Comparison between conventional-dose and high-dose rocuronium use in general anesthesia for Cesarean section.

Background: There have been few studies comparing the effects of high- and low-dose rocuronium during cesarean section by directly measuring the concentration. Therefore, we conducted a study to examine the blood concentrations and clinical effects of both doses of rocuronium on mothers and fetuses. Methods: Eighteen patients were randomly assigned to two groups: C Group (0.6 mg/kg), and H Group, (1.0 mg/kg). The primary outcome was the comparison of umbilical vein rocuronium concentration between two groups. We assessed ease of intubation, time from rocuronium administration to some TOF points, post-anesthesia care unit (PACU) stay time, infused remifentanil dose, maternal rocuronium concentration, and Apgar scores. Results: No differences were observed in demographic data, ease of intubation, PACU stay time, 1 min Apgar scores, umbilical venous blood gas analysis between both groups. However, the time from rocuronium administration to T3 disappearance was shorter (p=0.009) and time to T1 and T2 reappearance were longer (p=0.003, p=0.009) in H group than that in C group. The administered remifentanil dose (p=0.042) was lower in the H group than in the C group. Rocuronium concentrations in the umbilical vein (p=0.004) and maternal vein before cord clamping (p=0.002) and at discharge (p<0.001) were also found to be higher in the H group than in the C group. Conclusions: We observed no prolongation of PACU stay, and no differences in Apgar scores in H group compared to C group. It suggests that 1.0 mg/kg of rocuronium has no negative effects on the fetus and mother in cesarean section.

Dynamic Mitochondrial Migratory Features Associated with Calcium Responses during T Cell Antigen Recognition.

A T cell clone is able to distinguish Ags in the form of peptide-MHC complexes with high specificity and sensitivity; however, how subtle differences in peptide-MHC structures translate to distinct T cell effector functions remains unknown. We hypothesized that mitochondrial positioning and associated calcium responses play an important role in T cell Ag recognition. We engineered a microfluidic system to precisely manipulate and synchronize a large number of cell-cell pairing events, which provided simultaneous real-time signaling imaging and organelle tracking with temporal precision. In addition, we developed image-derived metrics to quantify calcium response and mitochondria movement. Using myelin proteolipid altered peptide ligands and a hybridoma T cell line derived from a mouse model of experimental autoimmune encephalomyelitis, we observed that Ag potency modulates calcium response at the single-cell level. We further developed a partial least squares regression model, which highlighted mitochondrial positioning as a strong predictor of calcium response. The model revealed T cell mitochondria sharply alter direction within minutes following exposure to agonist peptide Ag, changing from accumulation at the immunological synapse to retrograde movement toward the distal end of the T cell body. By quantifying mitochondria movement as a highly dynamic process with rapidly changing phases, our result reconciles conflicting prior reports of mitochondria positioning during T cell Ag recognition. We envision applying this pipeline of methodology to study cell interactions between other immune cell types to reveal important signaling phenomenon that is inaccessible because of data-limited experimental design.

Evaluation of MARK BTM for Quantitative Measurement of Three Tumor Markers: Prostate Specific Antigen, Alpha Fetoprotein, and Carcinoembryonic Antigen.

BACKGROUND: Tumor marker assays have played a crucial role for screening cancers and monitoring cancer patients, as they reflect the status and prognosis of patients. Alpha fetoprotein (AFP), prostate specific antigen (PSA), and carcinoembryonic antigen (CEA) are the most commonly used tumor marker proteins. The MARK BTM immunoassay system is a novel platform based on magnetic nanoparticles and electrochemical immunoassay. METHODS: The analytical performance of MARK BTM immunoassay system for determination of AFP, PSA, and CEA are evaluated. Comparisons of methods are also conducted by comparing the assay results of MARK BTM immunoassay system to that of cobas e 801 system. RESULTS: The MARK BTM immunoassay system provides within-run, between-run, and between-day precisions for the three tumor markers, ranging from 1.13 - 7.46%. Data measured by the MARK BTM immunoassay system show high correlation with that of the cobas e 801 system, with a linear slope ranging from 0.966 to 1.042 and a correlation coefficient of r > 0.996 for the three markers. CONCLUSIONS: These results show that the MARK BTM immunoassay system can be used for the quantitative measurements of AFP, PSA, and CEA in clinical practice.

