Mechanical spectroscopy and relaxometry on alginate hydrogels: a comparative analysis for structural characterization and network mesh size determination

The structure of calcium-saturated alginate hydrogels has been studied by combining rheological determinations and relaxometry measurements. The mechanical spectroscopy analyses performed on alginate gel cylinders at different polysaccharide concentration allowed estimating their main structural features such as the average mesh size. The calculation was based on the introduction of a front factor in the classical rubber elasticity approach which was correlated to the average length of the Guluronic acid blocks along the polysaccharide chain. Transverse relaxation time (T(2)) determinations performed on the cylinders revealed the presence of two relaxation rates of the water entrapped within the hydrogel network. The cross-correlation of the latter data with the rheological measurements allowed estimating the mesh size distribution of the hydrogel network. The results obtained for the hydrogel cylinders were found to be consistent with the relaxometric analysis performed on the alginate microbeads where, however, only one type of water bound into the network structure was detected. A good correlation was found in the average mesh size determined by means of relaxometric measurements on alginate microbeads and by a statistical analysis performed on TEM micrographs. Finally, the addition of a solution containing calcium ions allowed investigating further the different water relaxation modes within alginate hydrogels.     

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 μmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K _m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

     

Label-Free Single-Particle Imaging of the Influenza Virus by Objective-Type Total Internal Reflection Dark-Field Microscopy

Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.2×10⁴ plaque-forming units per milliliter. Our label-free optical imaging method does require neither elaborated sample preparation nor complex optical systems, offering a good platform for rapid and sensitive counting of viral particles.     

Integration of Acoustic Radiation Force and Optical Imaging for Blood Plasma Clot Stiffness Measurement

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.     

Preparation,Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.     

Synthesis and utilization of E. coli‐encapsulated PEG‐based microdroplet using a microfluidic chip for biological application

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single‐step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non‐toxic ways of fabricating microorganism‐encapsulated hydrogel microbeads. Due to these unique properties of micro‐sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl‐β‐D ‐thiogalactopyranoside induction, suggesting that there is no low‐molecular weight substrate transfer limitation inside of microbeads. Furthermore, non‐toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells. Biotechnol. Bioeng. 2010;107:747–751. © 2010 Wiley Periodicals, Inc.     

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.     

Light-activated immobilization of biomolecules to agarose hydrogels for controlled cellular response

We describe a new method of synthesizing photolabile hydrogel materials for convenient photoimmobilization of biomolecules on surfaces or in 3-D matrixes. Dissolved agarose was modified with photolabile S-(2-nitrobenzyl)cysteine (S-NBC) via 1,1'-carbonyldiimidazole (CDI) activation of primary hydroxyl groups. S-NBC-modified agarose remained soluble and gelable with up to 5% S-NBC substitution, yet gelation was slower and the elastic modulus of the resulting gel was lower than those of unmodified agarose. Irradiating S-NBC-grafted agarose resulted in the loss of the protecting 2-nitrobenzyl groups, thereby exposing free sulfhydryl groups for biomolecular coupling. When appropriately activated with sulfhydryl-reactive groups, either peptides or proteins were effectively immobilized to the photoirradiated hydrogel matrixes, with the irradiation energy dose (i.e., irradiation time) used to control the amount of biomolecule immobilization. When the GRGDS peptide was immobilized on agarose, it was shown to be cell-adhesive and to promote neurite outgrowth from primary, embryonic chick dorsal root ganglion neurons. The immobilized GRGDS surface ligand concentration affected the cellular response: neurite length and density increased with GRGDS surface concentration at low adhesion ligand concentration and then plateaued at higher GRGDS concentration. Grafting 2-nitrobenzyl-protected compounds to hydrogel materials is useful for creating new photolabile hydrogel substrates for light-activated functional group generation and biomolecular immobilization.     

