High Strength Buffer Research Articles

High-Throughput Strategy for Enhancing Aptamer Performance across Different Environmental Conditions.

Aptamers selected under specific environmental conditions (e.g., pH, ion concentration, and temperature) often exhibit greatly reduced affinity when used in other contexts. This can be especially problematic for biomedical applications in which aptamers are exposed to sample matrices with distinctive chemical properties, such as blood, sweat, or urine. We present a high-throughput screening procedure for adapting existing aptamers for use in samples whose chemical composition differs considerably from the original selection conditions. Building on prior work from our group, we have utilized a modified DNA sequencer capable of screening up to 107 unique aptamer mutants for target binding under the desired assay conditions. As an exemplar, we screened all 11,628 single- and double-substitution mutants for a previously reported glucose aptamer that was originally selected in high-ionic strength buffer and exhibited relatively low affinity in physiological conditions. After a single round of screening, we identified aptamer mutants with ∼four-fold increased affinity in physiological conditions. Interestingly, we found that the impact of single-base substitutions was relatively modest but observed considerably greater binding improvements among the double mutants, highlighting the importance of cooperative effects between mutations. This approach should be generalizable to other aptamers and environmental conditions for a range of applications.

Reply to the 'Comment on "Fluorimetric sensing of ATP in water by an imidazolium hydrazone based sensor"' by S. Farshbaf and P. Anzenbacher Jr., Chem. Commun., 2019, 55, 1770.

Bisantrene is marginally soluble in water, insoluble in high-ionic strength buffers and may require organic co-solvent such as DMSO to dissolve. Counter-ions and ionic strength of the medium must be considered when designing biological experiments.

Nanoparticle-Protein Interaction: Demystifying the Correlation between Protein Corona and Aggregation Phenomena.

Protein corona formation and nanoparticles' aggregation have been heavily discussed over the past years since the lack of fine-mapping of these two combined effects has hindered the targeted delivery evolution and the personalized nanomedicine development. We present a multitechnique approach that combines dynamic light and small-angle X-ray scattering techniques with cryotransmission electron microscopy in a given fashion that efficiently distinguishes protein corona from aggregates formation. This methodology was tested using ∼25 nm model silica nanoparticles incubated with either model proteins or biologically relevant proteomes (such as fetal bovine serum and human plasma) in low and high ionic strength buffers to precisely tune particle-to-protein interactions. In this work, we were able to differentiate protein corona, small aggregates formation, and massive aggregation, as well as obtain fractal information on the aggregates reliably and straightforwardly. The strategy presented here can be expanded to other particle-to-protein mixtures and might be employed as a quality control platform for samples that undergo biological tests.

Electrophoretic study of G-quadruplex aptamer interactions with different short single-strand complementary oligonucleotides

Aptamers are single-stranded nucleic acids, typically 20-80 nucleobases (nb) in length, which can bind different compounds with high affinity and selectively. Their ligand-binding properties can be attenuated by adding short complementary strands. These interactions open new opportunities for aptamer-based assays. Strong dependence between the length and electrophoretic mobility of short nucleic acids makes polyacrylamide gel electrophoresis a powerful tool for studying their complexes. The interactions between the 36 nb DNA G-quadruplex aptamer (5’-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3’) specific to ochratoxin A and 9 complementary single-stranded DNA (ssDNA) were studied. The length of ssDNA varied from 5 to 9 nb. To maintain ligand-binding conformation of the aptamer, a high ionic strength buffer was used. The best resolution between the aptamer and its complex was provided for the gel with 15% monomer and a monomer/cross-linker ratio of 15:1. Bands of free aptamer and ssDNA were observed for all studied variants. If the ssDNA length was less than 9 nb, the position of the aptamer’s band remained unchanged, independent of the aptamer/ssDNA ratios, and additional bands did not appear. The longest ssDNA (5’-CGC-CAC-CCA-3’) did not lead to the appearance of a new band, but it slowed the aptamer’s migration depending on the ssDNA concentration. Under a 27-fold excess of the given ssDNA, the relative mobility of the aptamer band changed from 0.566 to 0.468. Thus, electrophoresis visualizes aptamer-ssDNA interactions and can be used in the development of aptamer-based analytical systems.

Gold Nanostars Bioconjugation for Selective Targeting and SERS Detection of Biofluids.

