Cellular subpopulations identified using an ensemble average of multiple dielectrophoresis measurements.

While the average value measurement approach can successfully analyze and predict the general behavior and biophysical properties of an isogenic cell population, it fails when significant differences among individual cells are generated in the population by intracellular changes such as the cell cycle, or different cellular responses to certain stimuli. Detecting such single-cell differences in a cell population has remained elusive. Here, we describe an easy-to-implement and generalizable platform that measures the dielectrophoretic cross-over frequency of individual cells by decreasing measurement noise with a stochastic method and computing ensemble average statistics. This platform enables multiple, real-time, label-free detection of individual cells with significant dielectric variations over time within an isogenic cell population. Using a stochastic method in combination with the platform, we distinguished cell subpopulations from a mixture of drug-untreated and -treated isogenic cells. Furthermore, we demonstrate that our platform can identify drug-treated isogenic cells with different recovery rates.

The spatial distribution of physicochemical parameters in coastal sediments along the Bay of Bengal Coastal Zone with statistical analysis.

The current study focused on the monitoring of pollution loads in the Kalpakkam coastal zone of India in terms of physico-chemical characteristics of sediment. The investigation took place at 12 sampling points around the Kalpakkam coastal zone for one year beginning from 2019. The seasonal change of nutrients in the sediment, such as nitrogen, phosphorus, potassium, total organic carbon, and particles size distribution, was calculated. Throughout the study period, the pH (7.55 to 8.99), EC (0.99 to 4.98 dS/m), nitrogen (21.74 to 58.12 kg/ha), phosphorus (7.5 to 12.9 kg/ha), potassium (218 to 399 kg/ha), total organic carbon (0.11 to 0.88%), and particle size cumulative percent of sediments (from 9.01 to 9.39%) was observed. A number of multivariate statistical techniques were used to examine the changes in sediment quality. The population means were substantially different according to the three-way ANOVA test at the 0.05 level. Principal component analysis and cluster analysis showed a substantial association with all indicators throughout all seasons, implying contamination from both natural and anthropogenic causes. The ecosystem of the Kalpakkam coastal zone has been affected by nutrient contamination.

Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds.

Formed by aberrant cell division, minicells possess functional metabolism despite their inability to grow and divide. Minicells exhibit not only superior stability when compared with bacterial cells but also exceptional tolerance-characteristics that are essential for a de novo bioreactor platform. Accordingly, we engineered minicells to accumulate protein, ensuring sufficient production capability. When tested with chemicals regarded as toxic against cells, the engineered minicells produced titers of C6-C10 alcohols and esters, far surpassing the corresponding production from bacterial cells. Additionally, microbial autoinducer production that is limited in expanding bacterial population was conducted in the minicells. Because bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-responsive gene circuits. When bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-based gene circuits with the minicells. This study demonstrates the potential of minicells as bioreactors suitable for products with known limitations in microbial production, thus providing new possibilities for bioreactor engineering.

DC corral trapping of single nanoparticles and macromolecules in solution.

Progress in sorting, separating, and characterizing ever smaller amounts of chemical and biological material depends on the availability of methods for the controlled interaction with nanoscale and molecular-size objects. Here, we report on the reversible, tunable trapping of single DNA molecules and other charged micro- and nanoparticles in aqueous solution using a direct-current (DC) corral trap setup. The trap consists of a circular, non-conductive void in a metal-coated surface that, when charged, generates an electrostatic potential well in the proximate solution. Our results demonstrate that stable, nanoscale confinement of charged objects is achievable over extended periods of time, that trap stiffness is controlled by the applied voltage, and that simultaneous trapping of multiple objects is feasible. The approach shows great promise for lab-on-a-chip systems and biomedical applications due to its simplicity, scalability, selectivity, and the capability to manipulate single DNA molecules in standard buffer solutions.

Ultra-sensitive dielectrophoretic surface charge multiplex detection inside a micro-dielectrophoretic device.

