Selective Protein Binding Research Articles

Diamond Surfaces with Clickable Antifouling Polymer Coating for Microarray‐Based Biosensing

AbstractDiamond enables the construction of various (bio)sensors, including those with quantum‐based detection. However, bare diamond interfaces are susceptible to unspecific adhesion of proteins and other macromolecules from biological media or complex samples. This impairs selectivity in biosensing, leads to low signal‐to‐noise ratio in fluorescence‐based applications, and introduces the need for blocking steps in incubation protocols. Here, a stable, protein‐repellent, and clickable reactive polymer coating is introduced, abolishing unspecific protein adhesion while concurrently enabling covalent immobilization of functional compounds as recognition elements. The polymer coating has two segments, an antifouling poly(N‐(2‐hydroxypropyl) methacrylamide) and an alkyne‐terminated poly(propargyl methacrylamide) providing the click functionality. The antifouling properties and click‐reactivity of the polymers are demonstrated by selective protein binding assays on micropatterns written by microchannel cantilever spotting (µCS). The assays demonstrated the successful functionalization of both diamond and glass surfaces and the excellent antifouling properties of the polymer coating. The coating procedure is compatible with oxidized diamond surfaces thus well‐suitable for diamond‐based quantum technology. The results can directly impact applications of diamond materials in optically detected quantum sensing or fluorescence sensing in general. The polymer functionalization can also be used for any case where highly specific interaction with low fouling is desired.

Preparation of nickel-chelated iminodiacetate-functionalized macroporous agarose monolith using modular and clickable building blocks for affinity separation of histidine-tagged recombinant proteins

Selective separation and purification of protein from complex medium is required to completely investigate the structure and function of the target protein. In this study, a composite macroporous agarose monolith containing iminodiacetate-chelated Ni2+ ligands was synthesized for selective separation and purification of histidine-tagged recombinant proteins. The large and interconnected pores in the monolith enabled fast binding of proteins with high matrix tolerance in treating complex mediums. To realize the selective protein binding, the iminodiacetate was directly conjugated to epoxy-functionalized agarose monolith via simple chemical reactions between epoxy and imino groups. After chelated Ni2+, the composite monolith could bind histidine-tagged recombinant proteins through the coordination interaction between transition metal ions and the imidazole ring of histidine. To further increase the binding capacities of the monolith, a hydrophilic intermediate polymer chain containing multiple iminodiacetate immobilization sites was conjugated to the azide-functionalized agarose monolith via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The morphology and chemical composition of the composite agarose monolith were characterized systematically. The protein binding capacities of the obtained composite agarose monolith were subsequently investigated. The binding capacities of the composite agarose monolith towards the model proteins Gp10 and Lys84 were 0.93 and 0.51 mg/mL, respectively. The protein binding of the composite agarose monolith could be manipulated by adjusting the temperature and concentrations of imidazole. These results demonstrate that the composite agarose monolith could be used as an affinity medium for rapid separation and purification of histidine-tagged recombinant proteins from biological samples.

Tuning Proapoptotic Activity of a Phosphoric‐Acid‐Tethered Tetraphenylethene by Visible‐Light‐Triggered Isomerization and Switchable Protein Interactions for Cancer Therapy

AbstractWe herein report a phosphoric‐acid‐substituted tetraphenylethene (T‐P) capable of adapting its geometric configuration and biological activity to the microenvironment upon light irradiation for apoptosis modulation. Different from most ultraviolet‐responsive isomerization, T‐P undergoes cis‐trans isomerization under visible light irradiation, which is biocompatible and thus photo‐modulation is possible in living biosystems. By using alkaline phosphatase (ALP) and albumin as dual targets, T‐P isomers display different protein binding selectivity, cancer‐cell internalization efficiency and apoptosis‐inducing ability. The proapoptotic activity was found to be kinetically controlled by the enzymatic reaction with ALP and regulated by co‐existing albumin. Motivated by these findings, two‐way modulation of proapoptotic effect and on‐demand boosting anticancer efficacy were realized in vitro and in vivo using light and endogenous proteins as multiple non‐invasive switching stimuli.

