Thin-layer Gel Research Articles

Effect of a Thin Fluid Layer on Surface Wave Speed Measurements: A Lung Phantom Study.

Lung ultrasound (US) surface wave elastography (SWE) is a novel technique that measures superficial lung tissue elastic properties. A thin pleural fluid layer covers a lung, but its effect on lung measurements in SWE is unknown. We modeled a lung and pleural fluid with sponges and a thin layer of US transmission gel. Sponge surface wave speeds measured from SWE were compared for sponges without and with the thin US gel layer at 3 wave excitation frequencies. The comparison showed that the sponge surface wave speed measurements were not affected by the thin gel layer.

Extraction, isolation and characterization of depsidones from Usnea baileyi (Stirt.) Zahlbr collected from tree barks in Tam Bo Mountain of Di Linh, Lam Dong Province, Viet Nam

Chemical data on the lichen Usnea baileyi are scarce. The current investigation reports the extraction, isolation and characterization of depsidones from this lichen collected in Lam Dong Province. Eight depsidones were isolated and elucidated, including methylstictic acid (1), stictic acid (2), cryptostictic acid (3), 8’-O-methylmenegazziaic acid (4), menegazziaic acid (5), 8’- O-methylconstictic acid (6), constictic acid (7), and hypoconstictic acid (8). Multi chromatographic methods comprising normal phase silica gel column chromatography and thin layer chromatography were applied to the crude acetone extract to isolate and purify compounds 1-8. Their chemical structures were elucidated by spectroscopic analysis as well as comparing their data with ones in the literature.

Preparation and characterization of PDMS-D2EHPA extraction gel membrane for metal ions extraction and stability enhancement

A novel extraction gel membrane (EGM) was prepared via gelation technique by forming polydimethylsiloxane-di (2-ethylhexyl) phosphoric acid (PDMS-D2EHPA) extraction gel layer on the outer surface of PVDF ultrafiltration hollow fibers. This special design aims to achieve both efficient mass transfer within the thin extraction gel layer and improvement in operating stability by preventing the extractant (D2EHPA) loss from EGM. The physical and chemical properties of the prepared EGM were studied via a series of characterization techniques, and the fixed-site jumping carrier mechanism for EGM was confirmed. The influences of the EGM preparation parameters on its performance in nickel separation were investigated systematically. The optimal extraction efficiency and stability of EGM were achieved by the EGM prepared from the ratio of 10:8.0:0.2 for PDMS / TEOS / DBTL, 24 wt% PDMS concentration, 40 min-coating and a low level (15–25%) of relative humidity. Using EGM prepared from these conditions, long-term operating stability was tested and the flux results were compared to that of the conventional supported liquid membrane (SLM) process. It was shown that the flux attenuation of the EGM was only 34.11% within 120 h, while that of the conventional SLM was 100% within 45 h. Simultaneously, the initial flux of EGM was as high as 2436.30 mg m−2 h−1 which was 7.1 times of that of the conventional SLM. The results of this study indicate evident advantages of the novel EGM process over the traditional SLM by showing higher flux and operating stability.

Pleural fluid may pose no measurable effect on lung stiffness in lung ultrasound surface wave elastography

Lung ultrasound surface wave elastography (LUSWE) was developed as a non-invasive tool for assessing pulmonary fibrosis. In this research, the effect of the fleural fluid on surface wave speed measurement of the lung is studied. Low-cost sponges [J. Ultrasound Med. 2017; 36, 2133], as good lung phantoms, were used in this study. The ultrasound transmission gel was used to simulate the pleural fluid. A harmonic vibration was generated at sponge surfaces using a shaker at 100, 150, and 200 Hz. The sponge surface wave speeds were measured with a 6 MHz ultrasound probe for both with and without a thin layer gel. The ultrasound features of A and B-lines were clearly observed in every sponge model in our experiment. The surface wave speeds for the sponges with the gel were indistinguishable from those without the gel for each frequency. The surface wave speeds were faster at higher excitation frequencies which is consistent with our patient study results. It was conclude that the pleural fluid may not affect the lung surface wave speed measurement based upon our lung phantom sponge models.