Determining the Variability of Lesion Size Measurements from CT Patient Data Sets Acquired under “No Change” Conditions

PURPOSE: To determine the variability of lesion size measurements in computed tomography data sets of patients imaged under a “no change” (“coffee break”) condition and to determine the impact of two reading paradigms on measurement variability. METHOD AND MATERIALS: Using data sets from 32 non-small cell lung cancer patients scanned twice within 15 minutes (“no change”), measurements were performed by five radiologists in two phases: (1) independent reading of each computed tomography dataset (timepoint): (2) a locked, sequential reading of datasets. Readers performed measurements using several sizing methods, including one-dimensional (1D) longest in-slice dimension and 3D semi-automated segmented volume. Change in size was estimated by comparing measurements performed on both timepoints for the same lesion, for each reader and each measurement method. For each reading paradigm, results were pooled across lesions, across readers, and across both readers and lesions, for each measurement method. RESULTS: The mean percent difference (± SD) when pooled across both readers and lesions for 1D and 3D measurements extracted from contours was 2.8 ± 22.2% and 23.4 ± 105.0%, respectively, for the independent reads. For the locked, sequential reads, the mean percent differences (± SD) reduced to 2.52 ± 14.2% and 7.4 ± 44.2% for the 1D and 3D measurements, respectively. CONCLUSION: Even under a “no change” condition between scans, there is variation in lesion size measurements due to repeat scans and variations in reader, lesion, and measurement method. This variation is reduced when using a locked, sequential reading paradigm compared to an independent reading paradigm.     

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.     

Methods for animal cell immobilization using electrostatic droplet generation

Summary A technique for the production of small polymer microbeads (less than 150 m diam.) utilizing electrostatic spraying is presented. Video and image analysis were performed to reveal details of the mechanism of droplet formation. Microbeads coated with poly-L-lysine were prepared and found to be effective as microcarriers for culturing surface immobilized insect cells to a high cell density (1.55 × 10⁷ cells/ml microbeads).     

Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells

In subcellular light-sheet fluorescence microscopy (LSFM) of adherent cells, glass substrates are advantageously rotated relative to the excitation and emission light paths to avoid glass-induced optical aberrations. Because cells are spread across the sample volume, three-dimensional imaging requires a light-sheet with a long propagation length, or rapid sample scanning. However, the former degrades axial resolution and/or optical sectioning, while the latter mechanically perturbs sensitive biological specimens on pliant biomimetic substrates (e.g., collagen and basement membrane). Here, we use aberration-free remote focusing to diagonally sweep a narrow light-sheet along the sample surface, enabling multicolor imaging with high spatiotemporal resolution. Further, we implement a dithered Gaussian lattice to minimize sample-induced illumination heterogeneities, significantly improving signal uniformity. Compared with mechanical sample scanning, we drastically reduce sample oscillations, allowing us to achieve volumetric imaging at speeds of up to 3.5 Hz for thousands of Z-stacks. We demonstrate the optical performance with live-cell imaging of microtubule and actin cytoskeletal dynamics, phosphoinositide signaling, clathrin-mediated endocytosis, polarized blebbing, and endocytic vesicle sorting. We achieve three-dimensional particle tracking of clathrin-associated structures with velocities up to 4.5 μm/s in a dense intracellular environment, and show that such dynamics cannot be recovered reliably at lower volumetric image acquisition rates using experimental data, numerical simulations, and theoretical modeling.     

Protein A immobilized polyhydroxyethylmethacrylate beads for affinity sorption of human immunoglobulin G

Protein A immobilized polyhydroxylmethyacrylate (PHEMA) microbeads were investigated for the specific removal of HIgG from aqueous solutions and from human plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by CNBr in an alkaline medium (pH 11.5). Protein A was then immobilized by covalent binding onto these microbeads. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The maximum protein A immobilization was observed at pH 9.5. Up to 3.5 mg protein A/g PHEMA was immobilized on the CNBr activated PHEMA microbeads. The maximum HIgG adsorption on the protein A immobilized PHEMA microbeads was observed at pH 8.0. The non-specific HIgG adsorption onto the plain PHEMA microbeads was low (about 0.167 mg of HIgG/g PHEMA). Higher adsorption values (up to 6.0 mg of HIgG/g PHEMA) were obtained in which the protein A immobilized PHEMA microbeads were used. Much higher amounts of HIgG (up to 24.0 mg of HIgG/g PHEMA) were adsorbed from human plasma.     