Label-free dielectrophoretic force-based surface charge detection has shown great potential for highly sensitive and selective sensing of metal ions and small biomolecules. However, this method suffers from a complex calibration process and measurement signal interference in simultaneous multi-analyte detection, thus creating difficulties in multiplex detection. We have developed a method to overcome these issues based on the optical discrimination of the dielectrophoretic behaviors of multiple microparticle probes considering the surface charge difference before and after self-assembling conjugation. In this report, we demonstrate and characterize this dielectrophoretic force-based surface charge detection method with particle probes functionalized by various biomolecules. This technique achieved an attomolar limit of detection (LOD) for Hg2+ in distilled water and a femtomolar LOD in drinking water using DNA aptamer-functionalized particle probes. More importantly, using two different DNA aptamer-functionalized particle probes for Hg2+ and Ag+, label-free dielectrophoretic multiplex detection of these species in drinking water with a femtomolar and a nanomolar LOD was achieved for the first time.

Effect of geometry on dielectrophoretic trap stiffness in microparticle trapping.

Dielectrophoresis, an electrokinetic technique, can be used for contactless manipulation of micro- and nano-size particles suspended in a fluid. We present a 3-D microfluidic DEP device with an orthogonal electrode configuration that uses negative dielectrophoresis to trap spherical polystyrene micro-particles. Traps with three different basic geometric shapes, i.e. triangular, square, and circular, and a fixed trap area of around 900 µm2 were investigated to determine the effect of trap shape on dynamics and strength of particle trapping. Effects of trap geometry were quantitatively investigated by means of trap stiffness, with applied electric potentials from 6 VP-P to 10 VP-P at 1 MHz. Analyzing the trap stiffness with a trapped 4.42 µm spherical particle showed that the triangular trap is the strongest, while the square shape trap is the weakest. The trap stiffness grew more than eight times in triangular traps and six times in both square and circular traps when the potential of the applied electric field was increased from 6 VP-P to 10 VP-P at 1 MHz. With the maximum applied potential, i.e. 10 VP-P at 1 MHz, the stiffness of the triangular trap was 60% and 26% stronger than the square and circular trap, respectively. A finite element model of the microfluidic DEP device was developed to numerically compute the DEP force for these trap shapes. The findings from the numerical computation demonstrate good agreement with the experimental analysis. The analysis of three different trap shapes provides important insights to predict trapping location, strength of the trapping zone, and optimized geometry for high throughput particle trapping.

Metabolic engineering of Methylorubrum extorquens AM1 for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production using formate.

Formate is a promising environmentally friendly and sustainable feedstock synthesized from syngas or carbon dioxide. Methylorubrum extorquens is a type II methylotroph that can use formate as a carbon source. It accumulates polyhydroxyalkanoates (PHAs) inside the cell, mainly producing poly-3-hydroxybutyrate (PHB), a degradable biopolymer. Owing to its high melting point and stiff nature, however, mechanical property improvement is warranted in the form of copolymerization. To produce the PHA copolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the endogenous gene phaC was deleted and the pathway genes bktB, phaJ1, and phaC2, with broader substrate specificities, were heterologously expressed. To improve the incorporation of 3-hydroxyvalerate (3HV), the expression level of bktB was improved by untranslated region (UTR) engineering, and the endogenous gene phaA was deleted. The engineered M. extorquens produced PHBV with 8.9% 3HV using formate as the sole carbon source. In addition, when propionate and butyrate were supplemented, PHBVs with 3HV portions of up to 70.6% were produced. This study shows that a PHBV copolymer with a high proportion of 3HV can be synthesized using formate, a C1 carbon source, through metabolic engineering and supplementation with short-chain fatty acids.

Quantitative analysis of the three-dimensional trap stiffness of a dielectrophoretic corral trap.

