Droplet-based Microfluidic Chip Research Articles

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus.

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per μL to 2500 copies per μL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.

Analysis of Phosphatase Activity in a Droplet-Based Microfluidic Chip.

We report analysis of phosphatase activity and inhibition on droplet-based microfluidic chips. Phosphatases are such attractive potential drug targets because abnormal phosphatase activity has been implicated in a variety of diseases including cancer, neurological disorders, diabetes, osteoporosis, and obesity. So far, several methods for assessing phosphatase activity have been reported. However, they require a large sample volume and additional chemical modifications such as fluorescent dye conjugation and nanomaterial conjugation, and are not cost-effective. In this study, we used an artificial phosphatase substrate 3-O-methylfluorescein phosphate as a fluorescent reporter and dual specificity phosphatase 22. Using these materials, the phosphatase assay was performed from approximately 340.4 picoliter (pL) droplets generated at a frequency of ~40 hertz (Hz) in a droplet-based microfluidic chip. To evaluate the suitability of droplet-based platform for screening phosphatase inhibitors, a dose–response inhibition study was performed with ethyl-3,4-dephostatin and the half-maximal inhibitory concentration (IC50) was calculated as 5.79 ± 1.09 μM. The droplet-based results were compared to microplate-based experiments, which showed agreement. The droplet-based phosphatase assay proposed here is simple, reproducible, and generates enormous data sets within the limited sample and reagent volumes.

Easy-to-Operate Co-Flow Step Emulsification Device for High-Throughput Three-Dimensional Cell Culture.

Cell culture plays an essential role in tissue engineering and high-throughput drug screening. Compared with two-dimensional (2D) in vitro culture, three-dimensional (3D) in vitro culture can mimic cells in vivo more accurately, including complex cellular organizations, heterogeneity, and cell–extracellular matrix (ECM) interactions. This article presents a droplet-based microfluidic chip that integrates cell distribution, 3D in vitro cell culture, and in situ cell monitoring in a single device. Using the microfluidic “co-flow step emulsification” approach, we have successfully prepared close-packed droplet arrays with an ultra-high-volume fraction (72%) which can prevent cells from adhering to the chip surface so as to achieve a 3D cell culture and make scalable and high-throughput cell culture possible. The proposed device could produce droplets from 55.29 ± 1.52 to 95.64 ± 3.35 μm, enabling the diverse encapsulation of cells of different sizes and quantities. Furthermore, the cost for each microfluidic CFSE chip is approximately USD 3, making it a low-cost approach for 3D cell culture. The proposed device is successfully applied in the 3D culture of saccharomyces cerevisiae cells with an occurrence rate for proliferation of 80.34 ± 3.77%. With low-cost, easy-to-operate, high-throughput, and miniaturization characteristics, the proposed device meets the requirements for 3D in vitro cell culture and is expected to be applied in biological fields such as drug toxicology and pharmacokinetics.

Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification.

DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.

Facile fabrication of drug-loaded PEGDA microcapsules for drug evaluation using droplet-based microchip

Droplet-based microfluidic technology can be utilized as a microreactor to prepare novel functional monodisperse microcapsules. In this study, a droplet-based microfluidic chip with surface modification, which allowed the one-step preparation of double emulsion microcapsules. An O/W/O double emulsion using polyethylene (glycol) diacrylate (PEGDA) solution as the intermediate water phase was prepared by regulating the hydrophilicity and hydrophobicity of the chip surface, with PEGDA microcapsules prepared using UV polymerization. And then anti-tumor drug paclitaxel and neurotoxin 6-OHDA were encapsulated in microcapsules for drug and toxicology evaluation, respectively. Compared to controls, drug-loaded microcapsules caused a significant increase in the death rate of PC12 cells. This indicates that the obtained drug-loaded microcapsules could be used in drug evaluation and potentially in drug screening and delivery.

Droplet-based Synthesis of Homogeneous Gold Nanoparticles for Enhancing HRP-based ELISA Signals

Gold nanoparticles (AuNPs) have widely used for various biological applications, such as drug screening, photo-thermal therapy, and biosensing. In particular, the synthesis of AuNPs with narrow size distribution plays an important role in increasing the efficiency of nanoparticle-mediated biosensors. However, the conventional synthesis methods (e.g., citrate reduction method) still suffer from controlling the sizes of the nanoparticles. In this paper, we present the synthesis method of homogeneous AuNPs using a droplet-based microfluidic chip. Prior to experiments, we optimized the size of droplets using a simulation software for stable droplet generation. We demonstrated that the nanoparticles synthesized in our microfluidic chip system showed a narrower size distribution and a higher reproducibility compared to conventional batch synthesis. Furthermore, we observed that the signal of anti-horseradish peroxidase (HRP) was significantly enhanced by the droplet-based microfluidic chip. Therefore, our synthesis method of homogeneous AuNPs could play an important role in improving the efficiency of AuNPs-based sensing signals.