Rapid and separation-free sandwich immunosensing based on accumulation of microbeads by negative-dielectrophoresis

We report here a rapid and separation-free immunoassay using a dielectrophoresis (DEP) device consisting of an interdigitated microarray (IDA) electrode and a polydimethylsiloxane (PDMS) substrate. On applying an AC voltage to the IDA in a negative-DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microbeads moved to the surface of the PDMS substrate placed above the IDA. The microbeads accumulated at designated areas of the PDMS surface that had been precoated with anti-mouse IgG. When the fluorescence microbeads bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microbeads trapped the analyte to form immunocomplexes on microbeads. The microbeads reacted with mouse IgG accumulated and were captured at the designated areas of the PDMS surface via an antibody-antigen-antibody (sandwich) reaction. The captured microbeads were detected selectively by fluorescence measurements at the focused designated areas, regardless of the presence of uncaptured microbeads suspended in solution. Thus, the separation and washing-out steps usually required for conventional immunoassay are eliminated in the presented procedure. Since the formation of the sandwich structures was accelerated significantly by n-DEP, a period as short as 30s was sufficient to detect the immunoreaction at the surface. The fluorescence intensity of the captured microbeads at the designated areas increased with analyte concentration in the range 0.01-10ng/mL. The present procedure therefore yields a rapid, sensitive, and separation-free immunoassay in a simple device.     

Data-Driven Method to Estimate Nonlinear Chemical Equivalence

There is great need to express the impacts of chemicals found in the environment in terms of effects from alternative chemicals of interest. Methods currently employed in fields such as life-cycle assessment, risk assessment, mixtures toxicology, and pharmacology rely mostly on heuristic arguments to justify the use of linear relationships in the construction of “equivalency factors,” which aim to model these concentration-concentration correlations. However, the use of linear models, even at low concentrations, oversimplifies the nonlinear nature of the concentration-response curve, therefore introducing error into calculations involving these factors. We address this problem by reporting a method to determine a concentration-concentration relationship between two chemicals based on the full extent of experimentally derived concentration-response curves. Although this method can be easily generalized, we develop and illustrate it from the perspective of toxicology, in which we provide equations relating the sigmoid and non-monotone, or “biphasic,” responses typical of the field. The resulting concentration-concentration relationships are manifestly nonlinear for nearly any chemical level, even at the very low concentrations common to environmental measurements. We demonstrate the method using real-world examples of toxicological data which may exhibit sigmoid and biphasic mortality curves. Finally, we use our models to calculate equivalency factors, and show that traditional results are recovered only when the concentration-response curves are “parallel,” which has been noted before, but we make formal here by providing mathematical conditions on the validity of this approach.     

A蛋白定向固定抗体用于椭偏光学生物传感器免疫检测

椭偏光学生物传感器是在椭偏光学显微成像技术的基础上发展的一项生物传感技术。它能够直接观测固体表面上的生物分子面密度,毋需任何标记辅助,适合发展成为一种无标记免疫检测技术。研究了在硅片表面上通过A蛋白定向固定抗体分子用于椭偏光学生物传感器免疫检测的可能性。实验结果表明,通过A蛋白固定抗体得到的抗体膜层的均一性和固定量的重复性能够保证椭偏光学生物传感器免疫检测结果的质量。通过A蛋白定向固定的抗体的抗原结合位点趋向一致,显著提高了抗体与抗原结合的能力。此外,通过蛋白A固定的免疫球蛋白G分子能够结合更多的多克隆抗体分子说明通过A蛋白固定的蛋白质分子能够较好地保持其空间构象。     