Diffusional and migrational transport of ionic species affected by electrostatic interactions with an oppositely charged hydrogel layer attached to an electrode surface

The effect of the presence of a charged thin gel layer on the electrode surface on mass transport of electroactive species of opposite charge was investigated. The layer was deposited using the electrochemically-induced free-radical polymerization method. The ferrocenyltrimethylammonium cation (FcN+) served as the electroactive probe. Ionic accumulation of the anodic electrode product was observed in experiments with normal sized electrodes. Microelectrodes were used without supporting electrolyte to study the contribution of migration to the overall mass transport to the modified surface.

In vitro effects of two silicate-based materials, Biodentine and BioRoot RCS, on dental pulp stem cells in models of reactionary and reparative dentinogenesis

BackgroundCalcium silicate-based cements are biomaterials with calcium oxide and carbonate filler additives. Their properties are close to those of dentin, making them useful in restorative dentistry and endodontics. The aim of this study was to evaluate the in vitro biological effects of two such calcium silicate cements, Biodentine (BD) and Bioroot (BR), on dental stem cells in both direct and indirect contact models. The two models used aimed to mimic reparative dentin formation (direct contact) and reactionary dentin formation (indirect contact). An original aspect of this study is the use of an interposed thin agarose gel layer to assess the effects of diffusible components from the materials.ResultsThe two biomaterials were compared and did not modify dental pulp stem cell (DPSC) proliferation. BD and BR showed no significant cytotoxicity, although some cell death occurred in direct contact. No apoptosis or inflammation induction was detected. A striking increase of mineralization induction was observed in the presence of BD and BR, and this effect was greater in direct contact. Surprisingly, biomineralization occurred even in the absence of mineralization medium. This differentiation was accompanied by expression of odontoblast-associated genes. Exposure by indirect contact did not stimulate the induction to such a level.ConclusionThese two biomaterials both seem to be bioactive and biocompatible, preserving DPSC proliferation, migration and adhesion. The observed strong mineralization induction through direct contact highlights the potential of these biomaterials for clinical application in dentin-pulp complex regeneration.

The effect of experimental hyperoxia on erythrocytes’ oxygen-transport function

ABSTRACTThe aim of this study was to investigate the effect of hyperoxia, calcium ions and pH value on the composition of major phospholipids in human erythrocyte membranes and erythrocytes’ oxygen-transport function. To create a model of hyperoxia, we saturated the incubated mixture with oxygen by constant passing of oxygen–air mixture through the incubation medium. To assess the effect of elevated calcium ion concentrations, CaCl2 was added to the incubation medium. An incubation medium with different pH was used to study the effect of various pH values. Lipids were extracted from erythrocytes and chromatographic separation was carried out in a thin layer of silica gel deposited on a glass plate. The thiobarbituric acid (TBA)-active products and the content of diene conjugates (DC) in erythrocytes were determined. The oxygen-binding capacity of haemoglobin was evaluated using Raman spectroscopy. The obtained results indicated that hyperoxia causes deep changes both in the composition and character of bilayer lipids of erythrocyte membranes, which affects the functional characteristics of erythrocytes, primarily the oxygen-transport properties of erythrocyte haemoglobin. It should be noted that a combination of Ca2+ ions and change in the pH value intensify the processes associated with disruption of phospholipids’ composition. The findings indicate that the lipid phase is one of the key elements in the functioning of erythrocytes in norm as well as during development of various pathological processes.

Attenuation of the Acoustic Signal Propagating Through a Bubbly Liquid Layer

The acoustic signal dynamics in a five-layer medium containing two liquid layers with polydisperse gas bubbles has been investigated. Calculations have been made for the interaction between the pulse perturbation of smallamplitude pressure and a multilayer sample containing two layers of industrial gel with polydisperse air bubbles. It has been shown that a small content of bubbles (about 0.1 vol. %) in a thin gel layer decreases tenfold or more the amplitude of acoustic waves with frequencies close to the resonance frequency of natural oscillations of bubbles. There are frequency ranges thereby where the influence of the bubbly layer is insignificant.

