Low Levels Of Nonspecific Binding Research Articles

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

Next Generation Immunotherapies – Emerging Strategies for Immune Modulation against Cancer, Infections, and Beyond

Next Generation Immunotherapies – Emerging Strategies for Immune Modulation against Cancer, Infections, and Beyond

Aqueous quantum dots with high fluorescence, colloidal stability and biocompatibility encapsulated by an amphiphilic fluorine copolymer

Quantum dots (QDs) encapsulated by amphiphilic fluorine copolymers have shown great promise as molecular probes for bio-imaging. Here, a series of amphiphilic fluorine copolymers, methoxypolyethylene glycols-block-poly (2-(diethylamino)ethyl methacrylate)-block-poly (2,2,3,4,4,4-hexafluorobutyl methacrylate) (OmAnFp), were successfully coated on the surface of semi conductive quantum dots (QDs) to overcome their stability issues in aqueous solution. The hydrodynamic diameters (Dh,DLS) of these OmAnFp encapsulated QDs (OmAnFp@QDs) can be tuned between 40 nm and 61 nm by choosing amphiphilic fluorine copolymers with different hydrophobic portions. The obtained OmAnFp@QDs colloids are found stable over a wide pH range (4.0–12.0) with no sign of aggregation. In the pH range of 4.0–7.0, the repulsion of block O and cationic block A on OmAnFp@QDs contributes to the colloids’ stability in aqueous solution. The protonation of block A also leads to the decrease of Dh,DLS from 61 to 51 nm and the enhancement of the transmittance of O113A11F19@QDs solution from 78% to 92%. In the pH range from 8.0 to 12.0, the repulsion between the strong hydrated block O makes OmAnFp@QDs nanoparticles stable with deprotonated hydrophobic block A. The fluorescent quantum yield properties of QDs are well preserved in OmAnFp@QDs aqueous solution due to the protection of block F wrapping tightly around the QDs cores. The photoluminescence quantum yield (PL QY) is found reserved better under basic conditions due to the co-encapsulation of hydrophobic block A. The PL intensity of OmAnFp@QDs in 400 mM NaCl solution changes only slightly. Block O and F in the polymer offer OmAnFp@QDs with good biocompatibility and low levels of nonspecific binding to bovine serum albumin proteins (BSA). These advantages render OmAnFp@QDs an attractive candidate as ideal fluorescent probes in advanced biomedical imaging studies.

Nature-inspired design and evolution of anti-amyloid antibodies

Antibodies that recognize amyloidogenic aggregates with high conformational and sequence specificity are important for detecting and potentially treating a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, these types of antibodies are challenging to generate because of the large size, hydrophobicity, and heterogeneity of protein aggregates. To address this challenge, we developed a method for generating antibodies specific for amyloid aggregates. First, we grafted amyloidogenic peptide segments from the target polypeptide [Alzheimer's amyloid-β (Aβ) peptide] into the complementarity-determining regions (CDRs) of a stable antibody scaffold. Next, we diversified the grafted and neighboring CDR sites using focused mutagenesis to sample each WT or grafted residue, as well as one to five of the most commonly occurring amino acids at each site in human antibodies. Finally, we displayed these antibody libraries on the surface of yeast cells and selected antibodies that strongly recognize Aβ-amyloid fibrils and only weakly recognize soluble Aβ. We found that this approach enables the generation of monovalent and bivalent antibodies with nanomolar affinity for Aβ fibrils. These antibodies display high conformational and sequence specificity as well as low levels of nonspecific binding and recognize a conformational epitope at the extreme N terminus of human Aβ. We expect that this systematic approach will be useful for generating antibodies with conformational and sequence specificity against a wide range of peptide and protein aggregates associated with neurodegenerative disorders.

Magnetic hybrid magnetite/metal organic framework nanoparticles: facile preparation, post-synthetic biofunctionalization and tracking in vivo with magnetic methods

Multifunctional hybrid nanocomposites remain to be of great interest in biomedicine as a universal tool in a number of applications. As a promising example, the nanoparticles with magnetic core and porous shell have a potential as theranostic agents combining both the diagnostics probe and drug delivery vehicle properties. However, reported methods of the nanostructure preparation are complex and include tedious time-consuming growth of porous shell by means of layer by layer assembly technique. In this study, we develop new way of fabrication of the superparamagnetic magnetite core @ porous metal organic framework shell nanoparticles and demonstrate their application both as a multimodal (MRI contrasting, magnetometric and optical labeling) and multifunctional (in vivo bioimaging, biotargeting by coupled receptors, lateral flow assay) agents. The easiness of fabrication, controllable bioconjugation properties and low level of non-specific binding indicate high potential of the nanoparticles to be employed as multifunctional agents in theranostics, advanced biosensing and bioimaging.

