RESUMEN
Ovarian cancer (OvCa) is the most fatal among gynecological cancers and affects many women worldwide. Since OvCa is prone to metastasis, which significantly increases chances of death, biomarkers for early-stage OvCa are greatly needed. This study develops an integrated microfluidic platform for isolating and quantifying one of the OvCa blood biomarkers. As a demonstration, microRNA-21 (miRNA-21), which is one of the important biomarkers for cancers, was isolated and measured in this study. Extracellular vesicles (EVs) in blood were first captured and isolated by anti-CD63-coated magnetic beads. Then, EV-encapsulated miRNA-21 was isolated by complementary DNA-coated magnetic beads, and finally the isolated miRNA-21 was quantified by digital polymerase chain reaction (digital PCR, dPCR). The integrated chip featured a sample treatment module and a miRNA quantification module that automated the entire process, and the limit of detection (LOD) was 11 copies per mL. The inaccuracy of the miRNA quantification module (i.e. dPCR) was found to be <12%. Additionally, spiked samples and clinical samples were used to test the performance of the developed platform. It is envisioned that the developed system can serve as a valuable and promising tool for OvCa biomarker measurements.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Biomarcadores de Tumor , Humanos , Separación Inmunomagnética , MicroARNs/genética , MicrofluídicaRESUMEN
Antimicrobial resistance stemming from indiscriminate usage of antibiotics has emerged as a global healthcare issue with substantial economic implications. The inefficacy of commonly used antibiotics combined with superfluous consumption has worsened the issue. Rapid antimicrobial susceptibility testing (AST) to antibiotics can be advantageous in thwarting bacterial infections. Therefore, this study developed a simple nanoliter array-based microfluidic platform for performing rapid AST, which can handle and manipulate liquids both in nanoliter and microliter volumes. The platform consisted of two microfluidic devices, one for performing AST and another for diluting antibiotics and these two were suitably integrated. The microfluidic device used for generating microarrays for AST experiments is single-layered (no air layer) and has no active microvalves and air hole, which makes the device easy to fabricate and use. The loading process ensures uniform distribution of bacteria and relies on displacing the air from microarrays through porous polydimethylsiloxane membranes. Furthermore, the chip for dilution consisted of active microfluidic components, and could prepare and test seven different concentrations of antibiotics, which make the platform multiplexed and be capable of evaluating minimum inhibitory concentrations (MICs), a clinically relevant parameter. MIC determination requires less number of bacteria (â¼2000) and hence shortens the pre-culture step, i.e. bacteria culture in blood and urine. This automated system demonstrated AST and evaluated MICs using Escherichia coli and two antibiotics, including ampicillin and streptomycin, and the results were ascertained using a gold standard method. It only took 8-9 h to perform AST, which is substantially less compared to a conventional process and hence is of high clinical utility.
Asunto(s)
Antibacterianos , Microfluídica , Antibacterianos/farmacología , Escherichia coli , Dispositivos Laboratorio en un Chip , Pruebas de Sensibilidad MicrobianaRESUMEN
Cell-released, membrane-encapsulated extracellular vesicles (EVs) serve as a means of intercellular communication by delivering bioactive cargos including proteins, nucleic acids and lipids. EVs have been widely used for a variety of biomedical applications such as biomarkers for disease diagnosis and drug delivery vehicles for therapy. Herein, this study reports a novel method for label-free, contact-free isolation and recovery of EVs via optically-induced dielectrophoresis (ODEP) on a pneumatically-driven microfluidic platform with minimal human intervention. At an optimal driving frequency of 20 kHz and a voltage of 20 Vpp, an ODEP force from a 75 µm moving light beam was characterized to be 23.5-97.7 fN in 0.2 M sucrose solution. Furthermore, rapid enrichment of EVs with a small volume of only 27 pL in 32 s achieved an increase of 272-fold by dynamically shrinking circular light patterns. Moreover, EVs could be automatically isolated and recovered within 25 min, while achieving a releasing efficiency of 99.8% and a recovery rate of 52.2% by using an integrated microfluidics-based optically-induced EV isolation (OIEV) platform. Given the capacity of label-free, contact-free EV isolation, and automatic, easy-releasing EV recovery, this integrated OIEV platform provides a unique approach for EV-based disease diagnosis and drug delivery applications.
Asunto(s)
Vesículas Extracelulares , Ácidos Nucleicos , Comunicación Celular , Humanos , Lípidos , MicrofluídicaRESUMEN
Since early diagnosis of sepsis may assist clinicians in initiating timely, effective, and prognosis-improving antibiotic therapy, we developed an integrated microfluidic chip (IMC) for rapid isolation of both Gram-positive and Gram-negative bacteria from blood. The device comprised a membrane-based filtration module (90 min operating time), a bacteria-capturing module using a micro-mixer containing magnetic beads coated with "flexible neck" regions of mannose-binding lectin proteins for bacteria capture (20 min), and a miniature polymerase chain reaction (PCR) module for bacteria identification (90 min via TaqMan® probe technology). The filter separated all white blood cells and 99.5% of red blood cells from bacteria, which were captured at rates approaching 85%. The PCR assay's limit of detection was 5 colony-forming units (CFU) per reaction, and the entire process was completed in only 4 h. Since this is far less than that for culture-based approaches, this IMC may serve as a promising device for detection of sepsis.
