RESUMO
Natural organic matter (NOM) readily interacts with nanoparticles, leading to the formation of NOM corona structures on their surface. NOM corona formation is closely related to the surface coatings and bioavailability of nanoparticles. However, the mechanism underlying NOM corona formation on silver nanoparticles (AgNPs) remains largely unknown due to the lack of effective analytical methods for identifying the changes in the AgNP surface. Herein, the separation ability of biased cyclical electrical field-flow fractionation (BCyElFFF) for same-sized polyvinyl pyrrolidone-coated and poly(ethylene glycol)-coated silver nanoparticles (AgNPs) with different electrophoretic mobilities was evaluated under various electrical conditions. Then, the mechanism behind the NOM corona formation on these AgNP surfaces was elucidated based on the changes in the elution time and off-line characterization of the collected fractions during their elution time in a BCyElFFF run. Finally, the survival rates of E. coli exposed to polyvinyl pyrrolidone-coated and poly(ethylene glycol)-coated AgNPs with or without NOM collected during repeated BCyElFFF runs were observed to increase with increasing NOM concentration, clearly demonstrating the negative effect of NOM corona structures on the bioavailability of AgNPs. These findings highlight the powerful separation and isolation ability of BCyElFFF in studying the transformation and fate of nanoparticles in aqueous environments.
Assuntos
Nanopartículas Metálicas , Prata , Escherichia coli , Polivinil , Polietilenoglicóis , PovidonaRESUMO
Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.
Assuntos
Exossomos , Vesículas Extracelulares , Glioblastoma , Centrifugação , Meios de Cultura , Humanos , UltracentrifugaçãoRESUMO
Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches, yet still results in low sperm retrieval. In this work, a high recovery, high throughput sperm separation process is proposed for the clinical biopsy sperm retrieval process. It is found that sperm have different focusing positions compared with non-sperm cells in the inertial flow, which is explained by a sperm alignment phenomenon. Separation in the spiral channel device results in a 95.6% sperm recovery in which 87.4% of non-sperm cells get removed. Rare sperm isolation from a clinical biopsy sample is performed with the current approach. The chance of finding sperm is shown to increase 8.2 fold in the treated samples. The achieved results highly support this method being used for the development of a rapid biopsy sperm sorting process. In addition, the mechanism was proposed and can be applied for the high-efficiency separation of non-spherical particles in general.
Assuntos
Espermatozoides , Biópsia , Separação Celular , MasculinoRESUMO
In this work, a new high-volume, continuous particle separation device that separates based upon size and charge is described. Two continuous flow-electrical-split-flow lateral transport thin (Fl-El-SPLITT) device architectures (a platinum electrode on a porous membrane and a porous graphite electrode under a membrane) were developed and shown to improve particle separations over a purely electrical-SPLITT device. The graphite FL-El-SPLITT device architecture achieved the best separation of approximately 60% of small (28 nm) vs large (1000 nm) polystyrene particles. Fl-El-SPLITT (platinum) achieved a 75% separation on a single pass using these same particles. Fl-El-SPLITT (platinum) achieved a moderate 26% continuous separation of U87 glioma cell-derived small extracellular vesicles (EVs) from medium EVs. Control parameter testing showed that El-SPLITT continuously directed particle motility within a channel to exit a selected port based upon the applied voltage using either direct current or alternating current. The transition from one port to the other was dependent upon the voltage applied. Both large and small polystyrene particles transitioned together rather than separating at each of the applied voltages. These data present the first ever validation of El-SPLITT in continuous versus batch format. The Fl-El-SPLITT device architecture, monitoring, and electrical and fluid interfacing systems are described in detail for the first time. Capabilities afforded to the system by the flow addition include enhanced particle separation as well as the ability to filter out small particles or desalinate fluids. High-throughput continuous separations based upon electrophoretic mobility will be streamlined by this new technique that combines electrical and flow fields into a single device.
Assuntos
Fracionamento Químico , Eletricidade , Tamanho da Partícula , Fenômenos FísicosRESUMO
Although many properties for small extracellular vesicles (sEVs, formerly termed "exosomes") isolated at â¼100â¯000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed "oncosomes") preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).
Assuntos
Centrifugação/métodos , Fracionamento Químico/métodos , Técnicas Eletroquímicas , Vesículas Extracelulares/química , Glioblastoma/química , Linhagem Celular Tumoral , HumanosRESUMO
Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It is characterized by a lack of spermatogenesis in the seminiferous tubules. Microdissection testicular sperm extraction (microTESE) has significantly improved testicular sperm retrieval rates compared to conventional techniques for NOA. Following testicular biopsy, the sperm is usually non-motile and contained within seminiferous tubules requiring extensive laboratory processing to find individual sperm sufficient for artificial reproductive technologies (ART). Current techniques include mechanical and enzymatic processing which is time-consuming and often damaging to sperm. We review novel techniques that may help improve sperm retrieval rates after microTESE including microfluidics (dielectrophoretic cell sorting, spiral channel sorting, and pinched flow fractionation), fluorescence-activated cell sorting (FACS), and magnetic-activated cell sorting (MACS).