Dielectrophoresis is a robust approach for the manipulation and separation of (bio)particles using microfluidic platforms. We developed a dielectrophoretic corral trap in a microfluidic device that utilizes negative dielectrophoresis to capture single spherical polystyrene particles. Circular-shaped micron-size traps were employed inside the device and the three-dimensional trap stiffness (restoring trapping force from equilibrium trapping location) was analyzed using 4.42 µm particles and 1 MHz of an alternating electric field from 6 VP-P to 10 VP-P . The trap stiffness increased exponentially in the x- and y-direction, and linearly in the z-direction. Image analysis of the trapped particle movements revealed that the trap stiffness is increased 608.4, 539.3, and 79.7% by increasing the voltage from 6 VP-P to 10 VP-P in the x-, y-, and z-direction, respectively. The trap stiffness calculated from a finite element simulation of the device confirmed the experimental results. This analysis provides important insights to predict the trapping location, strength of the trapping, and optimum geometry for single particle trapping and its applications such as single-molecule analysis and drug discovery.

Variable Membrane Dielectric Polarization Characteristic in Individual Live Cells.

Investigation of the dielectric properties of cell membranes plays an important role in understanding the biological activities that sustain cellular life and realize cellular functionalities. Herein, the variable dielectric polarization characteristics of cell membranes are reported. In controlling the dielectric polarization of a cell using dielectrophoresis force spectroscopy, different cellular crossover frequencies were observed by modulating both the direction and sweep rate of the frequency. The crossover frequencies were used for the extraction of the variable capacitance, which is involved in the dielectric polarization across the cell membranes. In addition, this variable phenomenon was investigated by examining cells whose membranes were cholesterol-depleted with methyl-ß-cyclodextrin, which verified a strong correlation between the variable dielectric polarization characteristics and membrane composition changes. This study presented the dielectric polarization properties in live cells' membranes that can be modified by the regulation of external stimuli and provided a powerful platform to explore cellular membrane dielectric polarization.

Localized Dielectric Loss Heating in Dielectrophoresis Devices.

Temperature increases during dielectrophoresis (DEP) can affect the response of biological entities, and ignoring the effect can result in misleading analysis. The heating mechanism of a DEP device is typically considered to be the result of Joule heating and is overlooked without an appropriate analysis. Our experiment and analysis indicate that the heating mechanism is due to the dielectric loss (Debye relaxation). A temperature increase between interdigitated electrodes (IDEs) has been measured with an integrated micro temperature sensor between IDEs to be as high as 70 °C at 1.5 MHz with a 30 Vpp applied voltage to our ultra-low thermal mass DEP device. Analytical and numerical analysis of the power dissipation due to the dielectric loss are in good agreement with the experiment data.

Nanoinjection system for precise direct delivery of biomolecules into single cells.

Intracellular delivery of functional molecules such as proteins, transcription factors and DNA is effective and promising in cell biology. However, existing transfection methods are often unsuitable to deliver big molecules into cells or require carriers such as viruses and peptides specific to the target molecules. In addition, the nature of bulk processing does not generally provide accurate dose control of individual cells. The concept of single-cell-based material injection based on electrokinetic pumping through nanocapillaries could overcome these problems, yet the fabrication and operation of nanoscale 3-dimensional structures have remained unsolved. In this research, a hybrid (PDMS/glass) microfluidic chip with a true 3-dimensional nanoinjection structure (called "nanoinjection system") is presented. The nanoinjection structure was fabricated by femtosecond-laser (fs-laser) ablation in a single solid glass, which showed very successful delivery of red fluorescent protein (RFP) and expression of plasmid DNA in several different types of cells. This system is promising in that the amount of molecules to be delivered is controllable and the processed cells are systematically separated into a harvesting chamber, which can radically improve the purity of the processed cells. In addition, it was confirmed that the cells were healthy even after the molecule injection for a few seconds, indicating that the injection time can be significantly elongated, further improving the delivery efficiency of biomolecules without affecting the cell viability. We envision that the nanoinjection system having the major features of being carrier-free and dose-controllable, having an unlimited injection period, and ease of harvesting will greatly contribute to the next-generation research studies in the fields of cell biology and cell therapeutics.

Development of a Protein Microarray Chip with Enhanced Fluorescence for Identification of Semen and Vaginal Fluid.