Mobile diagnostics: next-generation technologies for in vitro diagnostics.

The emergence of a wide range of applications of smartphones along with advances in 'liquid biopsy' has significantly propelled medical research particularly in the field of in vitro diagnostics (IVD). Herein, we have presented a detailed analysis of IVD, its associated critical concerns and probable solutions. It also demonstrates the transition in terms of analytes from minimally invasive (blood) to non-invasive (urine, saliva and sweat) and depicts how the different features of a smartphone can be integrated for specific diagnostic purposes. This review basically highlights recent advances in the applications of smartphone-based biosensors in IVD taking into account the following factors: accuracy and portability; quantitative and qualitative analysis; and centralization and decentralization tests. Furthermore, the critical concerns and future direction of diagnostics based on smartphones are also discussed.

Productive chemical interaction between a bacterial microcolony couple is enhanced by periodic relocation.

This paper describes a system to study how small physical perturbations can affect bacterial community behavior in unexpected ways through modulation of diffusion and convective transport of chemical communication molecules and resources. A culture environment that mimics the chemically open characteristic of natural bacterial habitats but with user-defined spatiotemporal control of bacteria microcolonies is realized through use of an aqueous two phase system (ATPS). The ATPS is formulated with nontoxic dextran (DEX) and poly(ethylene glycol) (PEG) dissolved in cell culture media. DEX-phase droplets formed within a bulk PEG-phase stably confine the bacteria within it while small molecules diffuse relatively freely. Bacteria-containing DEX droplets can also be magnetically relocated, without loss of its bacterial content, when DEX-conjugated magnetic particles are included. We found that decreasing the distance between quorum-sensing (QS)-coupled microcolonies increased green fluorescent protein (GFP) expression due to increased inter-colony chemical communication but with upper limits. Periodic relocation of the chemical signal receiver colony, however, increased GFP expression beyond these typical bounds predicted by quorum sensing concepts alone by maintaining inter-colony chemical communication while also relieving the colony of short-range resource depletion effects. Computer simulations suggest that such increased productive output in response to periodic nonlethal physical perturbations is a common feature of chemically activated interactive cell systems where there is also a short-range inhibitory effect. In addition to providing insights on the effect of bacteria relocation, the magnetic ATPS droplet manipulation capability should be broadly useful for bioanalyses applications where selective partitioning at the microscale in fully aqueous conditions is needed.

Lab-on-a-disc for simultaneous determination of nutrients in water.

In this study, we describe a novel platform based on centrifugal microfluidics for simultaneous determination of nitrite, nitrate and nitrite, ammonium, orthophosphate, and silicate in water samples. All processes from sample metering to detection were integrated and automatically conducted on a rotating disc-shaped device. Fluid transfer was controlled by laser irradiation on the ferrowax-based microvalves. Liquid samples and reagents were pumped by centrifugal force in the rotating disc, and their positions and movements were controlled through a programmable light from a laser diode. This novel water analysis platform required only 500 µL of sample (100 µL for each nutrient) and 10-30 µL of reagents for colorimetric detection. In addition, the fully automated parallel processes and efficient mixing in the rotating disc allowed for a significant reduction in total analysis time (∼7 min 40 s) and increased accuracy. Validation with a seawater certified reference material indicated that the platform accurately measured nutrient concentrations in water samples. In addition, we showed that the nutrients in the seawater collected from Chunsu Bay in Korea measured by the proposed lab-on-a-disc and by a commercialized autoanalyzer are comparable.

