Plasmonic heating-based portable digital PCR system.

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s-1 and maximum cooling rate of 8 °C s-1. Target DNA concentrations in the range from 101 ± 1.4 copies per µL to 260 000 ± 20 000 copies per µL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 µm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 µm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per µL to 25 889 ± 737 copies per µL.

Electro-responsive hydrogel-based microfluidic actuator platform for photothermal therapy.

Electrical stimuli play an important role in regulating the delivery of plasmonic nanomaterials with cancer targeting peptides. Here, we developed an electro-responsive hydrogel-based microfluidic actuator platform for brain tumor targeting and photothermal therapy (PTT) applications. The electro-responsive hydrogels consisted of highly conductive silver nanowires (AgNWs) and biocompatible collagen I gels. We confirmed that an electrically conductive hydrogel could be used as an effective actuator by applying an electrical signal in the microfluidic platform. Furthermore, we successfully demonstrated PTT efficacy for brain tumor cells using targetable Arg-Gly-Asp (RGD) peptide-conjugated gold nanorods (GNRs). Therefore, our electro-responsive hydrogel-based microfluidic actuator platform could be useful for electro-responsive intelligent nanomaterial delivery and PTT applications.

Automated droplet reactor for the synthesis of iron oxide/gold core-shell nanoparticles.

Core-shell nanoparticles are promising candidates for theranostic drugs, as they combine different intrinsic properties with a small size and large surface area. However, their controlled synthesis, or the screening and optimization of synthesis conditions are often difficult and labor intensive. Through the precise control over mass and heat transfer, and automatization possibilities, microfluidic devices could be a solution to this problem in a lab scale synthesis. Here, we demonstrate a microfluidic, capillary, droplet reactor for the multi-step synthesis of iron oxide/gold core-shell nanoparticles. Through the integration of a transmission measurement at the outlet of the reactor, synthesis results can be monitored in a real-time manner. This allowed for the implementation of an optimization algorithm. Starting from three separate initial guesses, the algorithm converged to the same synthesis conditions in less than 30 minutes for each initial guess. These conditions resulted in diameter for the iron oxide core of 5.8 ± 1.4 nm, a thickness for the gold shell of 3.5 ± 0.6 nm, and a total diameter of the core-shell particles of 13.1 ± 2.5 nm. Finally, applications of the iron oxide/gold core-shell nanoparticles were demonstrated for Surface Enhanced Raman Spectroscopy (SERS), photothermal therapy, and magnetic resonance imaging (MRI).

Generation of tumor spheroids using a droplet-based microfluidic device for photothermal therapy.

Despite their simplicity, monolayer cell cultures are not able to accurately predict drug behavior in vivo due to their inability to accurately mimic cell-cell and cell-matrix interactions. In contrast, cell spheroids are able to reproduce these interactions and thus would be a viable tool for testing drug behavior. However, the generation of homogenous and reproducible cell spheroids on a large scale is a labor intensive and slow process compared to monolayer cell cultures. Here, we present a droplet-based microfluidic device for the automated, large-scale generation of homogenous cell spheroids in a uniform manner. Using the microfluidic system, the size of the spheroids can be tuned to between 100 and 130 µm with generation frequencies of 70 Hz. We demonstrated the photothermal therapy (PTT) application of brain tumor spheroids generated by the microfluidic device using a reduced graphene oxide-branched polyethyleneimine-polyethylene glycol (rGO-BPEI-PEG) nanocomposite as the PTT agent. Furthermore, we generated uniformly sized neural stem cell (NSC)-derived neurospheres in the droplet-based microfluidic device. We also confirmed that the neurites were regulated by neurotoxins. Therefore, this droplet-based microfluidic device could be a powerful tool for photothermal therapy and drug screening applications.

Continuous separation of fungal spores in a microfluidic flow focusing device.

The research of fungi is of great importance in a number of fields, such as environmental and healthcare studies. While there are a large number of optical and molecular methods available for characterization and identification of fungi and their spores, their isolation is still conducted using slow and labor-intensive methods. Here, we develop a microfluidic device for the continuous separation of fungal spores from other eukaryotic cells. The spores were separated through the microfluidic device by expanding pinched flow fractionation (PFF) containing the spores, achieving a spatial separation perpendicular to the flow direction according to the spore size. Further branch flow fractionation (BFF) and co-flow of a Newtonian and viscoelastic fluid were used to enhance the separation performance. Using this microfluidic device, we demonstrated the separation of two different types of fungal spores and further separation of fungal spores from eukaryotic cells with a separation efficiency of above 90%. Compared to the existing conventional methods, our microfluidic flow focusing device requires little manual handling and uses small amounts of samples without any pre-treatment steps of the samples.

