RESUMEN
Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost, long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) and then filled inside the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained from Viola ignobilis by a low voltage electric field extraction method and characterized by HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can find widespread applications in drug discovery research.
Asunto(s)
Ciclotidas , Viola , Secuencia de Aminoácidos , Ciclotidas/análisis , Ciclotidas/química , Gel de Sílice , Microfluídica , Viola/química , Extractos VegetalesRESUMEN
Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.
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Alérgenos/análisis , Arachis/inmunología , Espectrometría de Masas/métodos , Microfluídica/métodos , Arachis/química , Análisis de los Alimentos/métodos , Hipersensibilidad al Cacahuete/etiología , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunologíaRESUMEN
Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary, air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes and can be used to selectively migrate target cells. As a proof of principle, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique expands the acoustic cell manipulation possibilities and offers cell-sorting solutions suited for applications at point of care.
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Acústica , Separación Celular/instrumentación , Dispositivos Laboratorio en un Chip , Microburbujas , Diseño de EquipoRESUMEN
We describe a tandem affinity microfluidic separation that measures the ratio of CD4+/CD8+ T lymphocytes from blood samples. It is performed by injecting 2 µL of lysed blood samples at 1800-2700 cells µL(-1) into a microfluidic device containing two serially linked affinity regions, followed with a stop flow incubation that captures CD4+/CD8+ T lymphocytes on the corresponding affinity regions. Fluorophore conjugated antibodies are then injected at a controlled shear stress of 1.7 dyn cm(-2) to label target cells while eluting non-specific cells; and at last the CD4/CD8 ratio is calculated after the cell enumeration. The ratio of CD4+/CD8+ T lymphocytes achieved by our tandem affinity microfluidic system was in close agreement with that performed using conventional flow cytometry (R (2) = 0.97) over a wide range (0.4-2.5) that covered the reference values from immune deficient patients to healthy people. This approach may represent an inexpensive and powerful tool in diagnosis of immunodeficiency disorders including HIV or mycobacterium tuberculosis.
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Relación CD4-CD8 , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Microfluídica/métodos , Humanos , Dispositivos Laboratorio en un Chip , Valores de ReferenciaRESUMEN
A new microfluidic method of particle separation was proposed and studied theoretically in this paper. This method is based on the induced charge electro-osmotic flow (ICEOF) and polarizability of dielectric particles. In this method, a pair of metal plates is embedded on the side channel walls to create a region of circulating flows under applied electric field. When a dielectric particle enters this region, the vortices produced by ICEOF around the particle will interact with the circulating flows produced by the metal plates. Such hydrodynamic interaction influences the particle's trajectory, and may result in the particle being trapped in the flow circulating zone or passing through this flow circulating zone. Because the hydrodynamic interaction is sensitive to the applied electric field, and the polarizability and the size of the particles, separation of different particles can be realized by controlling these parameters. Comparing with electrophoresis and dielectrophoresis methods, this strategy presented in this paper is simple and sensitive.
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Electroósmosis/métodos , Técnicas Analíticas Microfluídicas/métodos , Simulación por Computador , Electricidad , Modelos Teóricos , Tamaño de la PartículaRESUMEN
With the development of in vitro diagnostics, extracting submicron scale particles from mixed body fluids samples is crucial. In recent years, microfluidic separation has attracted much attention due to its high efficiency, label-free, and inexpensive nature. Among the microfluidic-based separation, the separation based on ultrasonic standing waves has gradually become a powerful tool. A microfluid environment containing a tilted-angle ultrasonic standing surface acoustic wave (taSSAW) field has been widely adapted and designed to separate submicron particles for biochemical applications. This paper investigated submicron particle defection in microfluidics using taSSAWs analytically. Particles with 0.1-1 µm diameters were analyzed under acoustic pressure, flow rate, tilted angle, and SSAW frequency. According to different acoustic radiation forces acting on the particles, the motion of large-diameter particles was more likely to deflect to the direction of the nodal lines. Decreasing the input flow rate or increasing acoustic pressure and acoustic wave frequency can improve particle deflection. The tilted angle can be optimized by analyzing the simulation results. Based on the simulation analysis, we experimentally showed the separation of polystyrene microspheres (100 nm) from the mixed particles and exosomes (30-150 nm) from human plasma. This research results can provide a certain reference for the practical design of bioparticle separation utilizing acoustofluidic devices.
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Exosomes, with diameters ranging from 30 to 150 nm, are saucer-shaped extracellular vesicles (EVs) secreted by various type of human cells. They are present in virtually all bodily fluids. Owing to their abundant nucleic acid and protein content, exosomes have emerged as promising biomarkers for noninvasive molecular diagnostics. However, the need for exosome separation purification presents tremendous technical challenges due to their minuscule size. In recent years, microfluidic technology has garnered substantial interest as a promising alternative capable of excellent separation performance, reduced reagent consumption, and lower overall device and operation costs. In this context, we hereby propose a novel microfluidic strategy based on thermally oxidized deterministic lateral displacement (DLD) arrays with tapered shapes to enhance separation performance. We have achieved more than 90% purity in both polystyrene nanoparticle and exosome experiments. The use of thermal oxidation also significantly reduces fabrication complexity by avoiding the use of high-precision lithography. Furthermore, in a simulation model, we attempt to integrate the use of dielectrophoresis (DEP) to overcome the size-based nature of DLD and distinguish particles that are close in size but differ in biochemical compositions (e.g., lipoproteins, exomeres, retroviruses). We believe the proposed strategy heralds a versatile and innovative platform poised to enhance exosome analysis across a spectrum of biochemical applications.