Analysis of deoxyribonuclease activity by conjugation-free fluorescence polarisation in sub-nanolitre droplets.

We report the analysis of deoxyribonuclease (DNase) activity by conjugation-free fluorescence polarisation in a droplet-based microfluidic chip. DNase is a DNA cleaving enzyme and its activity is important in the maintenance of normal cellular functions. Alterations in DNase activity have been implicated as the cause of various cancers and autoimmune diseases. To date, various methods for the analysis of DNase activity have been reported. However, they are not cost effective due to the requirement of large sample volumes and the need for the conjugation of fluorescent dyes. In this study, we have used ethidium bromide (EtBr), a DNA intercalating reagent, as a fluorescent reporter without any prior conjugation or modification of DNA. Degradation of DNA by DNase 1 was monitored at a steady state by making changes in the fluorescence polarisation of EtBr in droplets with a volume of 330 picolitre at a 40 hertz frequency under visible light. Using this technique, we successfully determined the half-maximal inhibitory concentration (IC50) of ethylenediaminetetraacetic acid (EDTA) for the inhibition of DNase 1 activity to be 1.56 ± 0.91 mM.

A single cell droplet microfluidic system for quantitative determination of food-borne pathogens

Single-cell detection methods are already of great significance for many bioanalysis applications, and droplet microfluidics technology is understood as particularly a powerful tool. Salmonella infection is a major hygienic problem worldwide that causes major public health and economic damage, and preventing Salmonella outbreaks requires detection food-borne detection methods that are rapid, portable, and reliable, ideally without the need for complicated pre-treatment protocol steps. Herein, we present a single-cell-level analysis method based on droplet microfluidics that can sensitively and rapidly detect Salmonella directly from food samples. Specifically, this method achieves single-cell encapsulation of Salmonella in droplets of a growth medium with resazurin that enables fluorescence-based detection of pathogens within 5 h. The ratio of positive droplets in a Poisson Distribution is used for quantitation, and the detection limit of our system determined to be 50 CFU/mL, a value lower than conventional analytical methods for assessing Salmonella contamination. Our experimental results demonstrate the precise and highly sensitive performance of a single-cell-precision, droplet-based microfluidic chip analytical method for monitoring pathogenic bacteria in food. Beyond our example case of Salmonella detection from milk samples, our work lays the foundation for a new generation of microfluidics-based analytical technologies for both public health and food safety applications which can undoubtedly benefit from increases in the sensitivity and rapidity of food-borne pathogen detection.

Governing factors for preparation of silver nanoparticles using droplet-based microfluidic device.

Droplet-based microfluidic devices are now more than ever used for the synthesis of nanoparticles with low polydispersity and well-defined properties suitable for various industrial applications. Very small reaction volumes (microlitre to femtolitre) and short diffusion lengths, provide superior mixing efficiency and heat transport. Both play the dominant role in case of ultra-fast chemical reactions triggered upon reactant mixing, e.g. preparation of colloidal silver by reduction of silver salt. The high sensitivity of these systems to process variables makes otherwise more straightforward batch-wise production prone to suffer from inconsistency and poor reproducibility, which has an adverse effect on the reliability of production and further particle utilisation. This work presents a rigorous description of microfluidic droplet formation, reactant mixing, and nanoparticle synthesis using CFD simulations and experimental methods. The reaction mixture inside of droplets was homogenized in less than 40 milliseconds, which has been confirmed by simulations. Silver nanoparticles produced by droplet-based microfluidic chip showed superior to batch-wise preparation in terms of both particle uniformity and polydispersity.

3D Droplet-Based Microfluidic Device Easily Assembled from Commercially Available Modules Online Coupled with ICPMS for Determination of Silver in Single Cell.