Indole-3-Acetic Acid Production by Colletotrichum siamense, An Endophytic Fungus from Piper nigrum Leaves

Plant endophytic fungi have been recognized as an important and a good source of natural bioactive products with potential application in agriculture, medicine and food industry. Chemical and biological studies of fungal metabolites originating from endophytic fungi isolated in Sri Lanka are relatively less studied. Therefore we investigated the chemistry and bioactivity of endophytic fungi present in leaves of Piper nigrum L. (black pepper) of the family Piperaceae famed as the spices king due to its pungent quality. A fungal endophyte isolated from the leaves of P. nigrum was identified as Colletotrichum siamense through molecular biological means using internal transcribed spacer (ITS) region of rDNA gene. This is the first report of the isolation of C. siamense from Piperaceae. The fungus was fermented in potato dextrose media and the fungal media were extracted with EtOAc. Chromatographic separation of the EtOAc extract over silica gel, Sephadex LH-20 and preparative thin layer chromatography furnished indole-3-acetic acid (IAA) and uracil. IAA showed high antifungal activity against the common plant pathogenic fungus Cladosporium cladosporiodies by TLC bioautography method and antioxidant activity against DPPH radical by spectrophotometry method.

The growth mechanism of CPED coating with zirconia sol addition on an Al-12Si alloy

A novel thermal-protective coating has been prepared by CPED process on Al-12%Si alloy with the ZrO2 sol addition. The microstructure characteristics and formation mechanism of the coating were investigated by SEM, TEM, EDS, XRD, fourier transform infrared spectrometer (FTIR) and laser particle size analyzer. It was confirmed that the sol particle adsorbed on the cathode surface at first, and then formed a thin zirconia gel layer, which can promote the cathode discharge. With ZrO2 sol addition, the cathode plasma discharge on the coating surface is more obvious than that without zirconia sol addition and the coating is more uniform and compact and the thickness of the coating increases. Compared with the electrolyte without ZrO2 sol addition, the crystalline degree of ceramic layer is higher in zirconia sol solution, which indicates that ZrO2 sol can promote the transition of coating from amorphous to crystalline. Under the high temperature of plasma discharge, gel particles entered the discharge channels and then were sintered into ZrO2 ceramic particles. ZrO2 combined with Al2O3 formed by barrier layer together to finally form the composite ceramic coating. With ZrO2 sol addition, the thermal insulation performance of the coating increases.

Abstract 4528: A novel transducer array design optimizing TTFields delivery to the thorax

Abstract Objective: The purpose of this study was to design transducer arrays for the delivery of Tumor Treating Fields (TTFields) to the thorax, generating high intensity in the upper torso while potentially improving patient comfort. Background: TTFields are low intensity, intermediate frequency, alternating electrical fields that disrupt cell division. The ongoing Phase II STELLAR trial [NCT02397928] is investigating the efficacy and safety of TTFields in combination with chemotherapy for the treatment of mesothelioma. A pivotal study to evaluate the efficacy of TTFields in treating NSCLC is planned for 2017. TTFields are delivered via capacitive transducer arrays, comprising ceramic disks covered with a thin layer of conductive medical-grade gel, which are arranged in an almost rectangular order. Two orthogonal pairs of arrays are placed on the skin in the vicinity of the treated region. One pair is placed on the anterior and posterior sides of the thorax. The other pair is placed on the lateral and contralateral sides of the thorax. This configuration has limitations: (a) Sub-optimal electric contact may be caused by poor attachment of the arrays on the chest due to body curvature; (b) TTFields intensity generated by anterior-posterior arrays may be attenuated in the thorax of females and obese individuals, due to high resistivity of the thick adipose layers in the breast; (c) the current lateral-contralateral arrays generate lower field intensities in the lung’s apex. This study presents novel array designs to overcome these limitations. Methods: Various concepts for array designs that avoid placement over regions of thick sub-cutaneous fat were designed. To evaluate the field distribution generated by these arrays and optimize their design, numerical simulations using realistic computational phantoms were used. Results: Novel array designs were developed for the delivery of TTFields to the lungs. These arrays consist of sets of interconnected small patches that adhere to the natural contours of the human body. Simulations showed that the proposed arrays deliver uniformly distributed, high electrical field to the lungs. One notable design is a pair of arrays shaped as circular rings, in which one array is placed around the neck and shoulders, whereas the second array is placed on the lower torso. This design yielded a highly uniform field directed longitudinally throughout the torso. Conclusion: The array designs presented in this study are expected to maintain patient comfort and will ensure high field intensity delivered to the thorax. Compliance and TTFields treatment delivery could both be improved by using these designs. Citation Format: Hadas S. Hershkovich, Sholi Strauss, Uri Weinberg, Eilon D. Kirson, Ze'ev Bomzon. A novel transducer array design optimizing TTFields delivery to the thorax [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4528. doi:10.1158/1538-7445.AM2017-4528

