Investigating influences of intravenous fluids on HUVEC and U937 monocyte cell lines using the magnetic levitation method.

Intravenous fluids are being widely used in patients of all ages for preventing or treating dehydration in the intensive care units, surgeries in the operation rooms, or administering chemotherapeutic drugs at hospitals. Dextrose, Ringer, and NaCl solutions are widely received as intravenous fluids by hospitalized patients. Despite their widespread administration for over 100 years, studies on their influences on different cell types have been very limited. Increasing evidence suggests that treatment outcomes might be altered by the choice of the administered intravenous fluids. In this study, we investigated the influences of intravenous fluids on human endothelial (HUVEC) and monocyte (U937) cell lines using the magnetic levitation technique. Our magnetic levitation platform provides label-free manipulation of single cells without altering their phenotypic or genetic properties. It allows for monitoring and quantifying behavior of single cells by measuring their levitation heights, deformation indices, and areas. Our results indicate that HUVEC and U937 cell lines respond differently to different intravenous fluids. Dextrose solution decreased the viability of both cell lines while increasing the heterogeneity of areas, deformation, and levitation heights of HUVEC cells. We strongly believe that improved outcomes can be achieved when the influences of intravenous fluids on different cell types are revealed using robust, label-free, and efficient methods.

Analysis of U87 glioma cells by dielectrophoresis.

Glioblastoma multiforme is the most aggressive and invasive brain cancer consisting of genetically and phenotypically altering glial cells. It has massive heterogeneity due to its highly complex and dynamic microenvironment. Here, electrophysiological properties of U87 human glioma cell line were measured based on a dielectrophoresis phenomenon to quantify the population heterogeneity of glioma cells. Dielectrophoretic forces were generated using a gold-microelectrode array within a microfluidic channel when 3 Vpp and 100, 200, 300, 400, 500 kHz, 1, 2, 5, and 10 MHz frequencies were applied. We analyzed the dielectrophoretic behavior of 500 glioma cells, and revealed that the crossover frequency of glioma cells was around 140 kHz. A quantifying dielectrophoretic movement of the glioma cells exhibited three distinct glioma subpopulations: 50% of the glioma cells experienced strong, 30% of the cells were spread in the microchannel by moderate, and the rest of the cells experienced very weak positive dielectrophoretic forces. Our results demonstrated the dielectrophoretic spectra of U87 glioma cell line. Dielectrophoretic responses of glioma cells linked population heterogeneity to membrane properties of glioma cells rather than their size distribution in the population.

A low-cost, portable, and practical LAMP device for point-of-diagnosis in the field.

Transition of rapid, ready-to-use, and low-cost nucleic acid-based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling through nucleic acid isolation and detection in the field. Here we developed a simple, portable, low-cost, colorimetric, and remotely controllable platform for reliable, high-throughput, and rapid diagnosis using loop-mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low-cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady-state temperature error of the system is <1%. We performed LAMP, Colony-LAMP, and Colony polymerase chain reactions (PCRs) using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65°C and 30 min. We detected the end-point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony-LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.

Long-term migratory velocity measurements of single glioma cells using microfluidics.

Microfluidic platforms enabling single-cell measurements notably contribute to the identification and observation of rare cancer cells that are involved in tumor invasion. Most aggressive, invasive, and heterogeneous glioblastoma cells cause incurable primary brain tumors. Infiltrating gliomas of a brain tumor microenvironment have been intensively studied using conventional assays. Still, quantitative, simple, and precise tools are required for long-term, steady-state migratory-velocity measurements of single glioma cells. To measure long-term velocity changes and investigate the heterogeneity of glioma cells under different growth conditions, we developed a microfluidic platform. We cultured U87 glioma cells in the microfluidic device using either regular growth medium or conditional medium composed of 50% basal medium and 50% macrophage-depleted medium. We microscopically monitored the behavior of 40 glioma cells for 5 days. Using acquired images, we calculated cellular circularity and determined the migratory velocities of glioma cells from 60 h to 120 h. The mean migratory velocity values of the glioma cells were 1.513 µm h-1 in the basal medium and 3.246 µm h-1 in the conditional medium. The circularity values of the glioma cells decreased from 0.20-0.25 to 0.15-0.20 when cultured in the conditional medium. Here, we clearly showed that the glioma cells lost their circularity and increased their steady-state velocities; in other words, they adopted an invasive glioma phenotype in the presence of macrophage-depleted medium. Besides, the heterogeneity of the circularity and the velocity of glioma cells were enhanced in the conditional medium.