More recently, single-cell analysis based on ICPMS has made considerable headway while a challenge remains to differentiate single cell from doublets during the analysis. One burgeoning solution is to encapsulate single cell into droplets on the platform of the microfluidic chip. However, the manufacture of the droplet-based microfluidic chip requires sophisticated fabrication and limits its potential application. In this paper, we presented an off-the-shelf three-dimensional (3D) microfluidic device by assembling commercially available parts without any proficient manufacturing process. Uniform monodisperse microdroplet was generated from the 3D microfluidic device with a size variation of 1.5%, and the innner diameter of the 3D microfluidic device was the same as the nebulizer (150 μm). The proposed 3D microfluidic device-time-resolved ICPMS system was applied to detect silver in single AgNPs (51 nm), and the result is in good agreement with conventional acid digestion method, demonstrating the accuracy of the method. Silver uptake behaviors in HepG2 cells were then studied by incubating with Ag+ or AgNPs under biocompatible conditions. The results revealed that the cell-to-cell variability in terms of the diversity of cells incubated with AgNPs was wider than those cells incubated with Ag+ from the aspect of the content distribution of silver at the single-cell level.

Highly reproducible and fast detection of 6-thioguanine in human serum using a droplet-based microfluidic SERS system

6-thioguanine is an anticancer drug with narrow safe dose range and intense toxic reactions, so it is urgently necessary to evaluate its clinical usage. Reproducibility and analysis speed are important factors to take into account when analyzing drug concentration in body fluids. In this work, we fabricated a droplet-based microfluidic chip for detecting 6-thioguanine in human serum by using the surface-enhanced Raman scattering (SERS) technique. The easily-prepared Au nanoparticles were used as SERS substrates, and reagent mixing and signal acquiring happened in droplets one by one. This assay device is endowed with the superiority in improving determination repeatability with coefficient of variation of no more than 5% (n = 25) and high speed of 10 s each assay. The SERS peak at 1295 cm−1 originated from N1-C6 stretching vibration mode of purine ring was selected for quantitating 6-thioguanine. The signals indicated a satisfied linear relationship with the concentrations of 6-thioguanine in the range of 0.010 μM to 10 μM, with a detection limit of 0.032 μM. The droplet-based microfluidic SERS system was successfully applied for credible and rapid analysis of 6-thioguanine in human serum, and is pretty promising for performing the therapeutic drug monitoring.

A droplet-based microfluidic chip as a platform for leukemia cell lysate identification using surface-enhanced Raman scattering.

A new approach is presented for cell lysate identification which uses SERS-active silver nanoparticles and a droplet-based microfluidic chip. Eighty-nanoliter droplets are generated by injecting silver nanoparticles, KCl as aggregation agent, and cell lysate containing cell constituents, such as nucleic acids, carbohydrates, metabolites, and proteins into a continuous flow of mineral oil. This platform enables accurate mixing of small volumes inside the meandering channels of the quartz chip and allows acquisition of thousands of SERS spectra with 785nm excitation at an integration time of 1s. Preparation of three batches of three leukemia cell lines demonstrated the experimental reproducibility. The main advantage of a high number of reproducible spectra is to apply statistics for large sample populations with robust classification results. A support vector machine with leave-one-batch-out cross-validation classified SERS spectra with sensitivities, specificities, and accuracies better than 99% to differentiate Jurkat, THP-1, and MONO-MAC-6 leukemia cell lysates. This approach is compared with previous published reports about Raman spectroscopy for leukemia detection, and an outlook is given for transfer to single cells. A quartz chip was designed for SERS at 785 nm excitation. Principal component analysis of SERS spectra clearly separates cell lysates using variations in band intensity ratios.

A Controllable and Integrated Pump-enabled Microfluidic Chip and Its Application in Droplets Generating

A microfluidic chip with a controllable and integrated piezoelectric pump was proposed and demonstrated, where the pump was designed as a micro-actuator based on polyvinylidene fluoride (PVDF) organic piezoelectric film. In this case, the pump should integrate with the microfluidics device very well into one chip. The flow rate can be precisely controlled in the range of 0–300 µl/min for water by tuning the Vpp and frequency of Alternating Current (AC) voltage applied on the diaphragm. To analyze the relationship between the flow rate and the deflection of diaphragm, the deformations of diaphragm at different voltages were researched. The displacement of diaphragm was defined as 17.2 µm at the voltages of 3.5 kV, 5 Hz when the pump chamber was full of water. We have used the integrated microfluidic chip with two pumps for droplet generation to demonstrate its great potential for application in droplet-based microfluidic chip.

