RESUMO
Digital PCR is a powerful method for absolute nucleic acid quantification and is widely used in the absolute quantification of viral copy numbers, tumor marker detection, and prenatal diagnosis. However, for most of the existing droplet-based dPCR systems, the droplet generation, PCR reaction, and droplet detection are performed separately using different instruments. Making digital PCR both easy to use and practical by integrating the qPCR workflow into a superior all-in-one walkaway solution is one of the core ideas. A new innovative and integrated digital droplet PCR platform was developed that utilizes cutting-edge microfluidics to integrate dPCR workflows onto a single consumable chip. This makes previously complex workflows fast and simple; the whole process of droplet generation, PCR amplification, and droplet detection is completed on one chip, which meets the clinical requirement of "sample in, result out". It provides high multiplexing capabilities and strong sensitivity while all measurements were within the 95% confidence interval. This study is the first validation of the DropXpert S6 system and focuses primarily on verifying its reliability, repeatability, and consistency. In addition, the accuracy, detection limit, linearity, and precision of the system were evaluated after sample collection. Among them, the accuracy assessment by calculating the absolute bias of each target gene yielded a range from -0.1 to 0.08, all within ±0.5 logarithmic orders of magnitude; the LOB for the assay was set at 0, and the LoD value calculated using probit curves is MR4.7 (0.002%); the linearity evaluation showed that the R2 value of the BCR-ABL was 0.9996, and the R2 value of the ABL metrics calculated using the ERM standard was 0.9999; and the precision evaluation showed that all samples had a CV of less than 4% for intra-day, inter-day, and inter-instrument variation. The CV of inter-batch variation was less than 7%. The total CV was less than 5%. The results of the study demonstrate that dd-PCR can be applied to molecular detection and the clinical evaluation of CML patients and provide more precise personal treatment guidance, and its reproducibility predicts the future development of a wide range of clinical applications.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Dispositivos Lab-On-A-Chip , Proteínas de Fusão bcr-abl/genética , Reação em Cadeia da Polimerase , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers' surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug's effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research.
Assuntos
Autofagia/efeitos dos fármacos , Técnicas de Cultura de Células/instrumentação , Dimetilpolisiloxanos/farmacologia , Técnicas Analíticas Microfluídicas/instrumentação , Tamoxifeno/farmacologia , Adsorção , Proliferação de Células/efeitos dos fármacos , Dimetilpolisiloxanos/química , Humanos , Limite de Detecção , Modelos Lineares , Células MCF-7 , Microscopia de Fluorescência , Polímeros/química , Polímeros/farmacologia , Reprodutibilidade dos Testes , Xilenos/química , Xilenos/farmacologiaRESUMO
We demonstrate that when using cell-laden core-shell hydrogel beads to support the generation of tumor spheroids, the shell structure reduces the out-of-bead and monolayer cell proliferation that occurs during long-term culture of tumor cells within core-only alginate beads. We fabricate core-shell beads in a two-step process using simple, one-layer microfluidic devices. Tumor cells encapsulated within the bead core will proliferate to form multicellular aggregates which can serve as three-dimensional (3-D) models of tumors in drug screening. Encapsulation in an alginate shell increased the time that cells could be maintained in three-dimensional culture for MCF-7 breast cancer cells prior to out-of-bead proliferation, permitting formation of spheroids over a period of 14 days without the need move the cell-laden beads to clean culture flasks to separate beads from underlying monolayers. Tamoxifen and docetaxel dose response shows decreased toxicity for multicellular aggregates in three-dimensional core-shell bead culture compared to monolayer. Using simple core-only beads gives mixed monolayer and 3-D culture during drug screening, and alters the treatment result compared to the 3-D core-shell or the 2-D monolayer groups, as measured by standard proliferation assay. By preventing the out-of-bead proliferation and subsequent monolayer formation that is observed with core-only beads, the core-shell structure can obviate the requirement to transfer the beads to a new culture flask during drug screening, an important consideration for cell-based drug screening and drugs which have high multicellular resistance index.
Assuntos
Alginatos/química , Técnicas de Cultura de Células/instrumentação , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Técnicas de Cultura de Células/métodos , Proliferação de Células , Docetaxel , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Desenho de Equipamento , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Hidrogéis , Dispositivos Lab-On-A-Chip , Células MCF-7/efeitos dos fármacos , Microesferas , Tamoxifeno/farmacologia , Taxoides/farmacologiaRESUMO
We have demonstrated an integrated platform for microfluidics and chemiluminescence (CL) detection that is capable of parallel cell culture, convenient liquid manipulation, and sensitive chemiluminescent detection. Luminol-dependent CL responses induced by three different stimuli, phytohemagglutinin (PHA), concanavalin A (ConA), and lipopolysaccharides (LPS), which can evoke a CL response in macrophages, were evaluated on this microfluidic chip. We studied the dose-dependent effect of these three stimuli on CL response in murine macrophages. PHA produced the highest CL response compared to LPS and ConA. The CL intensity produced by PHA was 6.85 and four times higher than that by LPS and ConA, respectively, at the low concentration of 100 µg/ml. We have found microfluidic based CL to be a very useful screening tool, which is less laborious and more sensitive. This microfluidic system is disposable and capable of rapid device prototyping; it may prove to be very useful in clinical and pharmaceutical applications.