Gold nanoparticles (AuNPs) show physicochemical and optical functionalities that are of great interest for spectroscopy-based detection techniques, and especially for surface enhanced Raman spectroscopy (SERS), which is capable of providing detailed information on the molecular content of analysed samples. Moreover, the introduction of different moieties combines the interesting plasmonic properties of the AuNPs with the specific and selective recognition capabilities of the antibodies (Ab) towards antigens. The conjugation of biomolecules to gold nanoparticles (AuNPs) has received considerable attention for analysis of liquid samples and in particular biological fluids (biofluids) in clinical diagnostic and therapeutic field. To date, gold nanostars (AuNSts) are gaining more and more attention as optimal enhancers for SERS signals due to the presence of sharp branches protruding from the core, providing a huge number of “hot spots”. To this end, we focused our attention on the design, optimization, and deep characterization of a bottom up-process for (i) AuNPs increasing stabilization in high ionic strength buffer, (ii) covalent conjugation with antibodies, while (iii) retaining the biofunctionality to specific tag analyte within the biofluids. In this work, a SERS-based substrate was developed for the recognition of a short fragment (HA) of the hemagglutinin protein, which is the major viral antigen inducing a neutralizing antibody response. The activity and specific targeting with high selectivity of the Ab-AuNPs was successfully tested in transfected neuroblastoma cells cultures. Then, SERS capabilities were assessed measuring Raman spectra of HA solution, thus opening interesting perspective for the development of novel versatile highly sensitive biofluids sensors.

Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (E-AB) sensors are a versatile sensing platform that can achieve rapid and robust target detection in complex matrices. However, the limited sensitivity of these sensors has impeded their translation from proof-of-concept to commercial products. Surface-bound aptamers must be sufficiently spaced to bind targets and subsequently fold for signal transduction. We hypothesized that electrodes fabricated using conventional methods result in sensing surfaces where only a fraction of aptamers are appropriately spaced to actively respond to the target. As an alternative, we presented a novel aptamer immobilization approach that favors sufficient spacing between aptamers at the microscale to achieve optimal target binding, folding, and signal transduction. We first demonstrated that immobilizing aptamers in their target-bound, folded state on gold electrode surfaces yields an aptamer monolayer that supports greater sensitivity and higher signal-to-noise ratio than traditionally prepared E-AB sensors. We also showed that performing aptamer immobilization under low ionic strength conditions rather than conventional high ionic strength buffer greatly improves E-AB sensor performance. We successfully tested our approach with three different small-molecule-binding aptamers, demonstrating its generalizability. On the basis of these results, we believe our electrode fabrication approach will accelerate development of high-performance sensors with the sensitivity required for real-world analytical applications.

Tunnel dielectrophoresis for ultra-high precision size-based cell separation.

In molecular and cellular biological research, cell isolation and sorting are required for accurate investigation of cell populations of specific physical or biological characteristics. By employing unique cell properties to distinguish between heterogeneous cell populations, rapid and accurate sorting with high efficiency is possible. Dielectrophoresis-based cell manipulation has significant promise for separation of cells based on their physical properties and is used in diverse areas ranging from cellular diagnostics to therapeutic applications. In this study, we present a microfluidic device that can achieve label-free and size-based cell separation with high size differential resolution from a mono-cellular population or complex sample matrices. It was realized by using the tunnel dielectrophoresis (TDEP) technique to manipulate the spatial position of individual cells three dimensionally with high resolution. Cells were processed in high speed flows in high ionic strength buffers. A mixture of different sizes of polystyrene micro-particles with a size difference as small as 1 μm can be separated with high purity (>90%). For the first time, high-pass, low-pass, and band-pass filtering within a mono-cellular mammalian cell population were demonstrated with a tunable bandwidth as small as 3 μm. In addition, leukocyte subtype separation was demonstrated by sorting monocytes out of peripheral blood mononuclear cells (PBMCs) from whole blood with high purity (>85%). Its ability to deliver real-time adjustable cut-off threshold size-based cell sorting and its capability to provide an arbitrary cell size pick-up band could potentially enable many research and clinical applications.

Narrower Nanoribbon Biosensors Fabricated by Chemical Lift-off Lithography Show Higher Sensitivity.

Wafer-scale nanoribbon field-effect transistor (FET) biosensors fabricated by straightforward top-down processes are demonstrated as sensing platforms with high sensitivity to a broad range of biological targets. Nanoribbons with 350 nm widths (700 nm pitch) were patterned by chemical lift-off lithography using high-throughput, low-cost commercial digital versatile disks (DVDs) as masters. Lift-off lithography was also used to pattern ribbons with 2 μm or 20 μm widths (4 or 40 μm pitches, respectively) using masters fabricated by photolithography. For all widths, highly aligned, quasi-one-dimensional (1D) ribbon arrays were produced over centimeter length scales by sputtering to deposit 20 nm thin-film In2O3 as the semiconductor. Compared to 20 μm wide microribbons, FET sensors with 350 nm wide nanoribbons showed higher sensitivity to pH over a broad range (pH 5 to 10). Nanoribbon FETs functionalized with a serotonin-specific aptamer demonstrated larger responses to equimolar serotonin in high ionic strength buffer than those of microribbon FETs. Field-effect transistors with 350 nm wide nanoribbons functionalized with single-stranded DNA showed greater sensitivity to detecting complementary DNA hybridization vs 20 μm microribbon FETs. In all, we illustrate facile fabrication and use of large-area, uniform In2O3 nanoribbon FETs for ion, small-molecule, and oligonucleotide detection where higher surface-to-volume ratios translate to better detection sensitivities.