Tuning Proapoptotic Activity of a Phosphoric-Acid-Tethered Tetraphenylethene by Visible-Light-Triggered Isomerization and Switchable Protein Interactions for Cancer Therapy.

We herein report a phosphoric-acid-substituted tetraphenylethene (T-P) capable of adapting its geometric configuration and biological activity to the microenvironment upon light irradiation for apoptosis modulation. Different from most ultraviolet-responsive isomerization, T-P undergoes cis-trans isomerization under visible light irradiation, which is biocompatible and thus photo-modulation is possible in living biosystems. By using alkaline phosphatase (ALP) and albumin as dual targets, T-P isomers display different protein binding selectivity, cancer-cell internalization efficiency and apoptosis-inducing ability. The proapoptotic activity was found to be kinetically controlled by the enzymatic reaction with ALP and regulated by co-existing albumin. Motivated by these findings, two-way modulation of proapoptotic effect and on-demand boosting anticancer efficacy were realized in vitro and in vivo using light and endogenous proteins as multiple non-invasive switching stimuli.

Selective Protein Separation Based on Charge Anisotropy by Spherical Polyelectrolyte Brushes.

Protein purification is of vital importance in the food industry, drug discovery, and other related fields. Among many separation methods, polyelectrolyte (PE)-based phase separation was developed and recognized as a low-cost purification technique. In this work, spherical polyelectrolyte brushes (SPBs) with a high specific surface area were utilized to study the protein accessibility and selective protein binding on highly charged nanoparticles (NPs) as well as the selective phase separation of proteins. The correlation between charge anisotropy, protein binding, and phase separation was investigated on various protein systems including those proteins with similar isoelectric points (pI) such as bovine serum albumin (BSA) and β-lactoglobulin (BLG), proteins with similar molecular weights such as BSA and hemoglobin (HB), and even protein variants (BLG-A and -B) with a tiny difference of amino acids. The nonspecific electrostatic interaction studied by turbidimetric titrations and isothermal calorimetry titration (ITC) indicates a specific binding between proteins and SPBs arising from the charge anisotropy of proteins. An optimized output based on selective protein binding on SPBs could be correlated for efficient protein separation through tuning external conditions including pH and ionic strength. These findings, therefore, proved that phase separation based on selective protein adsorption by SPBs was an efficient alternative for protein separation compared with the traditional practice.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Chemically Functionalized Phosphorene As a 2D Material Emerging Toward Sensitive Biosensors

The interest of phosphorene as 2D material is high due to its excellent mechanical, electrical and optical properties [1]. However, the possibility of its application in the area of biosensors is a result of low cytotoxic cell-viability effects and excellent cytocompatibility [2]. Phosphorene has much higher surface to volume ratio compared to graphene and transition metal dichalcogenides. This due to its “puckered” lattice structure. These properties are advantages in sensing applications [3]. But puckered layer is quite reactive to oxygen and water adsorbed on the surface of phosphorene [4]. One way to change it and improve the ambient stability of layered black phosphorus is to modify the surface so that covalently or non-covalently attached chemical compounds passivate the surface to prevent damage [4, 5].The biosensing device consists of chemical part and biological unit with unique specifities towards corresponding analytes for example proteins.In this work the biosensor of bacteria has been prepared in several steps: phosphorene preparation, functionalization with 4-azidobenzoic acid, followed by coating with an antibody layer.Phosphorene (few layered black phosphorus, FLBP) was prepared from pre-crushed black phosphorus (BP) dispersed in anhydrous dimethylformamide or toluene under an argon atmosphere by the liquid exfoliation. Afterwards, the resultant suspension was centrifuged to remove the residual un-exfoliated particles, yielding supernatant.We functionalized few layered black phosphorus (FLBP) by direct bonding of the phosphorus atom bearing lone electron pair with nitrogen during reaction with 4-azidobenzoic acid, leading to the formation of P=N double bonds, which passivate the reactive FLBP effectively. A covalent combining few-layered black phosphorus (FLBP) with 4-azidobenzoic acid can function as a bridge between FLBP and biomolecules. The functionalized phosphorene (f-FLBP) results in the possibility of using it as a biosensor platform for the detection of Heamophillus Influenza - one of the most common bacteria that cause infections in humans [6, 7]. It is very important to determine the presence of a given bacterial strain, because nowadays strains resistant to numerous antibiotics are more and more often observed. In the US alone, antibiotic-resistant bacteria cause at least 2 million infections and 23,000 deaths per year, which cause losses of 55-70 billion dollars a year [8].The efficient functionalization of BP flakes was revealed by infrared spectroscopy. The cyclic voltammetry and electrochemical impedance spectroscopy measurements were carried out in Na2SO4 solution containing reference redox systems: [Fe(CN)6]3−/4−.Heamophillus Influenza bacteria were detected by the impedimetric method. The changes of the charge transfer resistance (Rct) can be attributed to the selective protein binding on the surface of the electrode. This was preceded by the attachment of antibodies to f-FLBP and obtaining a stable sensory surface. The procedure is shown in Figure 1.Consequently:- the benzoic groups enhance electric conductivity delivering fast direct electron transfer.- antibody-coated functionalized phosphorene were used to detect a bacteria (Haemophilus influenzae).- modified FLBP can work as surface for determination of organic molecules, e.g., nucleic acid bases or peptides and proteins, while it offers the promising possibility for biosensing. ACKNOWLEDGEMENTS This work was supported by the Polish National Science Centre [2016/22/E/ST7/00102]; and the National Centre for Science and Development [347324/12/NCBR/2017]. The DS funds of the Faculty of Electronics, Telecommunications and Informatics of the Gdansk University of Technology are also acknowledged.

Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex.

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinβ1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.

Designing Selective Protein Binding Sites

Molecular recognition is a critical process for many biological functions and consists in non-covalent binding of different molecules, such as protein-protein, antigen-antibody and many others. The host-guest molecules involved often show a shape complementarity, and one of the main specification for molecular recognition is that the interaction must be selective, i.e. the host should bind strongly to one selected guest and poorly, if at all, to all other biomolecules. Our work focuses on the role played by the chemical heterogeneity and the steric compatibility on the selectivity power of the binding site between two proteins. We developed a computational design procedure, based on the caterpillar coarse-grained model, for a reference system where we fashioned the protein binding site as a protein-like surface which perfectly shapes a portion of the partner protein. The investigated range of surface area for the artificial binding sites falls in typical natural sizes (750 −1500 Å2). On the protein-like surface, we decorated anchoring points whose chemical functionalization is chosen so to optimise the binding with the protein. Using highly-efficient Monte Carlo simulations methods we explore the binding and folding properties of our artificial proteins. A significant result is that, despite the fact that protein and surface chemical sequences are interdependent and simultaneously generated to stabilise the bound folded structure, the protein is stable in the folded conformation even in the absence of the protein-like partner for all investigated systems. Moreover, we observe that an increase of the surface area results in a decrease of specificity for the binding, therefore imposing an upper limit for molecular recognition binding sites at the typical physiological temperature range.

Ultra-small v-shaped gold split ring resonators for biosensing using fundamental magnetic resonance in the visible spectrum

Strong light localization within metal nanostructures occurs by collective oscillations of plasmons in the form of electric and magnetic resonances. This so-called localized surface plasmon resonance (LSPR) has gained much interest in the development of low-cost sensing platforms in the visible spectrum. However, demonstrations of LSPR-based sensing are mostly limited to electric resonances due to the technological limitations for achieving magnetic resonances in the visible spectrum. In this work, we report the first demonstration of LSPR sensing based on fundamental magnetic resonance in the visible spectrum using ultrasmall gold v-shaped split ring resonators. Specifically, we show the ability for detecting adsorption of bovine serum albumin and cytochrome c biomolecules at monolayer levels, and the selective binding of protein A/G to immunoglobulin G.

Nanostructured Functional Polymers for Selective Protein Binding

Two-Photon Polymerization enables fabrication of two and three dimensional polymer structures. For structuring, a highly cross-linking acrylate monomer is mixed with a photo-initiator. Using femtosecond IR light as an excitation source, feature sizes down to 100 nm can be achieved. Applying stimulated emission depletion (STED) reduces the feature size even further [1, 2]. Recently, our group developed functional polymer structures below the diffraction limit by mixing metal oxo clusters into the acrylate-based photoresist [3].