Three dimensional multicellular co-cultures and anti-cancer drug assays in rapid prototyped multilevel microfluidic devices.

This report presents a multilevel microfluidic platform for robust construction of hydrogel scaffold in microchannels and its application to three dimensional (3D) multicellular co-cultures and assays. A new rapid prototyping method based on soft lithography using multi-layered adhesive tapes is also introduced. We have successfully cultured MCF-7 breast cancer cell line more than 11 days with > 98 % viability, and co-cultured MDA-MB-231 breast cancer cells and NIH/3T3 fibroblasts in separate compartments. This multilevel microfluidic device with a cell-laden hydrogel microstructure has also been applied for anticancer drug assays in multicellular niches. Here we tested the effect of estrogen receptor (ER) antagonist drug, tamoxifen, on the growth of ER positive MCF-7 cells in microchannels. The inhibitory effect of tamoxifen on the growth of MCF-7 cells was diminished when they were co-cultured with ER negative MDA-MB-231 cells. The rapid prototyped multilevel microfluidic devices would provide simple, easy to use, low cost, robust, and reproducible cell-based assay platforms for potential end-users such as biologists and pharmacists.

Lab-on-a-disc for fully integrated multiplex immunoassays.

This paper presents a cost-effective, rapid, and fully automated lab-on-a-disc for simultaneous detection of multiple protein biomarkers in raw samples such as whole blood or whole saliva. For the diagnosis of cardiovascular disease, here, a novel centrifugal microfluidic layout was designed to conduct the simultaneous detection of high sensitivity C-reactive protein, cardiac troponin I, and N-terminal pro-B type natriuretic peptide based on a bead-based sandwich type enzyme-linked immunosorbent assay (ELISA). Three reaction chambers are initially interconnected for the common processes such as sample injection, incubation, and washing and then isolated on-demand for the independent processes such as substrate incubation and final detection. The assay performances such as the limit of detection and the dynamic range were comparable with those of the conventional ELISA despite the significant reduction of the minimum sample volume (200 µL), the amount of washing buffer (700 µL), and the total process time (20 min).

Label-free cell separation using a tunable magnetophoretic repulsion force.

This paper describes a new label-free cell separation method using a magnetic repulsion force resulting from the magnetic susceptibility difference between cells and a paramagnetic buffer solution in a microchannel. The difference in the magnetic forces acting on different-sized cells is enhanced by adjusting the magnetic susceptibility of the surrounding medium, which depends on the concentration of paramagnetic salts, such as biocompatible gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), dissolved therein. As a proof-of-concept demonstration, Gd-DTPA solutions at concentrations of 0-80 mM were applied to separate U937 cells from red blood cells (RBCs) and to distinguish two different-sized polystyrene (PS) beads (8 and 10 µm in diameter). By increasing the Gd-DTPA concentration from 0 to 40 mM, the separation resolution of PS beads was increased from 0.08 to 0.91. Additionally, we successfully achieved label-free separation of U937 cells from RBCs with >90% purity and 1 × 10(5) cells/h throughput using a 40 mM Gd-DTPA solution.

Flexible fabrication and applications of polymer nanochannels and nanoslits.