RESUMO
OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.
Assuntos
Azoospermia , Microdissecção , Microfluídica , Técnicas de Reprodução Assistida , Recuperação Espermática/instrumentação , Testículo/patologia , Adulto , Azoospermia/complicações , Azoospermia/diagnóstico , Biópsia/métodos , Técnicas de Laboratório Clínico , Embriologia/métodos , Desenho de Equipamento , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/etiologia , Masculino , Microdissecção/instrumentação , Microdissecção/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Contagem de Espermatozoides , Motilidade dos EspermatozoidesRESUMO
The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at â¼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from â¼-1.6 to -0.1 µm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from â¼-5.5 to -0.1 µm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.
Assuntos
Exossomos/química , Solventes/química , 2-Propanol/química , Soluções Tampão , Linhagem Celular Tumoral , Fracionamento por Campo e Fluxo/métodos , Humanos , Nanopartículas/química , Concentração Osmolar , Fosfatos/química , Poliestirenos/química , Solução Salina/química , Trealose/química , Água/químicaRESUMO
Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively.
Assuntos
Terapia Genética/instrumentação , Terapia Genética/métodos , RNA Interferente Pequeno/farmacologia , Pele , Animais , Feminino , Camundongos , AgulhasRESUMO
Ovarian cancer is the fifth most common cancer affecting US women, killing more women each year than all other gynecologic cancers combined. Treatment of ovarian cancer is challenging with an overall 5-year survival rates of only 28-46% based on the metastatic state of the disease. While overall survival has improved with modern chemotherapy, poor outcomes have persisted. One of the greatest challenges in cancer therapeutic research remains that late-stage drug development trials for drug candidates have high attrition rates, up to 70% in Phase II and 59% in Phase III trials. The development of in vitro, high-throughput, cell based assays could provide a tool to overcome the challenges associated with high attrition rates by allowing for controlled cell deposition with a defined, controlled phenotype. Submerged, three-dimensional (3D) microfluidic printing technology is uniquely capable of controlling cell deposition without sacrificing the viability of cells for cell-based assays. Here, we investigate the phenotypic effects of tube length during printing on the cells. We observe that the length of the tube has minimal effects on the viability and density of A2780 ovarian cancer cells different cell lines. This study details foundational information for developing a high-throughput cell-based assays (CBA) for screening effective cancer drug candidates.
RESUMO
Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for "label-free" isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics.
Assuntos
Exossomos/patologia , Fracionamento por Campo e Fluxo/métodos , Melanoma/patologia , Animais , Linhagem Celular Tumoral , Luz , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Espalhamento de Radiação , Espectrofotometria Ultravioleta/métodosRESUMO
Rare variant enrichment and quantification was achieved by allele-specific, competitive blocker, digital PCR for aiming to provide a noninvasive method for detecting rare DNA variants from circulating cells. The allele-specific blocking chemistry improves sensitivity and lowers assay cost over previously described digital PCR methods while the instrumentation allowed for rapid thermal cycling for faster turnaround time. Because the digital counting of the amplified variants occurs in the presence of many wild-type templates in each well, the method is called "quasi-digital PCR". A spinning disk was used to separate samples into 1000 wells, followed by rapid-cycle, allele-specific amplification in the presence of a molecular beacon that serves as both a blocker and digital indicator. Monte Carlo simulations gave similar results to Poisson distribution statistics for mean number of template molecules and provided an upper and lower bound at a specified confidence level and accounted for input DNA concentration variation. A 111 bp genomic DNA fragment including the BRAF p.V600E mutation (c.T1799A) was amplified with quasi-digital PCR using cycle times of 23 s. Dilution series confirmed that wild-type amplification was suppressed and that the sensitivity for the mutant allele was <0.01 % (43 mutant alleles amongst 500,000 wild-type alleles). The Monte Carlo method presented here is publically available on the internet and can calculate target concentration given digital data or predict digital data given target concentration.
Assuntos
Variações do Número de Cópias de DNA , Primers do DNA/genética , DNA/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Alelos , Simulação por Computador , DNA/genética , Biblioteca Gênica , Genômica/métodos , Humanos , Método de Monte Carlo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Impressão Tridimensional , Análise Serial de Tecidos/métodos , Animais , Camundongos , Células NIH 3T3 , Análise Serial de Tecidos/instrumentaçãoRESUMO
Diagnostic assays implemented in microfluidic devices have developed rapidly over the past decade and are expected to become commonplace in the next few years. Hundreds of microfluidics-based approaches towards clinical diagnostics and pathogen detection have been reported with a general theme of rapid and customizable assays that are potentially cost-effective. This chapter reviews microfluidics in molecular diagnostics based on application areas with a concise review of microfluidics in general. Basic principles of microfabrication are briefly reviewed and the transition to polymer fabricated devices is discussed. Most current microfluidic diagnostic devices are designed to target a single disease, such as a given cancer or a variety of pathogens, and there will likely be a large market for these focused devices; however, the future of molecular diagnostics lies in highly multiplexed microfluidic devices that can screen for potentially hundreds of diseases simultaneously.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Animais , Bactérias/isolamento & purificação , Biomarcadores Tumorais/análise , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Saúde Pública/estatística & dados numéricos , Vírus/isolamento & purificaçãoRESUMO
Characterization of polymerized liposomes (PolyPIPosomes) was carried out using a combination of normal dc electrical field-flow fractionation and cyclical electrical field-flow fractionation (CyElFFF) as an analytical technique. The constant nature of the carrier fluid and channel configuration for this technique eliminates many variables associated with multidimensional analysis. CyElFFF uses an oscillating field to induce separation and is performed in the same channel as standard dc electrical field-flow fractionation separation. Theory and experimental methods to characterize nanoparticles in terms of their sizes and electrophoretic mobilities are discussed in this paper. Polystyrene nanoparticles are used for system calibration and characterization of the separation performance, whereas polymerized liposomes are used to demonstrate the applicability of the system to biomedical samples. This paper is also the first to report separation and a higher effective field when CyElFFF is operated at very low applied voltages. The technique is shown to have the ability to quantify both particle size and electrophoretic mobility distributions for colloidal polystyrene nanoparticles and PolyPIPosomes.