Versatile Bioconjugation Strategies of PEG-Modified Upconversion Nanoparticles for Bioanalytical Applications.

Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL).

Intact charge variant analysis of ziv-aflibercept by cationic exchange liquid chromatography as a proof of concept: Comparison between volatile and non-volatile salts in the mobile phase

Ziv-aflibercept (ziv-AFL) is a complex fusion protein which is widely used in hospitals for the treatment of colorectal metastatic cancer. Charge variants are critical attributes for assessing post-transitional modifications (PMTs) that have to be controlled during the development and manufacture of these proteins and until their administration to patients. Cation exchange (CEX) chromatography is a charge-sensitive analytical method that is well suited for analysing charge variants in proteins. The aim of this paper is to analyse the charge variants of ziv-AFL in the medicine (Zaltrap®) when fresh and when degraded. Two CEX chromatographic methods were compared for this purpose. The former was an adaptation of the method used in the first published study in which charge variants were analysed via pH gradient elution using volatile, low ionic strength buffers with direct coupling to high-resolution Orbitrap mass spectrometry. The second method was developed and optimized during our research using the salt-mediated pH gradient mode and classical non-volatile, high ionic strength buffers which were incompatible with direct coupling with mass detection. Fresh and controlled degraded samples of ziv-AFL were used to evaluate the capacity of both CEX chromatographic strategies for detecting charge variants in ziv-AFL. In the controlled degradation study the samples of the medicine were subjected to three stress factors: temperature of 60 °C for three hours, freeze/thaw process –two cycles-, and exposure to light for twelve hours. The CEX chromatographic method with non-volatile salts in the mobile phase enabled better detection of charge variants degraded ziv-AFL samples than the method using volatile salts with lower ionic strength. In addition, the complexity of the mass spectra data generated made it impossible to identify the multicharge variant species of ziv-AFL. Although charge variants were not separated in ziv-AFL fresh sample, our results indicate that the method with non-volatile salts in the mobile phase could be used to characterize and track changes in the charge variant UV chromatographic profile of ziv-AFL in fresh and degraded samples, even though it cannot be coupled to a mass detector and there is therefore no information about mass. The increase of basic protein degraded compounds were the most important degradation pattern detected in ziv-AFL (Zaltrap®).

High salt compatible oxyanion receptors by dual ion imprinting

Imprinting of an ion-pair in presence of mutually compatible anion and cation host monomers leads to polymers showing enhanced ion uptake in competitive high ionic strength buffers.

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins −1 and −4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.

The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism.

Amyloid fibrils, highly ordered protein aggregates, play an important role in the onset of several neurological disorders. Many studies have assessed amyloid fibril formation under specific solution conditions, but they all lack an important phenomena in biological solutions—buffer specific effects. We have focused on the formation of hen egg-white lysozyme (HEWL) fibrils in aqueous solutions of different buffers in both acidic and basic pH range. By means of UV-Vis spectroscopy, fluorescence measurements and CD spectroscopy, we have managed to show that fibrillization of HEWL is affected by buffer identity (glycine, TRIS, phosphate, KCl-HCl, cacodylate, HEPES, acetate), solution pH, sample incubation (agitated vs. static) and added excipients (NaCl and PEG). HEWL only forms amyloid fibrils at pH = 2.0 under agitated conditions in glycine and KCl-HCl buffers of high enough ionic strength. Phosphate buffer on the other hand stabilizes the HEWL molecules. Similar stabilization effect was achieved by addition of PEG12000 molecules to the solution.

Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid

The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.

Graphene‐Based Electronic Immunosensor with Femtomolar Detection Limit in Whole Serum

AbstractPurely electronic diagnostic devices that are sensitive to biomolecules' intrinsic charge present a very attractive platform for point‐of‐care (PoC) applications, since they can operate under label‐free conditions. In this report, a graphene‐based electrolyte‐gated field‐effect transistor is developed as an immunosensor capable of sensitive analyte detection, even in the complex environment of physiological samples. The coimmobilization of an antibody fragment (F(ab′)2) and polyethylene glycol on the graphene surface allows for a highly sensitive and selective detection of a protein analyte, with a limit of detection in the low femtomolar range, both in high ionic strength buffer and undiluted serum. Multiparametric analysis of the device's analyte‐dependent electronic response shows that the mechanism behind this very sensitive detection cannot be explained by a commonly reported electrostatic gating effect. Rather, the observed combination of charge neutrality point shifts and asymmetric mobility changes are attributed to the modulation of scattering by charged impurities, which seem to dominate the device's transfer characteristics. Furthermore, the reproducibility of the normalized signal response obtained from several different devices shows that this graphene‐based immunosensor is capable of direct and quantitative measurements of protein analytes in untreated serum, imperative for diagnostic tools geared toward PoC applications.