Liposome-Encapsulated NaLnF4 Nanoparticles for Mass Cytometry: Evaluating Nonspecific Binding to Cells

We are interested in developing lanthanide nanoparticles (NPs) as high sensitivity tagging reagents for antibodies to analyze cells by mass cytometry (MC). Two key prerequisites for this application are that the NPs have to be colloidally stable in phosphate-containing buffers and the free NPs must have very low levels of nonspecific binding to cells. These are the issues we address here. We describe the synthesis of 30 nm diameter NaYF4:Yb,Er nanoparticles, their transfer to aqueous solution via citrate exchange, and their encapsulation in liposomes to minimize their interaction with live cells. The lipid coating consisted of a 2:2:1 mol ratio mixture of dioleoylphosphatidyl choline (DOPC), egg sphingomyelin (ESM), and ovine cholesterol (Chol), referred to as DEC221. Since encapsulating 30 nm NPs in liposomes is an unprecedented challenge, we added varying amounts of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG-2000] (mPEG2K-DSPE) to the lipid formulation, both to promote curvature of th...

Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification

The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus. Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V. parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 105 colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5μg of IMNPs within 30min. Although a very low level of non-specific binding was seen among 8 non-V. parahaemolyticus Vibrio spp. and 5 non-Vibrio strains, the IMS–LAMP method identified 133 V. parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4×102CFU/mL in pure culture and was unaffected by the presence of 108CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9×103CFU/g without enrichment. After enrichment for 6–8h, the limit of detectability could be improved to 1.9 to 0.19CFU/g. Hence, the IMS–LAMP assay provided a rapid, simple, and cost-effective method for total V. parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution.

Allergen immobilisation and signal amplification by quantum dots for use in a biosensor assay of IgE in serum

The production of biosensors for point of care diagnostics usually requires the immobilisation and storage of protein (for example, antigen or antibody) on a sensor surface, in a manner that retains a high degree of activity and low levels of non-specific binding. These characteristics have been assessed for polymer immobilised antigens (allergens) using an IgG binding assay and demonstrated further by assay with serum containing reactive IgEs.The activity of allergens immobilised on sensor chips using copoly(DMA–NAS–MAPS) and a spotting technique, as well as the specificity of their binding interactions with cognate immunoglobulins was assessed using Dual Polarisation Interferometry (DPI). The data obtained indicate that the allergens studied remain stable over long periods of time (at least 114 days). This performance compared favourably with other immobilisation methods. Allergen coated chips were tested in an anti-casein IgE assay using human serum from allergic and non-allergic donors. Detection of both total Ig and specific IgE was demonstrated using a secondary anti-IgE antibody. Furthermore, optical signal enhancement with streptavidin conjugated quantum dots was shown to yield responses for samples below 0.84ng/mL (0.35KU/L) of IgE, which overlap with the industrial quasi-standard ImmunoCAP® and is the clinically relevant threshold used to classify serum samples from allergic individuals.

Stability of polymer encapsulated quantum dots in cell culture media

The unique optical properties of Quantum Dots have attracted a great interest to use these nanomaterials in diverse biological applications. The synthesis of QDs by methods from the literature permits one to obtain nanocrystals coated by hydrophobic alkyl coordinating ligands and soluble in most of the cases in organic solvents. The ideal biocompatible QD must be homogeneously dispersed and colloidally stable in aqueous solvents, exhibit pH and salt stability, show low levels of nonspecific binding to biological components, maintain a high quantum yield, and have a small hydrodynamic diameter. Polymer encapsulation represents an excellent scaffold on which to build additional biological function, allowing for a wide range of grafting approaches for biological ligands. As these QD are functionalized with poly(ethylene)glycol (PEG) derivatives on their surface, they show long term stability without any significant change in the optical properties, and they are also highly stable in the most common buffer solutions such as Phosphate Buffer Saline (PBS) or borate. However, as biological studies are normally done in more complex biological media which contain a mixture of amino acids, salts, glucose and vitamins, it is essential to determine the stability of our synthesized QDs under these conditions before tackling biological studies.