Fighting Cancer with Transition Metal Complexes: From Naked DNA to Protein and Chromatin Targeting Strategies.

Many transition metal complexes have unique physicochemical properties that can be efficiently exploited in medicinal chemistry for cancer treatment. Traditionally, double‐stranded DNA has been assumed to be the main binding target; however, recent studies have shown that nucleosomal DNA as well as proteins can act as dominant molecular binding partners. This has raised new questions about the molecular determinants that govern DNA versus protein binding selectivity, and has offered new ways to rationalize their biological activity and possible side effects. To address these questions, molecular simulations at an atomistic level of detail have been used to complement, support, and rationalize experimental data. Herein we review some relevant studies—focused on platinum and ruthenium compounds—to illustrate the power of state‐of‐the‐art molecular simulation techniques and to demonstrate how the interplay between molecular simulations and experiments can make important contributions to elucidating the target preferences of some promising transition metal anticancer agents. This contribution aims at providing relevant information that may help in the rational design of novel drug‐discovery strategies.

Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl−, Br− and I−) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

Pre- and Postfunctionalized Self-Assembled π-Conjugated Fluorescent Organic Nanoparticles for Dual Targeting

There is currently a high demand for novel approaches to engineer fluorescent nanoparticles with precise surface properties suitable for various applications, including imaging and sensing. To this end, we report a facile and highly reproducible one-step method for generating functionalized fluorescent organic nanoparticles via self-assembly of prefunctionalized π-conjugated oligomers. The engineered design of the nonionic amphiphilic oligomers enables the introduction of different ligands at the extremities of inert ethylene glycol side chains without interfering with the self-assembly process. The intrinsic fluorescence of the nanoparticles permits the measurement of their surface properties and binding to dye-labeled target molecules via Förster resonance energy transfer (FRET). Co-assembly of differently functionalized oligomers is also demonstrated, which enables the tuning of ligand composition and density. Furthermore, nanoparticle prefunctionalization has been combined with subsequent postmodification of azide-bearing oligomers via click chemistry. This allows for expanding ligand diversity at two independent stages in the nanoparticle fabrication process. The practicability of the different methods entails greater control over surface functionality. Through labeling with different ligands, selective binding of proteins, bacteria, and functionalized beads to the nanoparticles has been achieved. This, in combination with the absence of unspecific adsorption, clearly demonstrates the broad potential of these nanoparticles for selective targeting and sequestration. Therefore, controlled bifunctionalization of fluorescent π-conjugated oligomer nanoparticles represents a novel approach with high applicability to multitargeted imaging and sensing in biology and medicine.

Development of Prediction Method for GPCR-G-protein Coupling Selectivity Using Amino Acid Properties

We describe a novel method for predicting G-protein coupled receptor (GPCR) - G-protein coupling selectivity using amino acid properties of specific residues in GPCR sequences. We have evaluated various amino acid properties obtained with experimental or theoretical studies. The GPCRs having reliable G-protein binding information were collected from Guide to Receptors and Channels (GRAC) and gpDB databases, and these sequences were aligned with the amino acid sequence of bovine rhodopsin, whose structure is known, to identify positions of each amino acid residue and its secondary structure. The collected properties were used as feature values to calculate Fisher's ratio (FR) for each residue in GPCRs related with rhodopsin residue numbers. Some amino acid properties with high FR value were picked up as the effective characteristics for selecting G-protein type, and they were used as feature vectors in support vector machine (SVM) to predict GPCR - G-protein coupling selectivity. Applying this method to known GPCR sequences, each binding G-protein is predicted with high sensitivity and specificity of more than 96%. This result strongly suggests that the amino acid properties of specific residues are appreciably important for GPCR - G-protein coupling to determine G- protein binding selectivity and our method could be an effective tool to investigate the mechanism of GPCR - G-protein coupling through site directed mutagenesis experiments.