Dielectrophoretic characterization and separation of monocytes and macrophages using 3D carbon-electrodes.

Monocyte heterogeneity and its prevalence are revealed as indicator of several human diseases ranking from cardiovascular diseases to rheumatoid arthritis, chronic kidney diseases, autoimmune multiple sclerosis, and stroke injuries. When monocytes and macrophages are characterized and isolated with preserved genetic, phenotypic and functional properties, they can be used as label-free biomarkers for precise diagnostics and treatment of various diseases. Here, the dielectrophoretic responses of the monocytes and macrophages were examined. We present 3D carbon-electrode dielectrophoresis (carbon-DEP) as a separation tool for U937 monocytes and U937 monocyte-differentiated macrophages. The carbon-electrodes advanced the usability and throughput of DEP separation, presented wider electrochemical stability. Using the 3D carbon-DEP chip, we first identified the selective positive and negative DEP responses and specific crossover frequencies of monocytes and macrophages as their signatures for separation. The crossover frequency of monocytes and macrophages was 17 and 30 kHz, respectively. Next, we separated monocyte and macrophage subpopulations using their specific dielectrophoretic responses. Afterward, we used a fluorescence-activated cell sorter to confirm our results. Finally, we enriched 70% of monocyte cells from the mixed cell population, in other words, concentration of monocyte cells to macrophage cells was five times increased, using the 30-kHz, 10-Vpp electric field and 1 µL/min flow rate.

Microfabricated tools for quantitative plant biology.

The development of microfabricated devices that will provide high-throughput quantitative data and high resolution in a fast, repeatable and reproducible manner is essential for plant biology research. Plants have been intensely explored since the beginning of humanity, especially for medical needs. However, plant biology research is still laborious, lacking the latest technological advancements in the laboratory practices. Microfabricated tools can provide a significant contribution to plant biology research since they require small volumes of samples and reagents with minimal cost and labor. Besides, they minimize the wet lab requirements while providing a parallel measurement platform for high-throughput data. Here, we have reviewed the cutting-edge microfabricated technologies developed for plant biology research. The description of the microfabricated device components, their integration with plant science and their substitution with the conventional techniques are presented. Our discussion on the challenges and future opportunities for scientists working at the fascinating intersection between plant science and engineering concludes this study.

Dielectrophoretic Separation of Live and Dead Monocytes Using 3D Carbon-Electrodes.

Blood has been the most reliable body fluid commonly used for the diagnosis of diseases. Although there have been promising investigations for the development of novel lab-on-a-chip devices to utilize other body fluids such as urine and sweat samples in diagnosis, their stability remains a problem that limits the reliability and accuracy of readouts. Hence, accurate and quantitative separation and characterization of blood cells are still crucial. The first step in achieving high-resolution characteristics for specific cell subpopulations from the whole blood is the isolation of pure cell populations from a mixture of cell suspensions. Second, live cells need to be purified from dead cells; otherwise, dead cells might introduce biases in the measurements. In addition, the separation and characterization methods being used must preserve the genetic and phenotypic properties of the cells. Among the characterization and separation approaches, dielectrophoresis (DEP) is one of the oldest and most efficient label-free quantification methods, which directly purifies and characterizes cells using their intrinsic, physical properties. In this study, we present the dielectrophoretic separation and characterization of live and dead monocytes using 3D carbon-electrodes. Our approach successfully removed the dead monocytes while preserving the viability of the live monocytes. Therefore, when blood analyses and disease diagnosis are performed with enriched, live monocyte populations, this approach will reduce the dead-cell contamination risk and achieve more reliable and accurate test results.