Designing DNA Barcodes Orthogonal in Melting Temperature by Simulated Annealing Optimization

Molecular barcode arrays are widely employed in the analysis of large strain libraries, whereby probes linked to unique oligonucleotides ("antitags") are used to detect selected DNA targets ("tags") by highly specific hybridization. One of the major problems for such screen designs is thus insuring a high degree of probe-target specificity and a low level of nonspecific binding (in sum, "orthogonality") across the entire tag population ("collection"). Several approaches have been previously proposed for designing orthogonal DNA tags by-among others-focusing on their individual or pair-wise structures, such as Smith Waterman sequence similarity, the widely used nearest neighbor method, and full thermodynamic estimates of sequences. However, these methods generally involve imposing various heuristic constraints ("design rules") on possible tag/antitag (TaT) sequences in order to achieve probe-target specificity across the collection. The resulting lack of freedom in considering all putative sequences can lead to potentially suboptimal designs and to the ensuing reduction in the degree of orthogonality within the constructed TaT collections. Here, we demonstrate that a randomized-search algorithm based on simulated annealing optimization can be used in order to substantially free the design process from the limitations of sequence constraints-allowing for the elucidation of potentially more optimal DNA tag collections. The designed sets of DNA oligonucleotides are optimized for the highest degree of orthogonality as quantified by melting temperature Tm-an experimentally relevant system property, which could also be used as a theoretically meaningful thermodynamic metric for optimizing TaT binding specificity. That is, this work describes an approach to constructing tag/antitag libraries, which offer the greatest melting temperature separation between specific probe-target duplexes and other nonspecific structures. The proposed method finds, with high probability, the global solution that maximizes the difference in Tm between the specific and nonspecific tag-antitag hybridizations across a collection of given size for TaTs of specified length. An application of this approach is demonstrated using 2 different DNA probe sets.

Screening nonspecific interactions of peptides without background interference

The need to discover new peptide sequences to perform particular tasks has lead to a variety of peptide screening methods: phage display, yeast display, bacterial display and resin display. These are effective screening methods because the role of background binding is often insignificant. In the field of nonfouling materials, however, a premium is placed on chemistries that have extremely low levels of nonspecific binding. Due to the presence of background binding, it is not possible to use traditional peptide screening methods to select for nonfouling chemistries. Here, we developed a peptide screening method, as compared to traditional methods, that can successfully evaluate the effectiveness of nonfouling peptide sequences. We have tested the effect of different peptide lengths and chemistries on the adsorption of protein. The order of residues within a single sequence was also adjusted to determine the effect of charge segregation on protein adsorption.

Quantitative molecular profiling of biomarkers for pancreatic cancer with functionalized quantum dots

Applications in nanomedicine, such as diagnostics and targeted therapeutics, rely on the detection and targeting of membrane biomarkers. In this article we demonstrate absolute quantitative profiling, spatial mapping, and multiplexing of cancer biomarkers using functionalized quantum dots (QDs). We demonstrate highly selective targeting molecular markers for pancreatic cancer with extremely low levels of nonspecific binding. We confirm that we have saturated all biomarkers on the cell surface, and, in conjunction with control experiments, extract absolute quantitative values for the biomarker density in terms of the number of molecules per square micron on the cell surface. We show that we can obtain quantitative spatial information of biomarker distribution on a single cell, important because tumors' cell populations are inherently heterogeneous. We validate our quantitative measurements (number of molecules per square micron) using flow cytometry and demonstrate multiplexed quantitative profiling using color-coded QDs. From the Clinical EditorThis paper demonstrates a nice example for quantum dot-based molecular targeting of pancreatic cancer cells for advanced high sensitivity diagnostics and potential future selective therapeutic purposes.

Optimization of Phosphatase- and Redox Cycling-Based Immunosensors and Its Application to Ultrasensitive Detection of Troponin I

The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium-tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl(2), and the optimum conditions for antigen-antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen-antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (L-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (L-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.

Synthesis and functionalization of superparamagnetic poly-ε-caprolactone microparticles for the selective isolation of subpopulations of human adipose-derived stem cells.