Effect of gels containing chlorhexidine or epigallocatechin-3-gallate on the protein composition of the acquired enamel pellicle

ObjectiveThis study evaluated changes in protein profile of the acquired enamel pellicle (AEP) formed in vivo, after application of gels containing chlorhexidine or EGCG and further challenge with citric acid. DesignAEP was formed in 9 volunteers for 2h and then treated with one of the following gels: placebo, 400μM EGCG or 0.012% chlorhexidine. A thin layer of gel was applied and after 1min the excess was removed. One hour after gel application, the AEP was collected from the buccal surface (upper and lower jaw) of one of the sides with filter paper dipped in 3% citric acid. On the other side, erosive challenge was performed through gentle application of 1% citric acid (pH 2.5) for 20s (using a pipette) followed by washing with deionized water. The AEP was collected as mentioned before. Proteomic analysis was performed through liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS). The MS/MS spectra obtained were compared with human protein databases (SWISS-PROT). Label-free quantitation was done using the PLGS software. ResultsIn total, 223 proteins were identified. After treatment with EGCG and CHX gels, proteins with potential functions to protect against caries and erosion such as PRPs, calcium-bind proteins and Statherin were increased. When EGCG and CHX-treated AEPs were challenged with citric acid, there was increase in cystatins and Profilin-1. ConclusionCHX- and EGCG-treated AEPs, submitted to challenge with citric acid or not, had remarkable changes in their proteomic profiles.

Fabrication of micro-alginate gel tubes utilizing micro-gelatin fibers

Tissues engineered utilizing biofabrication techniques have recently been the focus of much attention, because these bioengineered tissues have great potential to improve the quality of life of patients with various hard-to-treat diseases. Most tissues contain micro-tubular structures including blood vessels, lymphatic vessels, and bile canaliculus. Therefore, we bioengineered a micro diameter tube using alginate gel to coat the core gelatin gel. Micro-gelatin fibers were fabricated by the coacervation method and then coated with a very thin alginate gel layer by dipping. A micro diameter alginate tube was produced by dissolving the core gelatin gel. Consequently, these procedures led to the formation of micro-alginate gel tubes of various shapes and sizes. This biofabrication technique should contribute to tissue engineering research fields.

Macrophages are Sensitive to Substrate Elasticity during Phagocytosis

Phagocytosis, the internalization of objects like living bacteria and dead cells by macrophages is a main function of the innate immune system. After the detection of foreign objects by membrane receptors, this process is driven by the reorganization of the actin cytoskeleton, which leads to a protrusion of the membrane around the target. Although many molecular players have been identified in the past, there is still little known about the role of mechanics during this process in general and about the role of the mechanical cellular environment in particular.In this work we investigate the influence of the underlying substrate rigidity on the phagocytic uptake efficiency and the uptake velocity. We cultured murine J774 macrophages on thin polymer gels with different stiffnesses in the physiological range. The uptake efficiency of antibody-coated microparticles was quantified with secondary antibody staining and the uptake speed was measured in live cell experiments by using optical tweezers. We found that the uptake efficiency as well as the uptake velocity depend on the rigidity of the substrate. Furthermore, we observed that cells were able to adapt to the various substrate stiffnesses over time. In addition to these cellular measurements, we also present a novel calibration technique that enables the simultaneous characterization of the elastic modulus and the Poisson's ratio of thin gel layers. The technique requires only a standard epifluorescence microscope and spherical indenters.Our results on the phagocytic uptake velocity and efficiency support the hypothesis that phagocytosis is a mechanosensitive process. Our findings might contribute to an understanding of the complex interplay between the immune system and disease states that come along with changes in tissue rigidity like cancer and atherosclerosis.

Open-Source-Based 3D Printing of Thin Silica Gel Layers in Planar Chromatography.

