A Thoroughgoing Design of a Rapid-cycle Microfluidic Droplet-based PCR Device to Amplify Rare DNA Strands

DNA is a molecule and assortment of fruitful information of organisms and a wide range of viruses. Polymerase chain reaction (PCR) is a process used to amplify DNA strands in order to generate millions of them and extract the applicable information. Although conventional methods for PCR are flourishing to a certain extent, they have such major drawbacks as contamination, high material consumption, and low-speed function. By the combination of PCR devices with the microfluidic approach and integrating them with droplet generation technology, the mentioned problems can be eliminated. In this study, a novel two-step rapid-cycle droplet-based PCR (dPCR) device, considering the design of microchannel and heat transfer system, has been presented. First, numerous studies have been conducted to select the proper droplet generator for the integration of the droplet generation with the PCR device. Then, with the careful attention to the requirements of a PCR device, the geometry of different zones of the PCR device has been, meticulously, designed. In the next and last step, the heat transfer system for the designed zones of the PCR device has been planned. Afterward, results are examined carefully which indicate that in a cycle of PCR, they are not any major discrepancies between the designed dPCR and the ideal one—the one that is intended to be created

Upregulation of PD-L1 expression in breast cancer cells through the formation of 3D multicellular cancer aggregates under different chemical and mechanical conditions

© 2019 Elsevier B.V. Expression of programmed death-ligand 1 (PD-L1) in cancer cells plays an important role in cancer-immune cell interaction. The emerging evidence suggests regulation of PD-L1 expression by several tumor microenvironmental cues. However, the association of PD-L1 expression with chemical and mechanical features of the tumor microenvironment, specifically epidermal growth factor receptor (EGFR) signaling and matrix stiffness, remains elusive. Herein, we determine whether EGFR targeting and substrate stiffness affect the regulation of PD-L1 expression. Breast carcinoma cell lines, MCF7 and MDA-MB-231, were cultured under different conditions targeting EGFR and exposing cells to distinct substrate stiffness to evaluate PD-L1 expression. Furthermore, the ability to form aggregates in short-term culture of breast carcinoma cells and its effect on expression level of PD-L1 was probed. Our results indicated that PD-L1 expression was altered in response to both EGFR inhibition and substrate stiffness. Additionally, a positive association between the formation of multicellular aggregates and PD-L1 expression was observed. MDA-MB-231 cells expressed the highest PD-L1 level on a stiff substrate, while inhibition of EGFR reduced expression of PD-L1. The results suggested that both physical and chemical features of tumor microenvironment regulate PD-L1 expression through alteration of tumor aggregate formation potential. In line with these results, the in-silico study highlighted a positive correlation between PD-L1 expression, EGFR signaling, epithelial to mesenchymal transition related transcription factors (EMT-TFs) and stemness markers in metastatic breast cancer. These findings improve our understanding of regulation of PD-L1 expression by tumor microenvironment leading to evasion of tumor cells from the immune system

Simultaneous Modeling of Young's Modulus, Yield Stress, and Rupture Strain of Gelatin/Cellulose Acetate Microfibrous/Nanofibrous Scaffolds Using RSM.

Electrospinning is a promising method to fabricate bioengineered scaffolds, thanks to utilizing various types of biopolymers, flexible structures, and also the diversity of output properties. Mechanical properties are one of the major components of scaffold design to fabricate an efficacious artificial substitute for the natural extracellular matrix. Additionally, fiber orientations, as one of the scaffold structural parameters, could play a crucial role in the application of fabricated fibrous scaffolds. In this study, gelatin was used as a highly biocompatible polymer in blend with cellulose acetate (CA), a polysaccharide, to enhance the achievable range of mechanical characteristics to fabricated fibrous electrospun scaffolds. By altering input variables, such as polymers concentration, weight ratio, and mandrel rotation speed, scaffolds with various mechanical and morphological properties could be achieved. As expected, the electrospun scaffold with a higher mandrel rotation speed shows higher fiber alignment. A wide range of mechanical properties were gained through different values of polymer ratio and total concentration. A general improvement in mechanical strength was observed by increasing the concentration and CA content in the solution, but contradictory effects, such as high viscosity in more concentrated solutions, influenced the mechanical characteristics as well. A response surface method was applied on experimental results in order to describe a continuous variation of Young's modulus, yield stress, and strain at rupture. A full quadratic version of equations with the 95% confidence level was applied for the response modeling. This model would be an aid for engineers to adjust mandrel rotation speed, solution concentration, and gelatin/CA ratio to achieve desired mechanical and structural properties