Fluidic devices that employ nanoscale structures (<100 nm in one or two dimensions, slits or channels, respectively) are generating great interest due to the unique properties afforded by this size domain compared to their micro-scale counterparts. Examples of interesting nanoscale phenomena include the ability to preconcentrate ionic species at extremely high levels due to ion selective migration, unique molecular separation modalities, confined environments to allow biopolymer stretching and elongation and solid-phase bioreactions that are not constrained by mass transport artifacts. Indeed, many examples in the literature have demonstrated these unique opportunities, although predominately using glass, fused silica or silicon as the substrate material. Polymer microfluidics has established itself as an alternative to glass, fused silica, or silicon-based fluidic devices. The primary advantages arising from the use of polymers are the diverse fabrication protocols that can be used to produce the desired structures, the extensive array of physiochemical properties associated with different polymeric materials, and the simple and robust modification strategies that can be employed to alter the substrate's surface chemistry. However, while the strengths of polymer microfluidics is currently being realized, the evolution of polymer-based nanofluidics has only recently been reported. In this critical review, the opportunities afforded by polymer-based nanofluidics will be discussed using both elastomeric and thermoplastic materials. In particular, various fabrication modalities will be discussed along with the nanometre size domains that they can achieve for both elastomer and thermoplastic materials. Different polymer substrates that can be used for nanofluidics will be presented along with comparisons to inorganic nanodevices and the consequences of material differences on the fabrication and operation of nanofluidic devices (257 references).

Evaluation of Analytical Performances of Magnetic Force-Assisted Electrochemical Sandwich Immunoassay for the Quantification of Carcinoembryonic Antigen.

Carcinoembryonic antigen (CEA) is a biomarker indicated in different cancers, targeted for quantitative analysis via immunoassay. Here we introduce a new technique called magnetic force-assisted electrochemical sandwich immunoassay (MESIA) for determination of CEA level in a drop of human serum using a fully automated point-of-care testing (POCT) device. The analytical performances of the assay are assessed based on precision, accuracy, limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ), linearity, Hook effect, interference, cross-reactivity, and method comparison following the guidelines of the Clinical Laboratory Standards Institute (CLSI). The LoD is 0.50 ng/ml. A linear relationship is shown in the range of 0.5-200 ng/ml. A high dose effect is not seen up to approximately 500,000 ng/ml. The recovery range is from 94.7 to 108.9%. The %CV of run-to-run and within-lab variations are less than 2.04 and 4.41% across the CEA concentrations, respectively, whereas reproducibility is 4.45-6.24%. Method comparison shows that the assay correlates well with the reference device (R 2 = 0.9884). The assay demonstrates acceptable precision, accuracy, LoB, LoD and LoQ, hook effect, linearity, interference, cross-reactivity, and high correlation with its reference device. Thus, the system is suitable for the quantification of CEA in clinical practices with a POCT manner.

Cyclic tangential flow filtration system for isolation of extracellular vesicles.

Size-based filtration techniques have been developed for high-throughput isolation of extracellular vesicles (EVs). Conventional direct filtration systems have limitations in that large particles generally not only block the pores of the membrane but also damage the particles because of the high fluid pressure. Here, we propose a cyclic tangential flow filtration (TFF) system that includes two membranes with pore sizes of 200 and 30 nm, connected to a peristaltic pump that feeds the stream flowing to the membrane for continuous circulation. The cyclic TFF system is better able to isolate the specific 30-200 nm size range in one step through dual cyclic filtration compared with direct filtration (DF) and single cyclic TFF (scTFF). We further introduced a buffer-exchange process to the dcTFF system after filtration to remove contaminants for more efficient purification. As a result of comparative evaluation of dcTFF and ExoQuick, EVs isolated by dcTFF had more abundant exosome markers and active EVs. The cyclic TFF system not only has great potential to separate EVs with high selectivity and separation efficiency in small volumes of samples but can also be used in clinical applications, including medical diagnostic procedures.

Optoelectrofluidic sandwich immunoassays for detection of human tumor marker using surface-enhanced Raman scattering.

A sandwich immunoassay is a powerful tool for identifying a specific substance in a biological sample. However, its heterogeneous strategy always requires repetitive liquid handlings and long processing time. Here an optoelectrofluidic immunoassay platform for simple, fast, and automated detection of human tumor marker based on surface-enhanced Raman scattering (SERS) has been developed. By using a conventional optoelectrofluidic device and a liquid crystal display module, simple and quantitative detection of human tumor marker, alpha-fetoprotein, in a ∼500 nL sample droplet has been automatically conducted with lower detection limit of about 0.1 ng/mL within 5 min. This study depicts the first practical application, for protein detection, of the optoelectrofluidic manipulation technology. This image-driven immunoassay platform opens a new way for simple, fast, automated, and highly sensitive detection of antigens.