Mathematical Model for Growth and Rifampicin-Dependent Killing Kinetics of Escherichia coli Cells.

Antibiotic resistance is a global health threat. We urgently need better strategies to improve antibiotic use to combat antibiotic resistance. Currently, there are a limited number of antibiotics in the treatment repertoire of existing bacterial infections. Among them, rifampicin is a broad-spectrum antibiotic against various bacterial pathogens. However, during rifampicin exposure, the appearance of persisters or resisters decreases its efficacy. Hence, to benefit more from rifampicin, its current standard dosage might be reconsidered and explored using both computational tools and experimental or clinical studies. In this study, we present the mathematical relationship between the concentration of rifampicin and the growth and killing kinetics of Escherichia coli cells. We generated time-killing curves of E. coli cells in the presence of 4, 16, and 32 µg/mL rifampicin exposures. We specifically focused on the oscillations with decreasing amplitude over time in the growth and killing kinetics of rifampicin-exposed E. coli cells. We propose the solution form of a second-order linear differential equation for a damped oscillator to represent the mathematical relationship. We applied a nonlinear curve fitting solver to time-killing curve data to obtain the model parameters. The results show a high fitting accuracy.

Quantifying Deformation and Migration Properties of U87 Glioma Cells Using Dielectrophoretic Forces.

Glioblastoma multiforme is one of the most aggressive malignant primary brain tumors. To design effective treatment strategies, we need to better understand the behavior of glioma cells while maintaining their genetic and phenotypic stability. Here, we investigated the deformation and migration profile of U87 Glioma cells under the influence of dielectrophoretic forces. We fabricated a gold microelectrode array within a microfluidic channel and applied sinusoidal wave AC potential at 3 Vpp, ranging from 30 kHz to 10 MHz frequencies, to generate DEP forces. We followed the dielectrophoretic movement and deformation changes of 100 glioma cells at each frequency. We observed that the mean dielectrophoretic displacements of glioma cells were significantly different at varying frequencies with the maximum and minimum traveling distances of 13.22 µm and 1.37 µm, respectively. The dielectrophoretic deformation indexes of U87 glioma cells altered between 0.027-0.040. It was 0.036 in the absence of dielectrophoretic forces. This approach presents a rapid, robust, and sensitive characterization method for quantifying membrane deformation of glioma cells to determine the state of the cells or efficacy of administrated drugs.

Optimization of Loop-Mediated Isothermal Amplification (LAMP) reaction mixture for biosensor applications.

Genetically Modified (GM) foods are becoming the future of agriculture on surviving global natural disasters and climate change by their enhanced production efficiency and improved functional properties. On the other hand, their adverse health and environmental effects, ample evidence on transgene leakage of Genetically Modified Organisms (GMOs) to crops have raised questions on their benefits and risks. Consequently, low-cost, reliable, rapid, and practical detection of GMOs have been important. GMO-detection platforms should be capable of stably storing detection reagents for long-delivery distances with varying ambient temperatures. In this study, we developed an event-specific, closed tube colorimetric GMO detection method based on Loop-Mediated Isothermal Amplification (LAMP) technique which can be integrated into GMO-detection platforms. The entire detection process optimized to 30 min and isothermally at 65 °C. The durability of the LAMP mixture in the test tubes showed that the LAMP reaction mixture, in which Bst polymerase and DNA sample was later included, yielded DNA amplicons for 3 days at room temperature, and for 6 days at 4 °C.•Simple, stable, and cheap storage method of LAMP reaction mixture for GMO-detection technologies.•GMO-detection platforms can stably store detection reagents for long-delivery distances with varying ambient temperatures.•Any DNA sample can be used in the field or resource-limited setting by untrained personnel.

Revealing Antibiotic Tolerance of the Mycobacterium smegmatis Xanthine/Uracil Permease Mutant Using Microfluidics and Single-Cell Analysis.