Point of Care Diagnostics in the Age of COVID-19.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated serious respiratory disease, coronavirus disease 2019 (COVID-19), poses a major threat to global public health. Owing to the lack of vaccine and effective treatments, many countries have been overwhelmed with an exponential spread of the virus and surge in the number of confirmed COVID-19 cases. Current standard diagnostic methods are inadequate for widespread testing as they suffer from prolonged turn-around times (>12 h) and mostly rely on high-biosafety-level laboratories and well-trained technicians. Point-of-care (POC) tests have the potential to vastly improve healthcare in several ways, ranging from enabling earlier detection and easier monitoring of disease to reaching remote populations. In recent years, the field of POC diagnostics has improved markedly with the advent of micro- and nanotechnologies. Due to the COVID-19 pandemic, POC technologies have been rapidly innovated to address key limitations faced in existing standard diagnostic methods. This review summarizes and compares the latest available POC immunoassay, nucleic acid-based and clustered regularly interspaced short palindromic repeats- (CRISPR)-mediated tests for SARS-CoV-2 detection that we anticipate aiding healthcare facilities to control virus infection and prevent subsequent spread

Circulating tumour cell clusters: Insights into tumour dissemination and metastasis.

INTRODUCTION:Metastasis results in more than 90% of cancer related deaths globally. The process is thought to be facilitated by metastatic precursor cells, commonly termed circulating tumour cells (CTCs). CTCs can exist as single cells or cell clusters and travel through the lymphovasculature to distant organs where they can form overt metastasis. Areas covered: Studies have highlighted that CTC clusters, which may be homotypic or heterotypic in composition, have a higher metastatic potential compared to single CTCs. The characterisation of CTC clusters is becoming important as heterotypic clusters can provide a mechanism for immune evasion. This review summarises the latest advances in CTC cluster mediated metastasis and clinical significance. Expert Opinion: Comprehensive characterisation of CTC clusters is needed to understand the cell types and interactions within clusters, in order to identify ways in which to reduce CTC cluster mediated metastasis. The role of CTC clusters in prognosticating disease progression needs to be determined by documenting CTC clusters from the time of diagnosis over the course of therapy

3D Printing of Inertial Microfluidic Devices.

Inertial microfluidics has been broadly investigated, resulting in the development of various applications, mainly for particle or cell separation. Lateral migrations of these particles within a microchannel strictly depend on the channel design and its cross-section. Nonetheless, the fabrication of these microchannels is a continuous challenging issue for the microfluidic community, where the most studied channel cross-sections are limited to only rectangular and more recently trapezoidal microchannels. As a result, a huge amount of potential remains intact for other geometries with cross-sections difficult to fabricate with standard microfabrication techniques. In this study, by leveraging on benefits of additive manufacturing, we have proposed a new method for the fabrication of inertial microfluidic devices. In our proposed workflow, parts are first printed via a high-resolution DLP/SLA 3D printer and then bonded to a transparent PMMA sheet using a double-coated pressure-sensitive adhesive tape. Using this method, we have fabricated and tested a plethora of existing inertial microfluidic devices, whether in a single or multiplexed manner, such as straight, spiral, serpentine, curvilinear, and contraction-expansion arrays. Our characterizations using both particles and cells revealed that the produced chips could withstand a pressure up to 150 psi with minimum interference of the tape to the total functionality of the device and viability of cells. As a showcase of the versatility of our method, we have proposed a new spiral microchannel with right-angled triangular cross-section which is technically impossible to fabricate using the standard lithography. We are of the opinion that the method proposed in this study will open the door for more complex geometries with the bespoke passive internal flow. Furthermore, the proposed fabrication workflow can be adopted at the production level, enabling large-scale manufacturing of inertial microfluidic devices

High-Throughput Particle Concentration Using Complex Cross-Section Microchannels.