On the basis of open-source packages, 3D printing of thin silica gel layers is demonstrated as proof-of-principle for use in planar chromatography. A slurry doser was designed to replace the plastic extruder of an open-source Prusa i3 printer. The optimal parameters for 3D printing of layers were studied, and the planar chromatographic separations on these printed layers were successfully demonstrated with a mixture of dyes. The layer printing process was fast. For printing a 0.2 mm layer on a 10 cm × 10 cm format, it took less than 5 min. It was affordable, i.e., the running costs for producing such a plate were less than 0.25 Euro and the investment costs for the modified hardware were 630 Euro. This approach demonstrated not only the potential of the 3D printing environment in planar chromatography but also opened new avenues and new perspectives for tailor-made plates, not only with regard to layer materials and their combinations (gradient plates) but also with regard to different layer shapes and patterns. As such an example, separations on a printed plane layer were compared with those obtained from a printed channeled layer. For the latter, 40 channels were printed in parallel on a 10 cm × 10 cm format for the separation of 40 samples. For producing such a channeled plate, the running costs were below 0.04 Euro and the printing process took only 2 min. All modifications of the device and software were released open-source to encourage reuse and improvements and to stimulate the users to contribute to this technology. By this proof-of-principle, another asset was demonstrated to be integrated into the Office Chromatography concept, in which all relevant steps for online miniaturized planar chromatography are performed by a single device.

Abstract A53: Probing forces and modulation of cancer cell mechanical properties during transendothelial migration

Abstract For all types of cancer, the survival rate while the tumor is still localized is significantly higher than when cancer has metastasized. Though different therapeutic methods can be applied to successfully treat primary tumors, treatment of metastasised cancer is a great challenge due to its complex and systemic nature. A deeper understanding of how cancer initiates, grows and migrates is essential for creating successful therapies. Particularly, extravasation — a process during which streaming tumor cells (TCs) adhere to the blood vessels and traverse through the vascular endothelium into the surrounding tissue — is one of the crucial steps of cancer metastasis. While cancer research has mostly focused on the initial processes of metastasis, little is known about mechanisms of transmigration of tumor cells through the vascular wall. We found that during transendothelial migration (TEM), cancers cells undergo substantial shape changes transforming from semi-spherical (with a 2D contact with the endothelium) to a fully spread 3D morphology inside the ECM. This significant morphological change occurs due to forces generated intracellularly and distributed externally. During these morphological changes in transmigration, the cytoskeleton provides the force for shape change but the rate at which shape change occurs is dictated by the degree of deformability of the tumor cell, endothelial cells (ECs) and subendothelial tissue. Hence, the strength and distribution of forces, as well as mechanical properties of the cells and ECM, play a central role in the process of TEM. In this project, we investigate modulation of TCs and ECs rheological properties and the role of forces generated by the cytoskeleton and their transmission to adjacent cells and the ECM during extravasation. To address the role of mechanical modulation during TEM, we have developed in vitro assays and combed them with optical/confocal microscopy, Traction Force Microscopy (TFM) and Brillouin Confocal Microscopy (BCM). In particular, we recently designed a simple system in which ECs are cultured on a thin layer of collagen. Using a combination of different coating treatments on glass to produce surface hydrophobicity and maintain strong adhesion, we created a thin (~80 μm) layer of gel. Following seeding by ECs (HUVECs) to produce a tight and uniform EC monolayer we introduced TCs on top of the EC monolayer and monitored extravasation in real-time with high spatiotemporal resolution. Measurements of HUVEC monolayer permeability in this system show physiologically relevant values (8±3 10-8 cm2/s). We further tested our system by using different cancer cells with different invasiveness potentials and investigated their extravasation efficiencies. A major advantage of this platform is that it allows integration of TFM and BCM so it enabled us to characterize forces and modulation of cell mechanical properties during TEM. In particular, we demonstrated the feasibility of our system for application of BCM on cancer cells extravasating through a monolayer. BCM is an optical technique in which the mechanical information can be read out optically via spectral analysis of the scattered light; thanks to the interaction of light with intrinsic mechanical vibrations (phonons) of the material. Our BCM measurements indicate a significant decrease in elasticity of TCs at different stages; from 260±20 Pa for TCs that were in the process of transmigration to 195±17 Pa for those that were fully transmigrated. Our results shed a new light on the fundamental understanding of the extravasation mechanics. Furthermore, we anticipate that these studies will potentially enhance our ability to identify and screen for new therapies to inhibit the tendency for metastatic spread of disease. Citation Format: Emad Moeendarbary, Roger Kamm, Giuliano Scarcelli. Probing forces and modulation of cancer cell mechanical properties during transendothelial migration. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A53.