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Electroforesis , Exosomas , Humanos , Técnicas Analíticas Microfluídicas , Microfluídica , Nanopartículas/química , Oxidación-ReducciónRESUMEN
Cyclotides as a cyclic peptide produced by different groups of plants have been a very attractive field of research due to their exceptional properties in biological activities and drug design applications. The importance of cyclotides as new biological activities from nature caused to attract researchers to develop new separation systems. Recent growth and development on chip-based technology for separation and bioassay especially for anticancer having sparklingly advantages comparison with common traditional methods. In this study, the microfluidic separation of Vigno 1-5 cyclotides extracted from Viola ignobilis by using polar and nonpolar forces as a liquid-liquid interaction was investigated through modified microfluidic chips and then the results were compared with a traditional counterpart technique of high-performance liquid chromatography (HPLC). The traditional process of separating cyclotides from plants is a costly and time-consuming procedure. The scientific novelty of this study is to accelerate the separation of cyclotides using modified microfluidic chips with low cost and high efficiency. The results revealed that a novel and simple microfluidic chip concept is an effective approach for separating the Vigno groups in the violet extract. We believe that the concept could potentially be utilized for further drug development process especially for anticancer studies by coupling bioassay chips as online procedures via reducing in time and cost compared with traditional offline methods.
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Length-based separation of DNA remains as relevant today as when gel electrophoresis was introduced almost 100 years ago. While new, long-read genomics technologies have revolutionised accessibility to powerful genomic data, the preparation of samples has not proceeded at the same pace, with sample preparation often constituting a considerable bottleneck, both in time and difficulty. Microfluidics holds great potential for automated, sample-to-answer analysis via the integration of preparatory and analytical steps, but for this to be fully realised, more versatile, powerful and integrable unit operations, such as separation, are essential. We demonstrate the displacement and separation of DNA with a throughput that is one to five orders of magnitude greater than other microfluidic techniques. Using a device with a small footprint (23 mm × 0.5 mm), and with feature sizes in the micrometre range, it is considerably easier to fabricate than parallelized nano-array-based approaches. We show the separation of 48.5 kbp and 166 kbp DNA strands achieving a significantly improved throughput of 760 ng/h, compared to previous work and the separation of low concentrations of 48.5 kbp DNA molecules from a massive background of sub 10 kbp fragments. We show that the extension of DNA molecules at high flow velocities, generally believed to make the length-based separation of long DNA difficult, does not place the ultimate limitation on our method. Instead, we explore the effects of polymer rotations and intermolecular interactions at extremely high DNA concentrations and postulate that these may have both negative and positive influences on the separation depending on the detailed experimental conditions.
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Isolation of microparticles and biological cells on microfluidic chips has received considerable attention due to their applications in numerous areas such as medical and engineering fields. Microparticles separation is of great importance in bioassays due to the need for smaller sample and device size and lower manufacturing costs. In this study, we first explain the concepts of separation and microfluidic science along with their applications in the medical sciences, and then, a conceptual design of a novel inertial microfluidic system is proposed and analyzed. The PDMS spiral microfluidic device was fabricated, and its effects on the separation of particles with sizes similar to biological particles were experimentally analyzed. This separation technique can be used to separate cancer cells from the normal ones in the blood samples. These components required for testing were selected, assembled, and finally, a very affordable microfluidic kit was provided. Different experiments were designed, and the results were analyzed using appropriate software and methods. Separator system tests with polydisperse hollow glass particles (diameter 2-20 µm), and monodisperse Polystyrene particles (diameter 5 & 15 µm), and the results exhibit an acceptable chip performance with 86% of efficiency for both monodisperse particles and polydisperse particles. The microchannel collects particles with an average diameter of 15.8, 9.4, and 5.9 µm at the proposed reservoirs. This chip can be integrated into a more extensive point-of-care diagnostic system to test blood samples.
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Dispositivos Laboratorio en un Chip , Microfluídica , Separación CelularRESUMEN
Isolation of circulating tumor cells (CTCs) from blood samples has important prognostic and therapeutic implications for cancer treatments, but the process is very challenging due to the low concentration of CTCs. In this study, we report a novel 3D printed microfluidic device functionalized with anti-EpCAM (epithelial cell adhesion molecule) antibodies to isolate CTCs from human blood samples. A 3D printing technology was utilized with specially designed interior structures to fabricate a microfluidic device with high surface area and fluid flow manipulation, increasing capture efficiency of tumor cells. These devices with the optimal flow rate (1 mL/h) and channel length (2 cm) were demonstrated to test three kinds of EpCAM positive cancer cell lines (MCF-7 breast cancer, SW480 colon cancer, and PC3 prostate cancer), and one kind of EpCAM negative cancer cell line (293T kidney cancer). Experimentally, the capture efficiency higher than 90% has been achieved, and the isolation of MCF-7 tumor cells from spiked human blood samples has also been demonstrated. Combined with DNA-based detection (e.g. polymerase chain reaction or DNA sequencing), the detection and analysis of released DNAs from captured tumor cells could be another future direction for clinical diagnosis and cancer treatment.
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Separación Celular/instrumentación , Dispositivos Laboratorio en un Chip , Células Neoplásicas Circulantes/patología , Anticuerpos Inmovilizados/química , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Humanos , Neoplasias/sangre , Neoplasias/patología , Células Neoplásicas Circulantes/química , Impresión TridimensionalRESUMEN
Isoelectric focusing (IEF) is an electrophoretic technique that enables the separation of proteins based on their isoelectric points. Until recently, this valuable method was not feasible for single-cell applications, which are necessary to interrogate heterogeneous cell populations. Herein we highlight a recently published method enabling the analysis of single-cell proteomics, which utilizes microfluidics coupled with IEF, photocapture, and immunoprobing of the protein in the same micro-gel, which can be stripped and reprobed multiple times.