Rapid and selective concentration of microparticles in an optoelectrofluidic platform.

We demonstrate rapid manipulation and selective concentration of microparticles using AC electrokinetics such as dielectrophoresis (DEP) and AC electro-osmosis (ACEO) in an optoelectrofluidic platform based on a liquid crystal display (LCD). When 10 V bias at 10 kHz was applied to the optoelectrofluidic device, only the 1 microm-diameter polystyrene particles were concentrated into the projected LCD image patterns and closely packed, forming the crystalline structure by ACEO flow, while the 6 microm-diameter particles were repelled by negative DEP forces. We have characterized this frequency-dependency of the optoelectrofluidic particle behavior according to the particle diameter. On the basis of these results, we can rapidly concentrate the 1 microm-diameter particles and separate them from the 6 microm particles, by applying an AC signal of 10 kHz frequency. This novel technique can be applied to rapidly concentrate, separate and pattern micro-/nanoparticles in many biological and chemical applications.

Direct rapid prototyping of PDMS from a photomask film for micropatterning of biomolecules and cells.

The soft lithographic technique is a collection of simple and cost-effective patterning techniques which applies an elastomeric stamp to transfer a nano/micro-scale pattern. Patterning biological materials using soft lithography provides procedurally simple control of the surface chemistry and the cell environments. However, conventional methods for generating microstructures on a substrate require expensive clean room facilities and skillful training. Here we report a simple and inexpensive clean-room free process using a conventional photomask film as a master to fabricate elastomeric stamps or microfluidic channels. This ultra rapid prototyping technique was applied to print FITC labeled poly-L-lysine with a 10 microm feature size on a glass substrate using soft lithographic processes, such as micro-contact printing and micromolding in capillaries, for patterning human hepatocellular carcinoma cells, human skin fibroblasts and hippocampal neurons from E-18 Sprague-Dawley rat. This novel technique using a photomask film as a master would be very useful 'hands-on' tool for the generation of micro-patterned chemical or biological assays using cells and proteins.

Dynamic light-activated control of local chemical concentration in a fluid.

This article reports a method for controlling the chemical concentration in a localized region of a fluid using optoelectrofluidic mechanisms. Optoelectrofluidic fluorescence microscopy (OFM), in which an optoelectrofluidic device is integrated into a conventional fluorescence microscope, allows both modulation and detection of local chemical concentration in an easy and simple way. Here, we present the first experimental investigation of the concentration change of polysaccharides, proteins, and fluorophores, due to the frequency-dependent ac electrokinetics and electrostatic interactions in an optoelectrofluidic device. The dynamic modulation of the local concentration of biomolecules such as dextran and serum albumin was demonstrated in a temporal and spatial manner. This OFM can be a useful tool for controlling the local chemical concentration in several chemical and biological applications.

Measurement of molecular diffusion based on optoelectrofluidic fluorescence microscopy.

This technical note reports a method for measuring the diffusion coefficient of molecules based on an optoelectrofluidic platform. Optoelectrofluidic fluorescence microscopy, which is constructed with an optoelectrofluidic device and a conventional fluorescence microscope, is a useful tool for controlling and detecting local molecular concentration in a fluid with a single light source. When we project a light for fluorescence excitation and apply an ac signal of a few hundred Hertz frequency around 100 Hz to the optoelectrofluidic device, a sudden decay of molecular concentration occurs within the illuminated area due to several mechanisms, including optically induced ac electroosmosis, electrostatic interaction forces among the polarized molecules, and interactions among the molecules and between the molecules and the electric double layer. After the applied voltage was turned off, the dispersed molecules diffuse into the molecular depletion area and the fluorescence signal is recovered. On the basis of these phenomena, we successfully measured the diffusion coefficient of various dextran molecules. A statistical analysis to determine the significance of our experimental values compared to the previously reported values measured using fluorescence recovery after photobleaching technology was also performed. This new technique demonstrates the first analytical measurement of diffusion based on the optoelectrofluidic platform and can be a useful tool for measuring the mobility of molecules in a simple and easy way.