The detection of body fluids has been used to identify a suspect and build a criminal case. As the amount of evidence collected at a crime site is limited, a multiplex identification system for body fluids using a small amount of sample is required. In this study, we proposed a multiplex detection platform using an Ag vertical nanorod metal enhanced fluorescence (MEF) substrate for semen and vaginal fluid (VF), which are important evidence in cases of sexual crime. The Ag nanorod MEF substrate with a length of 500 nm was fabricated by glancing angle deposition, and amino functionalization was conducted to improve binding ability. The effect of incubation time was analyzed, and an incubation time of 60 min was selected, at which the fluorescence signal was saturated. To assess the performance of the developed identification chip, the identification of semen and VF was carried out. The developed sensor could selectively identify semen and VF without any cross-reactivity. The limit of detection of the fabricated microarray chip was 10 times better than the commercially available rapid stain identification (RSID) Semen kit.

Automated Dielectrophoretic Tweezers-Based Force Spectroscopy System in a Microfluidic Device.

We reported an automated dielectrophoretic (DEP) tweezers-based force spectroscopy system to examine intermolecular weak binding interactions, which consists of three components: (1) interdigitated electrodes and micro-sized polystyrene particles used as DEP tweezers and probes inside a microfluidic device, along with an arbitrary function generator connected to a high voltage amplifier; (2) microscopy hooked up to a high-speed charge coupled device (CCD) camera with an image acquisition device; and (3) a computer aid control system based on the LabVIEW program. Using this automated system, we verified the measurement reliability by measuring intermolecular weak binding interactions, such as hydrogen bonds and Van der Waals interactions. In addition, we also observed the linearity of the force loading rates, which is applied to the probes by the DEP tweezers, by varying the number of voltage increment steps and thus affecting the linearity of the force loading rates. This system provides a simple and low-cost platform to investigate intermolecular weak binding interactions.

Probing the modulated formation of gold nanoparticles-beta-lactoglobulin corona complexes and their applications.

Understanding the interactions between proteins and nanoparticles (NPs) along with the underlying structural and dynamic information is of utmost importance to exploit nanotechnology for biomedical applications. Upon adsorption onto a NP surface, proteins form a well-organized layer, termed the corona, that dictates the identity of the NP-protein complex and governs its biological pathways. Given its high biological relevance, in-depth molecular investigations and applications of NPs-protein corona complexes are still scarce, especially since different proteins form unique corona patterns, making identification of the biomolecular motifs at the interface critical. In this work, we provide molecular insights and structural characterizations of the bio-nano interface of a popular food-based protein, namely bovine beta-lactoglobulin (ß-LG), with gold nanoparticles (AuNPs) and report on our investigations of the formation of corona complexes by combined molecular simulations and complementary experiments. Two major binding sites in ß-LG were identified as being driven by citrate-mediated electrostatic interactions, while the associated binding kinetics and conformational changes in the secondary structures were also characterized. More importantly, the superior stability of the corona led us to further explore its biomedical applications, such as in the smartphone-based point-of-care biosensing of Escherichia coli (E. coli) and in the computed tomography (CT) of the gastrointestinal (GI) tract through oral administration to probe GI tolerance and functions. Considering their biocompatibility, edible nature, and efficient excretion through defecation, AuNPs-ß-LG corona complexes have shown promising perspectives for future in vitro and in vivo clinical settings.

Difference in F-18 FDG Uptake After Esophagogastroduodenoscopy and Colonoscopy in Healthy Sedated Subjects.