High throughput particle/cell concentration is crucial for a wide variety of biomedical, clinical, and environmental applications. In this work, we have proposed a passive spiral microfluidic concentrator with a complex cross-sectional shape, i.e., a combination of rectangle and trapezoid, for high separation efficiency and a confinement ratio less than 0.07. Particle focusing in our microfluidic system was observed in a single, tight focusing line, in which higher particle concentration is possible, as compared with simple rectangular or trapezoidal cross-sections with similar flow area. The sharper focusing stems from the confinement of Dean vortices in the trapezoidal region of the complex cross-section. To quantify this effect, we introduce a new parameter, complex focusing number or CFN, which is indicative of the enhancement of inertial focusing of particles in these channels. Three spiral microchannels with various widths of 400 µm, 500 µm, and 600 µm, with the corresponding CFNs of 4.3, 4.5, and 6, respectively, were used. The device with the total width of 600 µm was shown to have a separation efficiency of ~98%, and by recirculating, the output concentration of the sample was 500 times higher than the initial input. Finally, the investigation of results showed that the magnitude of CFN relies entirely on the microchannel geometry, and it is independent of the overall width of the channel cross-section. We envision that this concept of particle focusing through complex cross-sections will prove useful in paving the way towards more efficient inertial microfluidic devices

Fabrication of unconventional inertial microfluidic channels using wax 3D printing.

Inertial microfluidics has emerged over the past decade as a powerful tool to accurately control cells and microparticles for diverse biological and medical applications. Many approaches have been proposed to date in order to increase the efficiency and accuracy of inertial microfluidic systems. However, the effects of channel cross-section and solution properties (Newtonian or non-Newtonian) have not been fully explored, primarily due to limitations in current microfabrication methods. In this study, we overcome many of these limitations using wax 3D printing technology and soft lithography through a novel workflow, which eliminates the need for the use of silicon lithography and polydimethylsiloxane (PDMS) bonding. We have shown that by adding dummy structures to reinforce the main channels, optimizing the gap between the dummy and main structures, and dissolving the support wax on a PDMS slab to minimize the additional handling steps, one can make various non-conventional microchannels. These substantially improve upon previous wax printed microfluidic devices where the working area falls into the realm of macrofluidics rather than microfluidics. Results revealed a surface roughness of 1.75 μm for the printed channels, which does not affect the performance of inertial microfluidic devices used in this study. Channels with complex cross-sections were fabricated and then analyzed to investigate the effects of viscoelasticity and superposition on the lateral migration of the particles. Finally, as a proof of concept, microcarriers were separated from human mesenchymal stem cells using an optimized channel with maximum cell-holding capacity, demonstrating the suitability of these microchannels in the bioprocessing industry

A Portable RT-LAMP/CRISPR Machine for Rapid COVID-19 Screening.

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary

Numerical and Experimental Study of Cross-Sectional Effects on the Mixing Performance of the Spiral Microfluidics.

Mixing at the microscale is of great importance for various applications ranging from biological and chemical synthesis to drug delivery. Among the numerous types of micromixers that have been developed, planar passive spiral micromixers have gained considerable interest due to their ease of fabrication and integration into complex miniaturized systems. However, less attention has been paid to non-planar spiral micromixers with various cross-sections and the effects of these cross-sections on the total performance of the micromixer. Here, mixing performance in a spiral micromixer with different channel cross-sections is evaluated experimentally and numerically in the Re range of 0.001 to 50. The accuracy of the 3D-finite element model was first verified at different flow rates by tracking the mixing index across the loops, which were directly proportional to the spiral radius and were hence also proportional to the Dean flow. It is shown that higher flow rates induce stronger vortices compared to lower flow rates; thus, fewer loops are required for efficient mixing. The numerical study revealed that a large-angle outward trapezoidal cross-section provides the highest mixing performance, reaching efficiencies of up to 95%. Moreover, the velocity/vorticity along the channel length was analyzed and discussed to evaluate channel mixing performance. A relatively low pressure drop (<130 kPa) makes these passive spiral micromixers ideal candidates for various lab-on-chip applications