Dual-neodymium magnet-based microfluidic separation device.

Microfluidic-based separation methods have been highlighted for a number of biological applications, such as single cell analysis, disease diagnostics, and therapeutics. Although a number of previous studies have been carried out to minimize the physical damage and chemical deformations of the sample during the separation process, it still remains a challenge. In this paper, we developed a microfluidic device with dual-neodymium magnet-based negative magnetophoresis for the separation of the microparticles and cells. The poly(ethylene oxide) (PEO) was added to the solution to increase the viscoelasticity of the medium which could assist the sorting of the microparticles in the microfluidic device even at low flow rates, while minimizing damage to the cells and microparticles. Following this method, it was possible to separate 10 and 16 µm microparticles with high efficiency of 99 ± 0.1%, and 97 ± 0.8%, respectively. We also demonstrated the separation of glioblastoma cancer cells and neural stem cells (NSCs) in the microfluidic device.

Micropillar-based microfluidic device to regulate neurite networks of uniform-sized neurospheres.

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar-based microfluidic device to trap the uniform-sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar-based microfluidic device could be a potential tool for screening of neurotoxins.

Generation of uniform-sized multicellular tumor spheroids using hydrogel microwells for advanced drug screening.

Even though in vitro co-culture tumor spheroid model plays an important role in screening drug candidates, its wide applications are currently limited due to the lack of reliable and high throughput methods for generating well-defined and 3D complex co-culture structures. Herein, we report the development of a hydrogel microwell array to generate uniform-sized multicellular tumor spheroids. Our developed multicellular tumor spheroids are structurally well-defined, robust and can be easily transferred into the widely used 2D culture substrates while maintaining our designed multicellular 3D-sphere structures. Moreover, to develop effective anti-cancer therapeutics we integrated our recently developed gold-graphene hybrid nanomaterial (Au@GO)-based photothermal cancer therapy into a series of multicellular tumor spheroid co-culture system. The multicellular tumor spheroids were harvested onto a two-dimensional (2D) substrate, under preservation of their three-dimensional (3D) structure, to evaluate the photothermal therapy effectiveness of graphene oxide (GO)-wrapped gold nanoparticles (Au@GO). From the model of co-culture spheroids of HeLa/Ovarian cancer and HeLa/human umbilical vein endothelial cell (HUVEC), we observed that Au@GO nanoparticles displayed selectivity towards the fast-dividing HeLa cells, which could not be observed to this extent in 2D cultures. Overall, our developed uniform-sized 3D multicellular tumor spheroid could be a powerful tool for anticancer drug screening applications.

Droplet-based synthesis of homogeneous magnetic iron oxide nanoparticles.

Nanoparticles have gained large interest in a number of different fields due to their unique properties. In medical applications, for example, magnetic nanoparticles can be used for targeting, imaging, magnetically induced thermotherapy, or for any combination of the three. However, it is still a challenge to obtain narrowly dispersed, reproducible particles through a typical lab-scale synthesis when researching these materials. Here, we present a droplet capillary reactor that can be used for the synthesis of magnetic iron oxide nanoparticles. Compared to conventional batch synthesis, the particles synthesized in our droplet reactor have a narrower size distribution and a higher reproducibility. Furthermore, we demonstrate how the particle size can be changed from 5.2 ± 0.9 nm to 11.8 ± 1.7 nm by changing the reaction temperature and droplet residence time in the droplet capillary reactor.

Dual-nozzle microfluidic droplet generator.

The droplet-generating microfluidics has become an important technique for a variety of applications ranging from single cell analysis to nanoparticle synthesis. Although there are a large number of methods for generating and experimenting with droplets on microfluidic devices, the dispensing of droplets from these microfluidic devices is a challenge due to aggregation and merging of droplets at the interface of microfluidic devices. Here, we present a microfluidic dual-nozzle device for the generation and dispensing of uniform-sized droplets. The first nozzle of the microfluidic device is used for the generation of the droplets, while the second nozzle can accelerate the droplets and increase the spacing between them, allowing for facile dispensing of droplets. Computational fluid dynamic simulations were conducted to optimize the design parameters of the microfluidic device.

Circular-shaped microfluidic device to study the effect of shear stress on cellular orientation.