Asunto(s)
Microfluídica , Sepsis , Antibacterianos , Bacterias , Diagnóstico Precoz , Bacterias Gramnegativas , Bacterias Grampositivas , Humanos , Sepsis/diagnósticoRESUMEN
Circulating extracellular vesicles (EVs), which can contain a wide variety of molecules such as proteins, messenger ribonucleic acids (mRNAs), micro ribonucleic acids (miRNAs) and deoxyribonucleic acids (DNAs) from cells or tissues of origin, have attracted great interest given their potential to serve as biomarkers that can be harvested in body fluids (i.e., relatively non-invasive). Since enrichment and detection of circulating EVs from whole blood have proven challenging, we report herein a fully integrated microfluidic system combining a membrane-based filtration module (i.e. pneumatically-driven microfluidic devices) and a magnetic-bead based immunoassay capable of automating blood treatment, EV enrichment, and EV quantification directly from human whole blood. Three functional modules were implemented; the first, a stirring-enhanced filtration module for separating plasma from blood cells, was characterized by a plasma recovery rate of 65%, a filtrate flow rate of 22 µL min-1, and a vesicle recovery rate of 94% within only 8 min (using 500 µL of blood). The second module, a magnetic bead-based EV enrichment device for immunocapture of circulating EVs from plasma, was characterized by a capture rate of 45%. The final module performed an on-chip enzyme-linked immunosorbent assay for plasma EV quantification in plasma. Given the automated capacity of this system, it could show promise in circulating EV research and clinical point-of-care applications.
Asunto(s)
Vesículas Extracelulares/química , Dispositivos Laboratorio en un Chip , ADN/sangre , ADN/química , Humanos , MicroARNs/sangre , MicroARNs/química , Pruebas en el Punto de Atención , ARN Mensajero/sangre , ARN Mensajero/químicaRESUMEN
An integrated microfluidic system combining 1) an optically-induced-dielectrophoresis (ODEP) module for manipulation of drug-containing particles and 2) an ultraviolet (UV) "direct writing" module capable of patterning hydrogels was established herein for automatic formulation of customized digital drug cocktails. Using the ODEP module, the drug-containing particles were assembled by using moving light patterns generated from a digital projector. The hydrogel, poly(ethylene glycol) diacrylate (PEGDA), was used as the medium in the ODEP module such that the assembled drug-containing particles could be UV-cured and consequently encapsulated in "pills" of specific sizes and shapes by using the UV direct writing module. At an optimal ODEP force of 335 pN, which was achieved in a solution of 15% PEGDA in 0.2 M sucrose, it was possible to manipulate and UV-cure the drug-containing particles. Furthermore, with a digital micromirror device inside the UV direct writing module, different UV patterns could be designed and projected, allowing for the digital drug cocktails to be packaged into different shapes in <60 s. As a demonstration, emulsion droplets containing two different anti-cancer drugs were further tested to show the capability of the developed device. This represents an automatic digital drug cocktail formulating device which stands to revolutionize personalized medicine.
Asunto(s)
Acrilatos/química , Antineoplásicos/química , Hidrogeles/química , Técnicas Analíticas Microfluídicas , Polietilenglicoles/química , Electroforesis , Etopósido/química , Fluorouracilo/química , Humanos , Imagen Óptica , Tamaño de la Partícula , Procesos Fotoquímicos , Medicina de Precisión , Propiedades de Superficie , Rayos UltravioletaRESUMEN
According to World Health Organization reports, cardiovascular diseases (CVDs) are amongst the major causes of death globally and are responsible for over 18 million deaths every year. Traditional detection methods for CVDs include cardiac computerized tomography scans, electrocardiography, and myocardial perfusion imaging scans. Although diagnosis of CVDs through such bio-imaging techniques is common, these methods are relatively costly and cannot detect CVDs in their earliest stages. In contrast, the levels of certain micro RNA (miRNA) biomarkers extracted from extracellular vesicles (EVs) in the bloodstream have been recognized as promising indicators for early CVD detection. However, detection and quantification of miRNA using existing methods are relatively labor-intensive and time-consuming. In this study, a new integrated microfluidic system equipped with highly sensitive field-effect transistors (FETs) was capable of performing EV extraction, EV lysis, target miRNA isolation and miRNA detection within 5 h. The limit of detection was within the physiological range (femtomolar) for two targeted miRNAs, miR-21 and miR-126, meaning that this integrated microfluidic system has the potential to be used as a tool for early detection of CVDs.