Assuntos
Luminol/química , Macrófagos/efeitos dos fármacos , Animais , Técnicas de Cultura de Células/métodos , Concanavalina A/farmacologia , Relação Dose-Resposta a Droga , Lipopolissacarídeos/farmacologia , Luminescência , Substâncias Luminescentes/química , Medições Luminescentes , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Fito-Hemaglutininas/farmacologiaRESUMO
Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was observed. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain constant location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Técnicas Analíticas Microfluídicas , Neoplasias/patologia , Esferoides Celulares/patologia , Alginatos/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Ensaios de Triagem em Larga Escala , Humanos , Microscopia Confocal , Microesferas , Esferoides Celulares/efeitos dos fármacosRESUMO
This paper demonstrated the chemical analysis of single cell on a cross PDMS microfluidic chip in a simple, fast, and high-throughput mode. The pre-stained cells were sequentially loaded into the cross section by hydrodynamic force, lysed by 0.2% SDS and subsequently the lysates were detected by LIF. Each cell can be lysed within 500 ms due to its high concentration of SDS at cross section resulted from the absence of electroosmosis after surface coating in microchannel. The reliability and quality of the analysis was confirmed by analysis of glutathione and rhodamine 123 in single K562 cells. In each run, approximately 100 cells could be analyzed in about 10 min, which demonstrated the comparatively high throughput. The proposed microfluidic method is simple, fast, and high throughput, which might be of significance in identifying the biological molecules involved in fast biochemical processes and studying heterogenous cells.
Assuntos
Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Nylons/química , Glutationa/análise , Humanos , Células K562 , Técnicas Analíticas Microfluídicas/instrumentação , Rodamina 123/análiseRESUMO
In this work, the electrophoretic mobility (EPM) measurement of individual cells was investigated by a simple on-chip electrophoresis system with LIF multipoint detection. The system enabled the characterization of cell electrophoresis behavior as well as the fluorescence signal from individual cells simultaneously. The measurement yielded the electropherograms of a large number of cells labeled with dye, in which the migration time and migration distance could be obtained easily. The EPM has been demonstrated to be different between the K562 cells and K562 cells treated with anticancer drug arsenic trioxide (As2O3). The K562 cells were found to exhibit a lower EPM compared to the cells after drug addition with different concentration. In this preliminary study, over 300 cells could be analyzed within 2 h, demonstrated a much higher analysis throughput compared with traditional methods. The established system is simple and fast, which is expected to be a promising method for evaluating cell surface properties and to be useful in clinical and pharmaceutical applications.
Assuntos
Eletroforese em Microchip/métodos , Espectrometria de Fluorescência/métodos , Humanos , Células K562 , LasersRESUMO
Microchip-based packed column SPE of DNA was performed using the microfabricated two-weir structure within a microchannel. We developed two methods to fabricate the two-weir structured glass chips: a "two-side etching/alignment" method and a simplified "one-side/two-step etching" method. The former method required a straightforward alignment step, while the latter approach comprised a simplified wet etching process using paraffin wax as the temporary protective layer. Both methods were convenient and rapid as compared to the previous approaches. Through a reversibly sealed bead-introduction channel, beads can be fed into or out of the chip columns, thus enabling refreshment of the packing materials. Using the proposed chip columns, highly efficient lambda-DNA extractions (average recovery >80%) were performed with good chip-to-chip reproducibility (RSD <10%). The on-chip SPE procedure was completed within 15 min at the flow rate of 3 microL/min and the bulk of the loaded DNA was eluted within a small volume of approximately 8 microL. Application of the microchip-based packed columns was demonstrated by purifying PCR-amplifiable genomic DNA from human hepatocellular carcinoma (HepG2) cells and human whole blood samples.
Assuntos
DNA/isolamento & purificação , Eletroforese em Microchip/instrumentação , Microextração em Fase Sólida/instrumentação , Bacteriófago lambda/química , Linhagem Celular , DNA/sangue , DNA Viral/isolamento & purificação , Eletroforese em Microchip/métodos , Desenho de Equipamento , Humanos , Microextração em Fase Sólida/métodosRESUMO
A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.