Understanding the effects of shear stress on mammalian cells is a crucial factor for understanding a number of biological processes and diseases. Here, we show the development of a circular-shaped microfluidic device for the facile generation of shear stress gradients. With this microfluidic device, the effect of shear stress on orientation of human umbilical vein endothelial cells was studied. This microfluidic device, which enables to control the alignment of human umbilical vein endothelial cells within a microchannel, can be a valuable tool to mimic blood vessels.

Poisson statistics-mediated particle/cell counting in microwell arrays.

Precise determination of particle or cell numbers is of importance for a wide array of applications in environmental studies, medical and biological applications, or manufacturing and monitoring applications in industrial production processes. A number of techniques ranging from manual counting to sophisticated equipment (e.g., flow cytometry) are available for this task. However, these methods are either labour intensive, prone to error, or require expensive equipment. Here, we present a fast, simple method for determining the number density of cells or microparticles using a microwell array. We analyze the light transmission of the microwells and categorize the microwells into two groups. As particles/cells contained in a microwell locally reduce the light transmission, these wells displayed a lower average transmission compared to unoccupied microwells. The number density of particles/cells can be calculated by Poisson statistics from the ratio of occupied to unoccupied microwells. Following this approach, the number densities of two different types of microparticles, as well as HeLa and E. Coli cells, ranging over four orders of magnitude were determined. Through the microwell array defined by microfabrication, a simple image recognition algorithm can be used with the formation of aggregates or irregular shaped samples providing no additional difficulty to the microwell recognition. Additionally, this method can be carried out using only simple equipment and data analysis automated by a computer program.

Prediction analysis and quality assessment of microwell array images.

Microwell arrays are widely used for the analysis of fluorescent-labelled biomaterials. For rapid detection and automated analysis of microwell arrays, the computational image analysis is required. Support Vector Machines (SVM) can be used for this task. Here, we present a SVM-based approach for the analysis of microwell arrays consisting of three distinct steps: labeling, training for feature selection, and classification into three classes. The three classes are filled, partially filled, and unfilled microwells. Next, the partially filled wells are analyzed by SVM and their tendency towards filled or unfilled tested through applying a Gaussian filter. Through this, all microwells can be categorized as either filled or unfilled by our algorithm. Therefore, this SVM-based computational image analysis allows for an accurate and simple classification of microwell arrays.

Analysis of 3D multi-layer microfluidic gradient generator.

We developed a three-dimensional (3D) simple multi-layer microfluidic gradient generator to create molecular gradients on the centimeter scale with a wide range of flow rates. To create the concentration gradients, a main channel (MC) was orthogonally intersected with vertical side microchannel (SC) in a 3D multi-layer microfluidic device. Through sequential dilution from the SC, a spatial gradient was generated in the MC. Two theoretical models were created to assist in the design of the 3D multi-layer microfluidic gradient generator and to compare its performance against a two-dimensional equivalent. A first mass balance model was used to predict the steady-state concentrations reached, while a second computational fluid dynamic model was employed to predict spatial development of the gradient by considering convective as well as diffusive mass transport. Furthermore, the theoretical simulations were verified through experiments to create molecular gradients in a 3D multi-layer microfluidic gradient generator.

Polymerase chain reaction in microfluidic devices.

The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.

Palm-Sized Device for Point-of-Care Ebola Detection.

We show the utilization of a recently developed cellphone-sized real-time polymerase chain reaction (PCR) device to detect Ebola virus RNA using single-step reverse transcription PCR (RT-PCR). The device was shown to concurrently perform four PCRs, each with a sample volume of 100 nL: one positive control with both Ebola and GAPDH RNA and one negative control. The last two positions were used to measure the GAPDH and the Ebola content of a sample. A comparison of threshold cycles (CT) from the two samples provided relative quantification. The entire process, which consisted of reverse transcription, PCR amplification, and melting curve analysis (MCA), was conducted in less than 37 min. The next step will be integration with a sample preparation unit to form an integrated sample-to-answer system for point-of-care infectious disease diagnostics.

Handheld real-time PCR device.

Here we report one of the smallest real-time polymerase chain reaction (PCR) systems to date with an approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in the form of virtual reaction chambers (VRCs) where a ≈200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate the PCR performance. The standard curve slope was -3.02 ± 0.16 cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of 0.91 ± 0.05 per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics.

Doubling Throughput of a Real-Time PCR.

The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.