MESIA: Magnetic force-assisted electrochemical sandwich immunoassays for quantification of prostate-specific antigen in human serum.

We propose a new immunoassay technique, called magnetic-force assisted electrochemical sandwich immunoassay (MESIA), where serum biomarkers can be determined by magnetic actuation and electrochemical detection of gold-coated iron oxide nanoparticles as probes for immunocomplex formation. In MESIA, neither washing buffer nor fluidic parts are necessary, because the formation of immunocomplexes and the removal of unbound probes are controlled by magnetic forces. Electrochemical pretreatment and measurement of the gold-coated magnetic probes allows highly sensitive, precise, and robust system for quantification of target analytes. Using MESIA, the concentration of prostate-specific antigen (PSA) in 10 µl of human serum is determined within 5 min. The limit of detection is 0.085 ng/mL, and the average coefficient of variance is 8.85% for five different PSA concentrations ranging from 0 to 25 ng/mL. This method shows good precision and reproducibility (<10%) and high correlation with cobas e 801 (r = 0.997) for clinical patient samples. We believe this technique to be useful in the development of a point-of-care testing platform for diagnosis and prognosis of various diseases, such as cancer, based on quantification of biomarkers in a drop of blood.

Experimental investigation of electrostatic particle-particle interactions in optoelectronic tweezers.

This paper reports experimental and theoretical investigation of electrostatic attraction and repulsion of microparticles in an optoelectronic tweezers (OET). When we manipulate dielectric particles suspended in a fluid using OET, the electrostatic interactions of the polarized particles occur, limiting the effective manipulation of microparticles using a light-induced dielectrophoresis. In this study, we first demonstrate the electrostatic particle-particle interactions in the OET device using a liquid crystal display. At the same time, the experimental investigation of the dipole interactions between two spherical particles has been performed using the OET device. On the basis of the point-dipole model, simulation studies on the dipole forces acting on the particles and their trajectories by the forces are also performed. The experimental results show good agreement with the previously reported numerical studies as well as the results of our simulation studies.

Magnetic force assisted electrochemical sensor for the detection of thrombin with aptamer-antibody sandwich formation.

A magnetic force assisted electrochemical aptamer-antibody sandwich assay (MESA) was developed for the detection of thrombin as a model protein in serum samples. The MESA using the formation of sandwich complexes on the electrochemical sensor probe for reaction and the removal of unbound bioconjugates from the sensor surface without washing are controlled by a magnetic field. Thrombin was determined by the cathodic currents of a toluidine blue O (TBO) attached with thrombin antibody modified magnetic nanoparticle (MNP) at the sensor surface. To detect thrombin in a serum sample, we applied a thrombin-specific aptamer as the capture molecule bound to the functionalized conducting polymer layer (poly-(2,2´:5´,5″-terthiophene-3´-p-benzoic acid) (pTBA)), and streptavidin and starch coated-MNP was conjugated with biotinylated thrombin antibodies (Ab) and TBO as the bioconjugate (MNP@Ab-TBO). The characterization of MNP@Ab-TBO and sensor probe was performed using voltammetry, impedance spectroscopy, XPS, and UV-VIS spectroscopy. The experimental conditions were optimized in terms of pH, binding time, removal time of unbound bioconjugates, and applied potential. The dynamic ranges of thrombin were from 1.0 to 500 nM with detection limit of 0.49 ( ±â€¯0.06) nM. The recovery test demonstrates the reliability of the proposed sensing system for a handheld device.