PURPOSE: We aimed to evaluate the difference in fluorodeoxyglucose (FDG) uptake in sedated healthy subjects after they underwent esophagogastroduodenoscopy (EGD) and colonoscopy procedures. METHODS: The endoscopy group (n = 29) included healthy subjects who underwent screening via F-18 FDG positron emission tomography/computed tomography (PET/CT) after an EGD and/or colonoscopy under sedation on the same day. The control group (n = 35) included healthy subjects who underwent screening via PET/CT only. FDG uptake in the tongue, uvula, epiglottis, vocal cords, esophagus, stomach, duodenum, liver, cecum, colon, anus, and muscle were compared between the two groups. RESULTS: Maximum standardized uptake value (SUVmax) in the tongue, pharynx, larynx, and esophagus did not significantly differ between the endoscopy and control groups. In contrast, mean SUVmax in the whole stomach was 18 % higher in the endoscopy group than in the control group (SUVmax: 2.96 vs. 2.51, P = 0.010). In the lower gastrointestinal track, SUVmax from the cecum to the rectum was not significantly different between the two groups, whereas SUVmax in the anus was 20 % higher in the endoscopy group than in the control group (SUVmax: 4.21 vs. 3.50, P = 0.002). SUVmax in the liver and muscle was not significantly different between the two groups. Mean volume of the stomach and mean cross section of the colon was significantly higher in the endoscopy group than in the control group (stomach: 313.28 cm3 vs. 209.93 cm3, P < 0.001, colon: 8.82 cm2 vs. 5.98 cm2, P = 0.001). CONCLUSIONS: EGD and colonoscopy under sedation does not lead to significant differences in SUVmax in most parts of the body. Only gastric FDG uptake in the EGD subjects and anal FDG uptake in the colonoscopy subjects was higher than uptake in those regions in the control subjects.

Fluorescence laminar optical tomography for brain imaging: system implementation and performance evaluation.

We present our effort in implementing a fluorescence laminar optical tomography scanner which is specifically designed for noninvasive three-dimensional imaging of fluorescence proteins in the brains of small rodents. A laser beam, after passing through a cylindrical lens, scans the brain tissue from the surface while the emission signal is captured by the epi-fluorescence optics and is recorded using an electron multiplication CCD sensor. Image reconstruction algorithms are developed based on Monte Carlo simulation to model light­tissue interaction and generate the sensitivity matrices. To solve the inverse problem, we used the iterative simultaneous algebraic reconstruction technique. The performance of the developed system was evaluated by imaging microfabricated silicon microchannels embedded inside a substrate with optical properties close to the brain as a tissue phantom and ultimately by scanning brain tissue in vivo. Details of the hardware design and reconstruction algorithms are discussed and several experimental results are presented. The developed system can specifically facilitate neuroscience experiments where fluorescence imaging and molecular genetic methods are used to study the dynamics of the brain circuitries.

Iridium Oxide-reduced Graphene Oxide Nanohybrid Thin Film Modified Screen-printed Electrodes as Disposable Electrochemical Paper Microfluidic pH Sensors.

A facile, controllable, inexpensive and green electrochemical synthesis of IrO2-graphene nanohybrid thin films is developed to fabricate an easy-to-use integrated paper microfluidic electrochemical pH sensor for resource-limited settings. Taking advantages from both pH meters and strips, the pH sensing platform is composed of hydrophobic barrier-patterned paper micropad (µPAD) using polydimethylsiloxane (PDMS), screen-printed electrode (SPE) modified with IrO2-graphene films and molded acrylonitrile butadiene styrene (ABS) plastic holder. Repetitive cathodic potential cycling was employed for graphene oxide (GO) reduction which can completely remove electrochemically unstable oxygenated groups and generate a 2D defect-free homogeneous graphene thin film with excellent stability and electronic properties. A uniform and smooth IrO2 film in nanoscale grain size is anodically electrodeposited onto the graphene film, without any observable cracks. The resulting IrO2-RGO electrode showed slightly super-Nernstian responses from pH 2-12 in Britton-Robinson (B-R) buffers with good linearity, small hysteresis, low response time and reproducibility in different buffers, as well as low sensitivities to different interfering ionic species and dissolved oxygen. A simple portable digital pH meter is fabricated, whose signal is measured with a multimeter, using high input-impedance operational amplifier and consumer batteries. The pH values measured with the portable electrochemical paper-microfluidic pH sensors were consistent with those measured using a commercial laboratory pH meter with a glass electrode.

