Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

© 2017, National Academy of Sciences. All rights reserved.Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity

Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX.Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients’ blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses.Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression.Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes

Dissolution and mixing of flavin mononucleotide in microfluidic chips for bioassay

Dissolution and mixing of flavin mononucleotide (FMN), which activates a luminescent reaction, were considered in various designs of microfluidic chip for pollution analysis of liquid samples. The aim was to determine the velocity mode of fluid flow ensured the uniform distribution of the FMN in the reaction chamber. Simulation of concentration distribution of FMN in various designs of microfluidic chips was conducted. It was shown that the passive mixing techniques based on the constant flow rate didn’t provide mixing of FMN in acceptable time (3 seconds). The most efficient mixing was achieved using variable flow rate with a gradually increasing frequency of oscillation

Si doped GaP layers grown on Si wafers by low temperature PE-ALD

International audienceLow-temperature plasma enhanced atomic layer deposition (PE-ALD) was successfully used to grow silicon (Si) doped amorphous and microcrystalline gallium phosphide (GaP) layers onto p-type Si wafers for the fabrication of n-GaP/p-Si heterojunction solar cells. PE-ALD was realized at 380 °C with continuous H2 plasma discharge and the alternate use of phosphine and trimethylgallium as sources of P and Ga atoms, respectively. The layers were doped with silicon thanks to silane (SiH4) diluted in H2 that was introduced as a separated step. High SiH4 dilution in H2 (0.1%) allows us to deposit stoichiometric GaP layers. Hall measurements performed on the GaP:Si/p-Si structures reveal the presence of an n-type layer with a sheet electron density of 6–10 × 1013 cm−2 and an electron mobility of 13–25 cm2 V−1 s−1 at 300 K. This is associated with the formation of a strong inversion layer in the p-Si substrate due to strong band bending at the GaP/Si interface. GaP:Si/p-Si heterostructures exhibit a clear photovoltaic effect, with the performance being currently limited by the poor quality of the p-Si wafers and reflection losses at the GaP surface. This opens interesting perspectives for Si doped GaP deposited by PE-ALD for the fabrication of p-Si based heterojunction solar cells

Analytical Enzymatic Reactions in Microfluidic Chips

Текст статьи не публикуется в открытом доступе в соответствии с политикой журнала.A number of approaches have been proposed and tested to transfer enzymatic reactions into the functional elements of microfluidic chips on the example of the bienzyme bioluminescent reaction involving NAD(P)H:FMN-oxidoreductase and luciferase. Measurement of the catalytic activity of these enzymes (under the influence of pollutants) is the basis of enzymatic bioassay of various liquids. It was found that all of the components of the reaction must be placed in the same cell of the chip to improve the reproducibility of the measurements. The use of starch gel as a carrier for immobilization and gelatin as a scaffold in the reactor of the chip enables the preservation of enzyme activity in the course of sealing the chip at room temperature. It is shown that the components of the reaction should be vigorously stirred in a microfluidic chip reactor to improve the efficiency of the analysis. As a result of the studies, a prototype of microfluidic chip based on the enzymatic bioluminescent reaction is proposed. It is characterized by a detection limit of copper sulfate of 3 μM that corresponds to the sensitivity of traditional lux-biosensors based on living cells. The analysis time is reduced to 1 min, and the analysis can be performed by individuals without special laboratory skills

Dissolution and mixing of flavin mononucleotide in microfluidic chips for bioassay

Dissolution and mixing of flavin mononucleotide (FMN), which activates a luminescent reaction, were considered in various designs of microfluidic chip for pollution analysis of liquid samples. The aim was to determine the velocity mode of fluid flow ensured the uniform distribution of the FMN in the reaction chamber. Simulation of concentration distribution of FMN in various designs of microfluidic chips was conducted. It was shown that the passive mixing techniques based on the constant flow rate didn't provide mixing of FMN in acceptable time (3 seconds). The most efficient mixing was achieved using variable flow rate with a gradually increasing frequency of oscillation

Dissolution and mixing of flavin mononucleotide in microfluidic chips for bioassay

Dissolution and mixing of flavin mononucleotide (FMN), which activates a luminescent reaction, were considered in various designs of microfluidic chip for pollution analysis of liquid samples. The aim was to determine the velocity mode of fluid flow ensured the uniform distribution of the FMN in the reaction chamber. Simulation of concentration distribution of FMN in various designs of microfluidic chips was conducted. It was shown that the passive mixing techniques based on the constant flow rate didn’t provide mixing of FMN in acceptable time (3 seconds). The most efficient mixing was achieved using variable flow rate with a gradually increasing frequency of oscillation