To reveal rare phenotypes in bacterial populations, conventional microbiology tools should be advanced to generate rapid, quantitative, accurate, and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations, microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells, we applied both conventional assays and a microfluidic-microscopy method to reveal the antibiotic tolerance mechanisms of wild-type and msm2570::Tn mutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared to wild-type cells. This is the first study to characterize the antibiotic tolerance phenotype of the msm2570::Tn mutant, which has a transposon insertion in the msm2570 gene-encoding a putative xanthine/uracil permease, which functions in the uptake of nitrogen compounds during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particular, antibiotics that target cell wall integrity.

Single-Cell Mechanophenotyping in Microfluidics to Evaluate Behavior of U87 Glioma Cells.

Integration of microfabricated, single-cell resolution and traditional, population-level biological assays will be the future of modern techniques in biology that will enroll in the evolution of biology into a precision scientific discipline. In this study, we developed a microfabricated cell culture platform to investigate the indirect influence of macrophages on glioma cell behavior. We quantified proliferation, morphology, motility, migration, and deformation properties of glioma cells at single-cell level and compared these results with population-level data. Our results showed that glioma cells obtained slightly slower proliferation, higher motility, and extremely significant deformation capability when cultured with 50% regular growth medium and 50% macrophage-depleted medium. When the expression levels of E-cadherin and Vimentin proteins were measured, it was verified that observed mechanophenotypic alterations in glioma cells were not due to epithelium to mesenchymal transition. Our results were consistent with previously reported enormous heterogeneity of U87 glioma cell line. Herein, for the first time, we quantified the change of deformation indexes of U87 glioma cells using microfluidic devices for single-cells analysis.

Quantifying Heterogeneity According to Deformation of the U937 Monocytes and U937-Differentiated Macrophages Using 3D Carbon Dielectrophoresis in Microfluidics.

A variety of force fields have thus far been demonstrated to investigate electromechanical properties of cells in a microfluidic platform which, however, are mostly based on fluid shear stress and may potentially cause irreversible cell damage. This work presents dielectric movement and deformation measurements of U937 monocytes and U937-differentiated macrophages in a low conductive medium inside a 3D carbon electrode array. Here, monocytes exhibited a crossover frequency around 150 kHz and presented maximum deformation index at 400 kHz and minimum deformation index at 1 MHz frequencies at 20 Vpeak-peak. Although macrophages were differentiated from monocytes, their crossover frequency was lower than 50 kHz at 10 Vpeak-peak. The change of the deformation index for macrophages was more constant and lower than the monocyte cells. Both dielectric mobility and deformation spectra revealed significant differences between the dielectric responses of U937 monocytes and U937-differentiated macrophages, which share the same origin. This method can be used for label-free, specific, and sensitive single-cell characterization. Besides, damage of the cells by aggressive shear forces can, hence, be eliminated and cells can be used for downstream analysis. Our results showed that dielectric mobility and deformation have a great potential as an electromechanical biomarker to reliably characterize and distinguish differentiated cell populations from their progenitors.

Quantitative Investigation into the influence of intravenous fluids on human immune and cancer cell lines.

The effect of intravenous fluids (IVF) has been investigated clinically through the assessment of post-treatment reactions. However, the responses to IVF vary from patient-to-patient. It is important to understand the response of IVF treatment to be able to provide optimal IVF care. Herein, we investigated the impact of commonly used IVFs, Dextrose, NaCl and Ringer on different human cancer (HepG2 (liver hepatocellular carcinoma) and MCF7 (breast adenocarcinoma)) and immune cell lines (U937 (lymphoma) monocyte and macrophages). The effect of IVF exposure on single cells was characterized using hemocytometer, fluorescence microscopy and flow cytometry. Quantitative data on the viability and morphology of the cells were obtained. Our results emphasize that different IVFs demonstrate important differences in how they influence distinct cell lines. Particularly, we observed that the lactated ringer and dextrose solutions altered the viability and nuclear size of cancer and immune cells differently. Our findings present valuable information to the knowledge of cellular-level IVF effects for further investigations in IVF usage on diverse patient populations and support the importance and necessity of developing optimal diluents not only for drug stability but also for patient benefits.

Single Cell Level Dielectrophoretic Responses & Dielectrophoretic Deformations of Monocytes to Quantify Population Heterogeneity.