Convex Grooves in Staggered Herringbone Mixer Improve Mixing Efficiency of Laminar Flow in Microchannel.

The liquid streams in a microchannel are hardly mixed to form laminar flow, and the mixing issue is well described by a low Reynolds number scheme. The staggered herringbone mixer (SHM) using repeated patterns of grooves in the microchannel have been proved to be an efficient passive micro-mixer. However, only a negative pattern of the staggered herringbone mixer has been used so far after it was first suggested, to the best of our knowledge. In this study, the mixing efficiencies from negative and positive staggered herringbone mixer patterns as well as from opposite flow directions were tested to investigate the effect of the micro-structure geometry on the surrounding laminar flow. The positive herringbone pattern showed better mixing efficiency than the conventionally used negative pattern. Also, generally used forward flow gives better mixing efficiency than reverse flow. The mixing was completed after two cycles of staggered herringbone mixer with both forward and reverse flow in a positive pattern. The traditional negative pattern showed complete mixing after four and five cycles in forward and reverse flow direction, respectively. The mixing effect in all geometries was numerically simulated, and the results confirmed more efficient mixing in the positive pattern than the negative. The results can further enable the design of a more efficient microfluidic mixer, as well as in depth understanding of the phenomena of positive and negative patterns existing in nature with regards to the surrounding fluids.

Biaxial Dielectrophoresis Force Spectroscopy: A Stoichiometric Approach for Examining Intermolecular Weak Binding Interactions.

The direct quantification of weak intermolecular binding interactions is very important for many applications in biology and medicine. Techniques that can be used to investigate such interactions under a controlled environment, while varying different parameters such as loading rate, pulling direction, rupture event measurements, and the use of different functionalized probes, are still lacking. Herein, we demonstrate a biaxial dielectrophoresis force spectroscopy (BDFS) method that can be used to investigate weak unbinding events in a high-throughput manner under controlled environments and by varying the pulling direction (i.e., transverse and/or vertical axes) as well as the loading rate. With the BDFS system, we can quantitatively analyze binding interactions related to hydrogen bonding or ionic attractions between functionalized microbeads and a surface within a microfluidic device. Our BDFS system allowed for the characterization of the number of bonds involved in an interaction, bond affinity, kinetic rates, and energy barrier heights and widths from different regimes of the energy landscape.

Characterizing exons and introns by regularity of nucleotide strings.

BACKGROUND: Translation of nucleotides into a numeric form has been approached in many ways and has allowed researchers to investigate the properties of protein-coding sequences and noncoding sequences. Typically, more pronounced long-range correlations and increased regularity were found in intron-containing genes and in non-transcribed regulatory DNA sequences, compared to cDNA sequences or intron-less genes. The regularity is assessed by spectral tools defined on numerical translates. In most popular approaches of numerical translation the resulting spectra depend on the assignment of numerical values to nucleotides. Our contribution is to propose and illustrate a spectra which remains invariant to the translation rules used in traditional approaches. RESULTS: We outline a methodology for representing sequences of DNA nucleotides as numeric matrices in order to analytically investigate important structural characteristics of DNA. This representation allows us to compute the 2-dimensional wavelet transformation and assess regularity characteristics of the sequence via the slope of the wavelet spectra. In addition to computing a global slope measure for a sequence, we can apply our methodology for overlapping sections of nucleotides to obtain an "evolutionary slope." To illustrate our methodology, we analyzed 376 gene sequences from the first chromosome of the honeybee. CONCLUSION: For the genes analyzed, we find that introns are significantly more regular (lead to more negative spectral slopes) than exons, which agrees with the results from the literature where regularity is measured on "DNA walks". However, unlike DNA walks where the nucleotides are assigned numerical values depending on nucleotide characteristics (purine-pyrimidine, weak-strong hydrogen bonds, keto-amino, etc.) or other spatial assignments, the proposed spectral tool is invariant to the assignment of nucleotides. Thus, ambiguity in numerical translation of nucleotides is eliminated.