液滴微流控芯片技术及其在药物筛选中的应用

液滴微流控芯片技术及其在药物筛选中的应用

Numerical simulation of droplet‐based microfluidic chip interfacing with laser desorption/ionisation mass spectrometry target substrate

Droplet-based microfluidic devices have shown great potential in biomedical applications. When proteomics bioanalysis or biomarker identification is required, off-line microfluidic chip interfacing with mass spectrometry (MS) is necessary. This reported work has focused on the use of numerical simulation of microdroplet formation and dripping for a droplet-based microfluidic-chip interfacing with off-line laser desorption/ionisation MS target substrate. Unlike most studies on the microdroplet formation at the liquid–liquid phase, this work has investigated the microdroplet formation at the liquid–gas phase at various injection flow rates and inner microchannel surface wettability to meet microfluidic-chip–MS interfacing requirements. After the microdroplet formation, subsequent microdroplet dripping analysis was performed. The effects of inner microchannel and outer orifice surface wettability are discussed.

Continuous monitoring of bisulfide variation in microdialysis effluents by on-line droplet-based microfluidic fluorescent sensor

We demonstrate a novel fluorescent sensor for real-time and continuous monitoring of the variation of bisulfide in microdialysis effluents by using a nanoparticle–glutathione-fluorescein isothiocyanate (AuNP–GSH-FITC) probe coupled with on-line droplet-based microfluidic chip. The AuNP–GSH-FITC fluorescent probe was firstly developed and used for bisulfide detection in bulk solution by quantitative real-time PCR, which achieved a linear working range from 0.1μM to 5.0μM and a limit of detection of ~50nM. The response time was less than 2min. With the aid of co-immobilized thiol-polyethylene glycol, the probe exhibited excellent stability and reproducibility in high salinity solutions, including artificial cerebrospinal fluids (aCSF). By adding 0.1% glyoxal to the probe solution, the assay allowed quantification of bisulfide in the presence of cysteine at the micro-molarity level. Using the AuNP–GSH-FITC probe, a droplet-based microfluidic fluorescent sensor was further constructed for online monitoring of bisulfide variation in the effluent of microdialysis. By using fluorescence microscope-charge-coupled device camera as the detector, the integrated microdialysis/microfluidic chip device achieved a detection limit of 2.0μM and a linear response from 5.0μM to 50μM for bisulfide in the tested sample. The method was successfully applied for the on-line measurement of bisulfide variation in aCSF and serum samples. It will be a very useful tool for tracking the variation of bisulfide or hydrogen sulfide in extracellular fluids.

Easy Route to Superhydrophobic Copper-Based Wire-Guided Droplet Microfluidic Systems

Droplet-based microfluidic systems are an expansion of the lab on a chip concept toward flexible, reconfigurable setups based on the modification and analysis of individual droplets. Superhydrophobic surfaces are one suitable candidate for the realization of droplet-based microfluidic systems as the high mobility of aqueous liquids on such surfaces offers possibilities to use novel or more efficient approaches to droplet movement. Here, copper-based superhydrophobic surfaces were produced either by the etching of polycrystalline copper samples along the grain boundaries using etchants common in the microelectronics industry, by electrodeposition of copper films with subsequent nanowire decoration based on thermal oxidization, or by a combination of both. The surfaces could be easily hydrophobized with thiol-modified fluorocarbons, after which the produced surfaces showed a water contact angle as high as 171 degrees +/- 2 degrees . As copper was chosen as the base material, established patterning techniques adopted from printed circuit board fabrication could be used to fabricate macrostructures on the surfaces with the intention to confine the droplets and, thus, to reduce the system's sensitivity to tilting and vibrations. A simple droplet-based microfluidic chip with inlets, outlets, sample storage, and mixing areas was produced. Wire guidance, a relatively new actuation method applicable to aqueous liquids on superhydrophobic surfaces, was applied to move the droplets.

Measuring Rapid Enzymatic Kinetics by Electrochemical Method in Droplet-Based Microfluidic Devices with Pneumatic Valves

This paper describes a droplet-based microfluidic chip with pneumatic valves for measuring millisecond enzyme kinetics using amperometric detection method. Aqueous streams containing reactants were injected to an oil flow to form droplets, and each droplet represented one microreactor. Pneumatic valves were used to control the moving distance and in turn the reaction time of the droplets. The reaction time was also fine-tuned by varying the flow rate of the droplets in microchannels. A complete Michaelis-Menten kinetics of catalase was successfully measured by amperometric method in a single-run experiment, and the total consumption of reagents was less than 50 microL. In the current experiment, the best time resolution was about 0.05 s, and the reaction time measured was from 0.05 to 25 s. This microfluidic system is applicable to many biochemical reactions, as long as one of the reactants or products is electrochemically active. With appropriate quenching method at the outlet, various detection methods can be integrated into the microfluidic system, further extending the application of the combination of pneumatic valves and droplets in microchannels.