Correlation of Electrical and Permeability Properties of Ion-Selective Membranes

The linear phenomenological equations giving particle and practical fluxes of a single electrolyte across an ion-selective membrane are stated and interrelated. It is shown that the experimental measurements commonly made in biological and synthetic membrane studies may be used, with minor modification, to obtain the phenomenological transport coefficients and their concentration dependences. It is demonstrated that the electrical properties of a homogeneous membrane may be obtained as functions of the bathing solution concentration by combining fluxes measured under open and short circuit. Attention is paid to the use of radiotracers when measuring ionic fluxes. To obtain all the phenomenological coefficients at least one measurement must be made under a pressure gradient. The experimental difficulties in such measurements are discussed and the merits and demerits of various experiments considered. The problems of measuring potentials and concentrations at the low pressure face of a supported membrane make several mathematically simple approaches experimentally unattractive. The best methods appear to be either the measurement of a succession of “apparent osmotic pressures” under concentration differences sufficiently small that the membrane does not require support or the study of “reverse osmosis”. Sets of equations are given which enable the phenomenological coefficients to be evaluated from convenient experiments. With a stable homogeneous membrane nine coefficients may be obtained thus enabling either the applicability of the reciprocal relations or the applicability of linear theory under the conditions of the experiments to be tested. For a discontinuous system the six independent coefficients may be obtained from experiments in a single membrane cell.     

PEG-Like Nanoprobes: Multimodal,Pharmacokinetically and Optically Tunable Nanomaterials

“PEG-like Nanoprobes” (PN’s) are pharmacokinetically and optically tunable nanomaterials whose disposition in biological systems can be determined by fluorescence or radioactivity. PN’s feature a unique design where a single PEG polymer surrounds a short fluorochrome and radiometal bearing peptide, and endows the resulting nanoprobe with pharmacokinetic control (based on molecular weight of the PEG selected) and optical tunability (based on the fluorochrome selected), while the chelate provides a radiolabeling option. PN’s were used to image brain capillary angiography (intravital 2-photon microscopy), tumor capillary permeability (intravital fluorescent microscopy), and the tumor enhanced permeability and retention (EPR) effect (¹¹¹In-PN and SPECT). Clinical applications of PN’s include use as long blood half-life fluorochromes for intraoperative angiography, for measurements of capillary permeability in breast cancer lesions, and to image EPR by SPECT, for stratifying patient candidates for long-circulating nanomedicines that may utilize the EPR mechanism.     

Alterations of Gray and White Matter Networks in Patients with Obsessive-Compulsive Disorder: A Multimodal Fusion Analysis of Structural MRI and DTI Using mCCA+jICA

Many of previous neuroimaging studies on neuronal structures in patients with obsessive-compulsive disorder (OCD) used univariate statistical tests on unimodal imaging measurements. Although the univariate methods revealed important aberrance of local morphometry in OCD patients, the covariance structure of the anatomical alterations remains unclear. Motivated by recent developments of multivariate techniques in the neuroimaging field, we applied a fusion method called “mCCA+jICA” on multimodal structural data of T1-weighted magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) of 30 unmedicated patients with OCD and 34 healthy controls. Amongst six highly correlated multimodal networks (p < 0.0001), we found significant alterations of the interrelated gray and white matter networks over occipital and parietal cortices, frontal interhemispheric connections and cerebella (False Discovery Rate q ≤ 0.05). In addition, we found white matter networks around basal ganglia that correlated with a subdimension of OC symptoms, namely ‘harm/checking’ (q ≤ 0.05). The present study not only agrees with the previous unimodal findings of OCD, but also quantifies the association of the altered networks across imaging modalities.     

Nutrient cycling is an important mechanism for homeostasis in plant cells

Homeostasis in living cells refers to the steady state of internal, physical, and chemical conditions. It is sustained by self-regulation of the dynamic cellular system. To gain insight into the homeostatic mechanisms that maintain cytosolic nutrient concentrations in plant cells within a homeostatic range, we performed computational cell biology experiments. We mathematically modeled membrane transporter systems and simulated their dynamics. Detailed analyses of ‘what-if’ scenarios demonstrated that a single transporter type for a nutrient, irrespective of whether it is a channel or a cotransporter, is not sufficient to calibrate a desired cytosolic concentration. A cell cannot flexibly react to different external conditions. Rather, at least two different transporter types for the same nutrient, which are energized differently, are required. The gain of flexibility in adjusting a cytosolic concentration was accompanied by the establishment of energy-consuming cycles at the membrane, suggesting that these putatively “futile” cycles are not as futile as they appear. Accounting for the complex interplay of transporter networks at the cellular level may help design strategies for increasing nutrient use efficiency of crop plants.

First principles of membrane transport explain why maintaining a constant cytosolic nutrient concentration is often accompanied by the “futile” cycling of the nutrient across the membrane.