There has been a growing interest in using biofunctionalized magnetic particles for cell isolation. This paper describes the synthesis and characterization of magnetite-polymer (Fe(3)O(4)-poly-ε-caprolactone, magnetite-PCL) microparticles surface functionalized with amino and epoxy groups allowing easy covalent attachment of specific antibodies and subsequent ability to bind target cells. Particles with different sizes (4-135 µm), spherical shape and superparamagnetic behaviour (magnetite content of about 13 wt%) were obtained. The functionalized microparticles presented high protein-binding capacity (coupling efficiency of 47% for epoxy- and 71% for amino-functionalized particles) with a low level of non-specific binding. We have further investigated the influence of initial protein concentration, pH, ionic strength, temperature and incubation time on the capacity of amino-functionalized particles to bind protein molecules. The results showed that maximum protein coupling is rapidly achieved (≤5 h) at pH 5.5 and low ionic strength (0.05 M NaCl). Furthermore, when cultured in direct contact with osteoblast-like cells (Saos-2) or human-derived adipose stem cells (ASCs), the amino-functionalized particles did not affect the proliferation and morphology of the cells. As a proof of principle for the application of magnetic microparticles for cell isolation, CD105 (endoglin) antibody was coupled to the magnetic particle surface to bind subpopulations of human ASCs expressing the CD105 antigen. The isolation of CD105+ ASCs from a heterogeneous cell population was confirmed by flow cytometry analysis. Given the demonstrated potential of functionalized magnetite-PCL microparticles for selective cell isolation, we expect that these particles may be further applied in immuno-magnetic cell separation owing to their versatility and ease of surface modification.

Synthesis and characterization of a ‘fluorous’ (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides

Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.

A heme fusion tag for protein affinity purification and quantification.

We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200-500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.

Microwave induced MIP synthesis: comparative analysis of thermal and microwave induced polymerisation of caffeine imprinted polymers

Caffeine templated molecularly imprinted polymers (MIPs) have been prepared using identical polymer formulations by thermal and microwave induced initiation, and the binding performance of the systems compared using solid phase extraction (SPE). While the binding capacity of MIP(Microwave) was found to be lower than MIP(Thermal), MIP(Microwave) recorded a higher imprinting factor (IF) due to comparatively low levels of non-specific binding. Selectivity against theophylline in cross-reactivity studies was also found to be greater for MIP(Microwave). While large time savings are achievable through the use of microwave irradiation conditions, physical analysis of the polymers (surface area analysis, thermal gravimetric analysis and differential scanning calorimetry) reveals that the different polymerisation methods lead to differences in both polymer structure and performance.

Platform for Highly Sensitive Alkaline Phosphatase‐Based Immunosensors Using 1‐Naphthyl Phosphate and an Avidin‐Modified Indium Tin Oxide Electrode

AbstractWe report a versatile platform for highly sensitive alkaline phosphatase (ALP)‐based electrochemical biosensors that uses an avidin‐modified indium tin oxide (ITO) electrode as a sensing electrode and 1‐naphthyl phosphate (NPP) as an ALP substrate. Almost no electrocatalytic activity of NPP and good electrocatalytic activity of 1‐naphthol (ALP product) on the ITO electrodes allow a high signal‐to‐background ratio. The effective surface covering of avidin on the ITO electrodes allows very low levels of nonspecific binding of proteins to the sensing electrodes. The platform technology is used to detect mouse IgG with a detection limit of 1.0 pg/mL.

The Preparation of Colloidally Stable, Water-Soluble, Biocompatible, Semiconductor Nanocrystals with a Small Hydrodynamic Diameter

We report a simple, economical method for generating water-soluble, biocompatible nanocrystals that are colloidally robust and have a small hydrodynamic diameter. The nanocrystal phase transfer technique utilizes a low molecular weight amphiphilic polymer that is formed via maleic anhydride coupling of poly(styrene-co-maleic anhydride) with either ethanolamine or Jeffamine M-1000 polyetheramine. The polymer encapsulated water-soluble nanocrystals exhibit the same optical spectra as those formed initially in organic solvents, preserve photoluminescence intensities, are colloidally stable over a wide pH range (pH 3-13), have a small hydrodynamic diameter, and exhibit low levels of nonspecific binding to cells.