High-density protein patterning through selective plasma-induced fluorocarbon deposition on Si substrates

A novel method for fabrication of protein microarrays through selective plasma-induced modification of patterned substrates is presented. Exposing Si substrates bearing SiO 2 or Si 3N 4 patterns to a c-C 4F 8 plasma in a high-density plasma reactor, a fluorocarbon (FC) film was selectively deposited on Si areas, whereas the SiO 2 or Si 3N 4 patterns were simultaneously etched. Optimizing the plasma parameters such as power, bias voltage and gas pressure, patterned substrates with highly selective protein adsorption capacities were obtained. More specifically, using fluorescently labeled protein solutions direct selective protein binding onto SiO 2 or Si 3N 4 areas versus the Si substrate was verified. In addition, model binding assays were demonstrated through proteins immobilization on the patterned substrate and their subsequent reaction with fluorescently labeled counterpart molecules. Patterned Si/SiO 2 or Si/Si 3N 4 surfaces not subjected to plasma treatment presented negligible protein adsorption. Following the proposed method, highly resolved protein spots with diameters down to 1 μm were created. The spots presented high intra-spot fluorescence homogeneity (coefficient variation (CV) ≤ 10%), inter-spot fluorescence repeatability (CV ≤ 5%) and excellent morphology. Thus, substrates prepared following the proposed method can be applied to the fabrication of high-density and high-quality protein microarrays.

Photoderivatized Polymer Thin Films at Quartz Crystal Microbalance Surfaces: Sensors for Carbohydrate−Protein Interactions

Photoderivatized polymer-coated gold surfaces have been developed following a perfluorophenylazide-based double ligation strategy. Gold-plated quartz crystal microbalance (QCM) crystals were initially covalently functionalized with a monolayer of poly(ethylene glycol) (PEG), using photo- or thermolytic nitrene formation and insertion. The polymer surfaces were subsequently used as substrates for photoinsertion of carbohydrate-derivatized photoprobes, yielding different recognition motifs for selective protein binding. The resulting robust and biocompatible sensor surfaces were applied to a flow-through QCM instrument for monitoring lectin-carbohydrate interactions in real time. The results clearly show the predicted lectin selectivity, demonstrating the applicability of the approach.

Global Proteomics in AML: Using SELDI-TOF MS on Diagnostic Samples To Identify Spectra and Protein Biomarkers Indicative of Prognosis.

With the object to identify key proteins related to treatment response in acute myeloid leukemia (AML), and to eventually elucidate new targets for therapy, we have investigated protein spectra from diagnostic AML samples. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to spot proteins in the molecular weight area of 2–40 kDa. The samples were applied to two different chip surfaces (CM10 and Q10) for selective protein binding, avoiding the necessity of extensive purification and separation procedures prior to analysis. Peripheral blood samples from a total of 67 acute leukemia patients at diagnosis were analyzed. The cells were collected, frozen and stored in biobanks between 1988 and 2003. This provided a good basis for correlating detected protein peaks to a large number of clinical diagnostic and therapeutic variables and outcomes. Among the patients, aged 18 to 86 yrs, response to induction therapy was observed with complete remission (CR) durations ranging between 12 and 3701 days. The duration of first CR was used as a dichotomic variable in order to compare protein spectra from groups of patients with “good” and “bad” response, respectively, to chemotherapy. A peak detection and annotation software was developed in-house1,2 and employed. The software will be presented and a comparison to commercially available MS-spectral analysis methods will be provided. Initial results from MS profiling of the two response groups showed 18 significantly (p>0.01) differently expressed peaks on anion exchange chips and 22 peaks on cation exchange surfaces. Ongoing work is focused on further development of the RS peak detection method, including the influence of normalisation and filtering of the SELDI data and supervised linear and non-linear modelling. In addition, identification of selected peaks and their relation to various clinical variables is in progress.

Functionalized porous silicon surfaces as MALDI-MS substrates for protein identification studies

Alkyl monolayer modified porous silicon functional surfaces are employed for selective binding of proteins from complex mixtures (through washing of the deposited mixture spot using appropriate buffer) and MALDI-MS is used to detect the components retained on the surface.

2P305 GPCRとGタンパク質の結合選択性予測(生命情報科学 B) 機能ゲノミクス))

2P305 GPCRとGタンパク質の結合選択性予測(生命情報科学 B) 機能ゲノミクス))