Single-cell dielectrophoretic movement and dielectrophoretic deformation of monocyte cells were interrogated applying 20 Vpp, 50 kHz to 1 MHz signal in the 3D carbon electrode array. Heterogeneity of the monocyte population is shown in terms of the crossover frequencies, translational movement, and deformation index of the cells. The results presented that crossover range for monocytes was 100 kHz - 200 kHz, the translational movement of the cells was rapidly altered when the initial positions of the cells were in the negative dielectrophoretic region. Finally, the deformation index of the monocyte population varied from 0.5 to 1.5.

THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress Analysis.

To elucidate dynamic developmental processes in plants, live tissues and organs must be visualised frequently and for extended periods. The development of roots is studied at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualisation and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualisation through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 h. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system's capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.

DaimonDNA: A portable, low-cost loop-mediated isothermal amplification platform for naked-eye detection of genetically modified organisms in resource-limited settings.

The steady increase in commercialization of genetically modified organisms (GMOs) demands low-cost, rapid and portable GMO-detection methods that are technically and economically sustainable. Traditional nucleic acid detection platforms are still expensive, immobile and generate complex read-outs to be analyzed by experienced personal. Herein, we report the development of a portable, rapid and user-friendly GMO-detection biosensor, DaimonDNA. The system specifically amplifies the target DNA using loop-mediated isothermal amplification (LAMP) and provides real-time, naked-eye detection with Hydroxynaphthol blue reagent in less than 30 min. The construction of the platform relies on 3D printing and off-the-shelf electronic components that makes it extremely low-cost (<25 Euro), light weight (108 g), mobile (6 × 6 × 3 cm) and suitable for field deployment. We present the detection of the soybean lectin gene as a species control, and P35S as a transgene element found in many GMO varieties. We confirmed specificity of the DaimonDNA biosensor using" RoundUp Ready (RRS)" and MON89788 soybean genomic DNA with P35S and lectin primer sets. We characterized sensitivity of our system using 76.92, 769.2 and 7692 copies of RRS soybean genomic DNA in a non-GMO background. We benchmarked the DNA amplification and detection efficiency of our system against a thermocycling machine by quantifying the images obtained from gel electrophoresis and showed that our system is comparable to most other reported isothermal amplification techniques. This system can also be used for widespread point-of-care or field-based testing that is infrequently performed due to the lack of rapid, inexpensive, user-friendly and portable methods.

On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy.

Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.

Quantitative Investigation for the Dielectrophoretic Effect of Fluorescent Dyes at Single-Cell Resolution.

Most of the microscopy-based, quantitative assays rely on fluorescent dyes. In this study, we investigated the impact of fluorescent dyes on the dielectrophoretic response of the mammalian cells. The dielectrophoretic measurements were performed to quantify whether the fluorescent dyes alter the dielectrophoretic properties of the cells at single-cell resolution. Our results present that when 10 Vpp electric field is applied, the fluorescent-labeled cells experienced the crossover frequency at 8-10 kHz, whereas the label-free cells exhibited at 16-18 kHz.

Isoniazid Killing of Mycobacterium smegmatis NADH Pyrophosphatase Mutant at Single-Cell Level using Microfluidics and Time-Lapse Microscopy.

We introduce single-cell analysis for isoniazid-treated Mycobacterium smegmatis mutant, msm1946-NADH pyrophosphatase, using microfluidics and automated time-lapse microscopy. Mycobacterial NADH pyrophosphatase isoforms play an important role for the mechanism of isoniazid and ethionamide activation. Our single-cell analysis revealed important insights on isoniazid killing mechanism that was masked by traditional killing assays, raised significant questions related to viable but non-culturable subpopulation of cells, and existing methods that defines minimum inhibitory concentration of drugs. The major goal of this study was quantitatively analyze bacterial cell parameters to obtain high-resolution data for the time evolution of antibiotic killing at the single-cell level. The presented tools and methods could be applied to the closely related organisms to provide more detailed information for the design and employment of antibiotic treatments.