Biomolecule Detection in High Ionic Strength With Enhanced Sensitivity Using Graphene Nanogrid FET Immunosensor

Detecting charges in high ionic strength solutions is a challenge due to the inherent problem of ionic screening in small Debye length. There have been some efforts to improve the sensing in high ionic strength buffer but the sensitivity and detection limit is poor. To address this challenge, we report high frequency sensing using smooth graphene nanogrid FET immunosensor. These sensors have certain intrinsic advantages: firstly, in the nanopores, the biomolecules usually reside within a distance shorter than the original pore radius which statistically raises the charge transfer probability between the biomolecules and the surface even at high ionic strength of the buffer and secondly, such a graphene nanostructure has a size dependent effective energy gap which increases the transconductance of the device. These features coupled with high frequency sensing have enabled detection down to 0.1 fMHep-B in a high ionic strength buffer (150mM) at 8 MHz with a sensitivity as high as 445.45%. This is an enhancement by more than 18 times compared to the existing reports.

If Squeezed, a Camel Passes Through the Eye of a Needle: Voltage-Mediated Stretching of Dendrimers Facilitates Passage Through a Nanopore.

Herein, we report uni-molecular observations of electric potential- and electrolyte-dependent elasticity of poly(amidoamine) (PAMAM)-G1.5 dendrimers containing sodium carboxylate surface groups, using the electric field-assisted migration through the α-hemolysin nanopore (α-HL). Although at moderate transmembrane potentials the dendrimer (~ 2.5nm in diameter) is sterically excluded from translocation across the constriction region of the nanopore (~ 1.5nm in diameter), we found a threshold for its translocation that depends on both the electrolyte pH and ionic strength. We posit that the decreased repulsive intramolecular interactions among dendrimer's branches at low when compared to neutral pH, caused mainly by the protonation of surface groups on the dendrimer, determine a larger propensity of the dendrimer to collapse and deform. This in turns enables the dendrimer to adopt more favorably conformations that facilitate its optimal squeezing through the α-HL's constriction region at low pH, despite the fact that the estimated net force acting on it becomes approximately one order of magnitude lower than at neutral pH. Experiments performed in a low ionic strength buffer, which decreases Coulombic screening, enhance the intramolecular forces on the dendrimer and renders the dendrimer stiffer than in high ionic strength buffer, confirming the dendrimer elastic properties-dependent threshold for deformation inside the nanopore.

Real‐time detection of condensin‐driven DNA compaction reveals a multistep binding mechanism

Condensin, a conserved member of the SMC protein family of ring‐shaped multi‐subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single‐molecule magnetic tweezers to measure, in real time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction can proceed in large steps, driving DNA molecules into a fully condensed state against forces of up to 2 pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt insensitive and for the subsequent compaction process. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

Development of a Spectrophotometric Method for Determining pH of Soil Extracts and Comparison with Glass Electrode Measurements

Core Ideas Soil pH is a critical parameter in soils as it influences many biogeochemical processes. There are problems in pH measurements in soils using glass electrodes. A new spectrophotometric method was developed to measure pH in soils. The method compared well to measurements using glass electrodes in a range of soils. A reflectance probe technique was also successfully trialed for in situ measurement. Soil pH measurement using conventional glass electrodes has several limitations. A spectrophotometric method was developed for determining soil pH involving indicator dye (bromocresol purple or phenol red) addition to soil extracts. Results were compared against values determined using a glass electrode for a range of soils (n = 13) with varying properties using different extraction conditions (1:1 w/v soil/water, 1:1 soil/0.01 mol L–1 CaCl2, 1:5 soil/water and 1:5 soil/0.01 mol L–1 CaCl2) and high and low ionic strength buffer calibrations of the electrode. For all extraction conditions, there was a strong relationship (r2 > 0.95, slope ≈ 1) between values of the spectrophotometric (pHspec) and glass electrode (pHelec) methods. The precision of pHspec was similar to pHelec measurements across the different extraction conditions (±0.02–0.08 average standard deviation of triplicate measurements, n = 39). Large and variable differences were observed between pHelec measured following calibration with high (µ = 0.1 mol L–1) and low (µ = 0.005 mol L–1) ionic strength buffers. In contrast, ionic strength effects on the indicator dye and resulting pHspec calculation are implicitly accounted for. A spectrophotometric reflectance probe in situ method was also successfully trialed. The spectrophotometric pH method circumvents many of the problems associated with the use of glass electrodes in soil solutions.

Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson’s disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies.