A ddPCR platform based on a microfluidic chip with a dual-function flow-focusing structure for sample-to-result DNA quantification analysis.

Droplet digital PCR (ddPCR) is a powerful method for absolute nucleic acid quantification with high precision and accuracy. However, complicated operational steps have hampered the use and diffusion of ddPCR. Therefore, an automated, easy-to-use, low-sample-consumption, and portable ddPCR platform is urgently needed. This paper proposes a microfluidic ddPCR platform based on a microfluidic chip that can realize the sample-to-result function by switching the rotary valve, achieving the dual function of the flow-focusing structure for droplet generation and readout. Sample, generation oil, and analysis oil were pre-added to the reservoirs. Droplets were generated due to focusing flow, and after passing through the integrated temporary storage bin in the rotary valve, the droplets and oil subsequently entered the collecting tube, improving the droplet-to-oil volume ratio for enhanced thermal cycle performance. Droplets with an average diameter of 107.44 µm and a CV of 2.38% were generated using our chip under the optimal pressures. High-performance thermal cycling was achieved through improvements of the droplet-to-oil volume ratio of the sample, the integrated heating lid, the pure copper heating base, and the temperature-controlling algorithm. Gradient quantification experiments were conducted for the HER2 and CEP17 genes extracted from breast cancer cells, yielding strong linear correlations with R2 values of 0.9996 for FAM and 0.9989 for CY5. Moreover, pronounced linearity was obtained between the detected concentrations of HER2 and CEP17, indicated by a slope of 1.0091 and an R2 of 0.9997, signifying consistent HER2 : CEP17 ratios across various sample dilutions. The outcomes of the quantitative analysis, encompassing the dynamic range and the consistency of the HER2 : CEP17 ratio using our ddPCR platform, meet the standards required for breast cancer assessment and therapy. Our ddPCR platform is automated, portable, and capable of stable droplet generation, high-efficiency amplification, realization of the sample-to-result function based on dual-function flow-focusing structure, and accuracy absolute quantification, underscoring its significant potential for ddPCR analysis in clinical diagnostics.

Establishment and Validation of an Integrated Microfluidic Step Emulsification Chip Supporting Droplet Digital Nucleic Acid Analysis.

Uniform and stable droplet generation is critical for accurate and efficient digital nucleic acid analysis (dNAA). In this study, an integrated microfluidic step emulsification device with wide-range droplet generation capability, small device dimensions, convenient fabrication strategy, low contamination and high robustness was developed. A tree-shaped droplet generation nozzle distribution design was proposed to increase the uniformity of droplet generation by equating flow rates, and the flow field in the design was numerically simulated. Theoretical analysis and comparative experiments on droplet size were performed regarding the influences of nozzle dimensions and surface properties. With incubation and hydrophobic reagent treatment, droplets as small as 73.1 µm were generated with multiplex nozzles of 18 µm (h) × 80 µm (w). The droplets were then collected into a standard PCR tube and an on-chip monolayer droplet collection chamber, without manual transfer and sample contamination. The oil-to-sample volume ratio in the PCR tube was recorded during collection. In the end, the droplets generated and collected using the microfluidic device proved to be stable and uniform for nucleic acid amplification and detection. This study provides reliable characteristic information for the design and fabrication of a micro-droplet generation device, and represents a promising approach for the realization of a three-in-one dNAA device under a step emulsification method.

Magnetic Core@Shell Fe3O4@Polypyrrole@Sodium Dodecyl Sulfate Composite for Enhanced Selective Removal of Dyestuffs and Heavy Metal Ions from Complex Wastewater.

Adsorption materials have demonstrated huge potential in treating sewage; however, it is a great challenge to fabricate an adsorbent effectively adsorbing multiple dyestuffs and heavy metal ions simultaneously. Here, a magnetic core@shell Fe3O4@polypyrrole@sodium dodecyl sulfate (Fe3O4@PPy@SDS) composite is prepared through the combination of a hydrothermal method, an in situ polymerization method, and modification, exhibiting enhanced selective removal of five dyestuffs (methylene blue (MB), malachite green (MG), rhodamine B (RhB), Congo red (CR), acid red 1 (AR1)), and heavy metal ions (Mn(VII)). The effects of adsorbent type, time, initial concentration of the adsorbate, and temperature on adsorption performances are investigated in detail. Kinetics and isotherm studies indicate that all adsorption processes are more in line with the pseudo-second-order kinetic model and the Langmuir model, the diffusion behavior is controlled by intraparticle diffusion and liquid film diffusion, and research of thermodynamics reveals a spontaneous endothermic behavior. The removal efficiency after five desorption-adsorption cycles can still reach more than 90%. The prepared Fe3O4@PPy@SDS composite is an efficient and promising renewable adsorbent for the treatment of dyestuffs and Mn(VII), exhibiting a wide range of applications in the field of adsorption.