The peculiar features of the distribution of 2-(dimethylamino)ethyl- (1-hydroxycyclopentyl) (phenyl) acetate in the organism of warm-blooded animals after its intravenous administration

The objective of the present study was to elucidate the dynamics of the distribution of 2-(dimethylamino)ethyl-(1-hydroxycyclopentyl)(phenyl) acetate in the organism of warm-blooded animals (rats) after its intravenous administration to the animals. The identification and the quantitative determination of 2-(dimethylamino)ethyl-(1-hydroxycyclopentyl)(phenyl) acetate present in the organs and blood of the warm-blooded animals were carried out with the use of chromatography in a thin layer of silica gel, chromogenic reaction for the synthesis of the acinitro salt, UV spectrophotometry, and gas chromatography mass-spectrometry (GCh-MS). The quantitation was performed on days 5, 20, 150, and 360 minutes after the intravenous administration of 2-(dimethylamino)ethyl-(1-hydroxycyclopentyl)(phenyl) acetate to the rats. The maximum amounts of cyclopentolate were found in the spleen, brain, lungs, and heart of the experimental animals.

Quartz Crystal Microbalance Electrode Modified with Thermoresponsive Crosslinked and Non-Crosslinked N- Isopropylacrylamide Polymers. Response to Changes in Temperature

Electrochemical quartz crystal microbalance (EQCM) electrode was modified with environmentally sensitive polymers. The polymer layers on the electrode were composed of either crosslinked or non-crosslinked thermoresponsive poly(N-isopropylacrylamide). For anchoring thin gel films on the electrode surface the electrochemically induced free-radical polymerization (EIFRP) was employed. The electroreduction of the peroxydisulfate anion led to generation of free radical. This radical initiated the free radical polymerization process that led to the formation of a thin gel layer attached to the electrode surface. To monitor in situ the growth of the polymer film the changes in resonant frequency of the quartz-crystal were recorded. A significant decrease in the frequency (growth of the layer) was seen in the potential range where the reduction of peroxydisulfate anion took place. The morphology of the layers was examined with a scanning electron microscopy (SEM). The phenomenon of volume phase transition (shrinking/swelling process) in the gel layers initiated by a change in temperature was investigated. Unexpected big, sharp and of negative sign minima appeared at the change-in-frequency vs. temperature plots. These minima were not seen in the plots obtained for the polymers without linker. That situation should be useful in the investigation of chemical interactions proceeding under the conditions of volume phase transition. The influence of volume phase transition on the transport of a member of a model red-ox system, ferro- and ferricyanide couple, and therefore on height of its voltammetric response was examined. The changes in electrochemical properties of the layers induced by volume phase transition were monitored with electrochemical impedance spectroscopy.

Single cell-resolution western blotting.

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

Quartz crystal microbalance electrode modified with thermoresponsive crosslinked and non-crosslinked N-isopropylacrylamide polymers. Response to changes in temperature

Electrochemical quartz crystal microbalance (EQCM) electrode was modified with environmentally sensitive polymers. The polymer layers on the electrode were composed of either crosslinked or non-crosslinked thermoresponsive poly(N-isopropylacrylamide). For anchoring thin gel films on the electrode surface, the electrochemically induced free radical polymerization (EIFRP) was employed. The electroreduction of the peroxydisulfate anion led to generation of free radical. This radical initiated the free radical polymerization process that led to the formation of a thin gel layer attached to the electrode surface. To monitor in situ the growth of the polymer film the changes in resonant frequency of the quartz crystal were recorded. A significant decrease in the frequency (growth of the layer) was seen in the potential range where the reduction of peroxydisulfate anion took place. The morphology of the layers was examined with a scanning electron microscopy (SEM). The phenomenon of volume phase transition (shrinking/swelling process) in the gel layers initiated by a change in temperature was investigated. Unexpected big, sharp, and of negative sign minima appeared at the change-in-frequency vs. temperature plots. These minima were not seen in the plots obtained for the polymers without linker. That situation should be useful in the investigation of chemical interactions proceeding under the conditions of volume phase transition. The influence of volume phase transition on the transport of a member of a model red-ox system, ferro- and ferricyanide couple, and therefore on height of its voltammetric response was examined. The changes in electrochemical properties of the layers induced by volume phase transition were monitored with electrochemical impedance spectroscopy.