of a non-invasive, specific diagnostic tool, along with profiling exosomal miRNA markers from urine, remains a significant challenge. Here, we present hydrogel-based hybridization chain reaction (HCR) for multiplex signal amplification to detect urinary exosomal miRNAs from human clinical samples. We succeeded in identifying small amounts (~amol) of exosomal miRNAs from 600 µL of urine with up to ~35-fold amplification and enhanced detection limits by over an order of magnitude for two miRNA biomarker candidates, hsa-miR-6090 and hsa-miR-3665. Furthermore, we proposed ratiometric analysis without requiring normalization to a reference miRNA and validated the clinical diagnostic potential toward differentiating prostate cancer patients from healthy controls. Our hydrogel-based HCR could serve as a new diagnostic platform for a non-invasive liquid biopsy before burdensome tissue biopsy of various diseases, including prostate cancer screening, complementing the PSA test.
Assuntos
Técnicas Biossensoriais , Exossomos , MicroRNAs , Neoplasias da Próstata , Biomarcadores Tumorais/genética , Detecção Precoce de Câncer , Exossomos/genética , Humanos , Hidrogéis , Masculino , MicroRNAs/genética , Antígeno Prostático Específico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genéticaRESUMO
Ion concentration polarization (ICP) is one of the preconcentration techniques which can acquire a high preconcentration factor. Still, the main hurdles of ICP are its instability and low efficiency under physiological conditions with high ionic strength and abundant biomolecules. Here, we suggested a sequentially driven ICP process, which enhanced the electrokinetic force required for preconcentration, enabling enrichment of highly ionic raw samples without increasing the electric field. We acquired a 13-fold preconcentration factor (PF) in human serum using a paper-based origami structure consisting of multiple layers for three-dimensional sequential ICP (3D seq-ICP). Moreover, we demonstrated a paper-based enzyme-linked immunosorbent assay (ELISA) by 3D seq-ICP using tau protein, showing a 6-fold increase in ELISA signals.
Assuntos
Técnicas Analíticas Microfluídicas , Humanos , Íons , Concentração OsmolarRESUMO
We present a 2-layer based microfluidic concentration generator by a hybrid of a serial and a volumetric dilution for dose-response experiments in drug screening. The hybrid dilution method using 2-layer based microfluidic network significantly reduces the total number of cascaded serial dilution stages. The proposed strategy is capable of generating a large number of universal stepwise monotonic concentrations with a wide range of logarithmic and linear scales. We have studied an equivalent electrical circuit to that of the 2-layer based microfluidic network, where the only variable parameter is channel length. We have designed a microfluidic dilution generator simultaneously covering 14 doses with a combination of 4-order logarithmic and 4-point linear concentrations. The design has been verified by a commercial circuit analysis software (e.g., P-Spice) for the electrical circuit analysis and a computational fluid dynamics software (e.g., CFD-ACE+) for the microfluidic circuit analysis. As a real-life application of the proposed dilution generator, we have successfully performed a dose-response experiment using MCF-7 human breast cancer cells. We expect that the proposed dilution method will be useful to study not only high throughput drug screening but also optimization in biology, chemistry, medicine, and material sciences.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Técnicas de Diluição do Indicador/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Relação Dose-Resposta a Droga , Humanos , Microfluídica/métodos , SoftwareRESUMO
As conventional bulky methods for extracellular vesicle (EV) separation are unsuitable for small volumes of samples, microfluidic devices are thought to offer a solution for the integrated and automatic processing of EV separation. This study demonstrates a simple microfluidic aqueous two-phase system (ATPS) for EV separation with high recovery efficiency to overcome the limitation of previous devices, which require complex external equipment or high cost manufacturing. With polyethylene glycol and dextran in the microfluidic channel, the isolation mechanism of the microfluidic ATPS was analyzed by comparison between two-phase and one-phase systems. Our device could facilitate continuous EV isolation with 83.4% recovery efficiency and remove 65.4% of the proteins from the EV-protein mixture. EVs were also successfully isolated from human plasma at high recovery efficiency.