Numerical Modeling of Temperature-Dependent Cell Membrane Permeability to Water Based on a Microfluidic System with Dynamic Temperature Control.

In order to describe temperature-dependent cell osmotic behaviors in a more reliable method, a novel mathematical mass transfer model coupled with dynamic temperature change has been established based on the combination of a time domain to temperature domain transformation equation and a constant temperature mass transfer model. This novel model is numerically simulated under multiple temperature changing rates and extracellular osmolarities. A microfluidic system that can achieve single-cell osmotic behavior observation and provide dynamic and swift on-chip temperature control was built and tested in this paper. Utilizing the temperature control system, the on-chip heating processes are recorded and then described as polynomial time-temperature relationships. These dynamic temperature changing profiles were performed by obtaining cell membrane properties by parameter fitting only one set of testing experimental data to the mathematical model with a constant temperature changing rate. The numerical modeling results show that predicting the osmotic cell volume change using selected dynamic temperature profiles is more suitable for studies concerning cell membrane permeability determination and cryopreservation process than tests using constant temperature changing rates.

Determination of the Membrane Transport Properties of Jurkat Cells with a Microfluidic Device.

The Jurkat cell is an immortalized line of human acute lymphocyte leukemia cells that is widely used in the study of adoptive cell therapy, a novel treatment of several advanced forms of cancer. The ability to transport water and solutes across the cell membrane under different temperatures is an important factor for deciding the specific protocol for cryopreservation of the Jurkat cell. In this study we propose a comprehensive process for determination of membrane transport properties of Jurkat cell. using a novel microfluidic controlled single cell-trapping system. The osmotic behavior of an individual Jurkat cell to water and dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA), under constant temperature, was recorded under a microscope utilizing the modified microfluidic system. The images of the Jurkat cell under osmotic change were processed to obtain a relationship between cell volume change and time. The experimental results were fitted using a two-parameter transport numeric model to calculate the Jurkat cell membrane permeability to water and DMSO at room temperature (22 °C). This model and the calculated parameters can help scientists optimize the cryopreservation protocol for any cell type with optimal cryoprotective agents and cooling rate for future experiments.

Fiber surface Bragg grating waveguide for refractive index measurements.

A fiber surface Bragg grating waveguide (BGW) fabricated in the surface of single-mode fiber by direct femtosecond laser inscription is demonstrated and successfully applied for refractive index (RI) measurements. Prior to laser inscription of the fiber surface BGW, an X-coupler is first inscribed across the fiber core to couple light from the core to the fiber surface. The light transmitted in the fiber surface BGW efficiently interacts with the surrounding medium due to a strong evanescent field, and obtains an acceptable RI sensitivity approaching ∼16 nm/RIU. The novel design efficiently couples the light guided in the core with the surrounding medium using a non-destructive, single-step micromachining process, and is expected to have potential applications in fiber biochemical sensing.

Automatic sequential fluid handling with multilayer microfluidic sample isolated pumping.

To sequentially handle fluids is of great significance in quantitative biology, analytical chemistry, and bioassays. However, the technological options are limited when building such microfluidic sequential processing systems, and one of the encountered challenges is the need for reliable, efficient, and mass-production available microfluidic pumping methods. Herein, we present a bubble-free and pumping-control unified liquid handling method that is compatible with large-scale manufacture, termed multilayer microfluidic sample isolated pumping (mµSIP). The core part of the mµSIP is the selective permeable membrane that isolates the fluidic layer from the pneumatic layer. The air diffusion from the fluidic channel network into the degassing pneumatic channel network leads to fluidic channel pressure variation, which further results in consistent bubble-free liquid pumping into the channels and the dead-end chambers. We characterize the mµSIP by comparing the fluidic actuation processes with different parameters and a flow rate range of 0.013 µl/s to 0.097 µl/s is observed in the experiments. As the proof of concept, we demonstrate an automatic sequential fluid handling system aiming at digital assays and immunoassays, which further proves the unified pumping-control and suggests that the mµSIP is suitable for functional microfluidic assays with minimal operations. We believe that the mµSIP technology and demonstrated automatic sequential fluid handling system would enrich the microfluidic toolbox and benefit further inventions.