Assuntos
Campos Eletromagnéticos , Fracionamento por Campo e Fluxo , Lipossomos/análise , Lipossomos/síntese química , Tamanho da Partícula , PolimerizaçãoRESUMO
A diffusion Split-Flow Thin Cell (SPLITT) system was used to partially remove small peptides such as ß2 microglobulin (ß2M) and parathyroid hormone (PTH) in a continuous manner from an input flow stream while preserving most (over 97%) of the larger protein in the sample, such as albumin. To help determine the operating conditions for this work, a two-dimensional numerical model based on the Navier-Stokes equation and convection-diffusion equations was developed for diffusional SPLITT using COMSOL multiphysics software (COMSOL Inc., MA). These simulations were used to obtain the relationship between important operational parameters and the purification efficiency for proteins of interest. The diffusion-based SPLITT system was fabricated using xurography and was used to demonstrate protein purification based on the differences in size or diffusion coefficient of the sample. The results obtained from the experiments are compared with the mathematical model and show good agreement, while the variations between these results are discussed. The results show that significant portions of small peptides (>25%) can be removed while preserving larger proteins (up to 95%) in the carrier stream. A potential application of this technique is to be used as an additional step in kidney dialysis to remove toxins that are not effectively removed by current dialysis protocols.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Proteínas/isolamento & purificação , Simulação por Computador , Diálise/instrumentação , Diálise/métodos , Difusão , Modelos Teóricos , Peptídeos/isolamento & purificaçãoRESUMO
We describe an approach to cataract phacoemulsification that uses the carouseling technique within the capsular bag. This is made possible by a newly designed phacoemulsification tip with 3 unique modifications: a 20-degree right bend in the tip, a semicircular opening, and a third irrigation port. These 3 features facilitate the carouseling technique of phacoemulsification without expressing the lens into the anterior chamber. The method decreases corneal endothelial injury by maximizing the distance between the delivered thermal energy and the corneal endothelium. The preoperative and postoperative pachymetry and endothelial cell counts in the first 8 patients treated using this technique are reported.
Assuntos
Facoemulsificação/métodos , Idoso , Contagem de Células , Perda de Células Endoteliais da Córnea/prevenção & controle , Endotélio Corneano/patologia , Feminino , Humanos , Complicações Intraoperatórias/prevenção & controle , Cápsula do Cristalino/cirurgia , Pessoa de Meia-Idade , Facoemulsificação/instrumentação , Estudos RetrospectivosRESUMO
Biomolecules, including protein A, albumin, and immunoglobulin G, are spotted on top of a nanoporous substrate by using a continuous-flow microspotter (CFM) system, which normally produces spots 3 to 4 orders of magnitude more sensitive than conventional biomolecule printing methods. The spots are observed with a fluorescence scanner. By using the CFM to print spots on nanoporous substrates, an additional order of magnitude increase in signal is observed, which leads to high signal-to-background ratios, highly saturated spots, and a measurable signal at printing concentrations as low as 1.6 ng mL(-1). This technique produces highly concentrated biomolecular spots from dilute samples and significantly increases the sensitivity of sensing platforms.
Assuntos
Óxido de Alumínio/química , Nanopartículas/química , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Reologia , Animais , Bovinos , Fluorescência , Tamanho da Partícula , Porosidade , Propilaminas , Soroalbumina Bovina/análise , Silanos/química , Proteína Estafilocócica A/análise , Propriedades de SuperfícieRESUMO
Treatment of age-macular degeneration requires monthly intravitreal injections, which are costly and have serious risks. The objective of this study was to develop a novel intraocular implant for drug delivery. The capsule drug ring is a reservoir inserted in the lens capsule during cataract surgery, refillable and capable of delivering multiple drugs. Avastin was the drug of interest in this study. Prototypes were manufactured using polymethylmethacrylate sheets as the reservoir material, a semi-permeable membrane for controlled delivery and silicone check valves for refilling. The device showed near zero-order release kinetics and Avastin stability was investigated with accelerated temperature studies.