Selective Protein Recognition Research Articles

A generic approach based on long-lifetime fluorophores for the assessment of protein binding to polymer nanoparticles by fluorescence anisotropy.

Quantitation of protein-nanoparticle interactions is essential for the investigation of the protein corona around NPs in vivo and when using synthetic polymer nanoparticles as affinity reagents for selective protein recognition in vitro. Here, a method based on steady-state fluorescence anisotropy measurement is presented as a novel, separation-free tool for the assessment of protein-nanoparticle interactions. For this purpose, a long-lifetime luminescent Ru-complex is used for protein labelling, which exhibits low anisotropy when conjugated to the protein but displays high anisotropy when the proteins are bound to the much larger polymer nanoparticles. As a proof of concept, the interaction of lysozyme with poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid) nanoparticles is studied, and fluorescence anisotropy measurements are used to establish the binding kinetics, binding isotherm and a competitive binding assay.

Molecularly imprinted CaCO3/polydopamine hybrid composite for selective protein recognition

Molecularly imprinted CaCO3/polydopamine hybrid composite for selective protein recognition

Abstract 2106: Role of 3-O-Sulfation-Induced Rare and Compact Topologies of Heparan Sulfate in Selective Recognition of Proteins

Abstract 2106: Role of 3-O-Sulfation-Induced Rare and Compact Topologies of Heparan Sulfate in Selective Recognition of Proteins

Future Prospects of Luminescent Silicon Nanowires Biosensors.

In this paper, we exploit the perspective of luminescent Si nanowires (NWs) in the growing field of commercial biosensing nanodevices for the selective recognition of proteins and pathogen genomes. We fabricated quantum confined fractal arrays of Si NWs with room temperature emission at 700 nm obtained by thin-film, metal-assisted, chemical etching with high production output at low cost. The fascinating optical features arising from multiple scattering and weak localization of light promote the use of Si NWs as optical biosensing platforms with high sensitivity and selectivity. In this work, label-free Si NW optical sensors are surface modified for the selective detection of C-reactive protein through antigen-gene interaction. In this case, we report the lowest limit of detection (LOD) of 1.6 fM, fostering the flexibility of different dynamic ranges for detection either in saliva or for serum analyses. By varying the NW surface functionalization with the specific antigen, the luminescence quenching of NW biosensors is used to measure the hepatitis B-virus pathogen genome without PCR-amplification, with an LOD of about 20 copies in real samples or blood matrix. The promising results show that NW optical biosensors can detect and isolate extracellular vesicles (EV) marked with CD81 protein with unprecedented sensitivity (LOD 2 × 105 sEV/mL), thus enabling their measurement even in a small amount of blastocoel fluid.

Facile synthesis of novel helical imprinted fibers based on zucchini-derived microcoils for efficient recognition of target protein in biological sample

Highly selective recognition and purification of target proteins from complex biological matrices remains a challenging subject in natural and life sciences. Compared with natural recognition receptors, artificial imprinted polymers are an ideal alternative candidate. In this study, we report a novel method to prepare helical protein imprinted fibers (HPIFs) with zucchini-derived microcoils as a carrier, firstly. Inspired by the self-polymerization of adhesive proteins in mussels, dopamine and 3,4-dihydroxyphenylacetic acid were chosen as bifunctional monomers for the first time to form a biocompatible imprinted layer. The chemical/physical properties and recognition performance of HPIFs were studied in a series of experiments. Additionally, the practicability of HPIFs was verified by specifically recognizing target protein in complex egg white sample. The one-step synthesis process and excellent binding performance of HPIFs make them a promising material for protein recognition and purification, and endow HPIFs with potential application value in the food, chemical and pharmaceutical fields.

Aggregation and Binding-Directed FRET Modulation of Conjugated Polymer Materials for Selective and Point-of-Care Monitoring of Serum Albumins

Nonspecific interactions of conjugated polymers (CPs) with various proteins prove to be a major impediment for researchers when designing a suitable CP-based probe for the amplified and selective recognition of particular proteins in complex body fluids. Herein, a new strategy is presented for the precise and specific monitoring of clinically important serum albumin (SA) proteins at the nanomolar level using fluorescence resonance energy transfer (FRET)-modulated CP-surfactant ensembles as superior sensing materials. In brief, the newly designed color-tunable CP PF-DBT-Im undergoes intense aggregation with the surfactant sodium dodecyl sulfate (SDS), enabling drastic change in the emission color from violet to deep red due to intermolecular FRET. The emission of PF-DBT-Im/SDS ensembles then changed from deep red to magenta specifically on addition of SAs owing to the exclusive reverse FRET facilitated by synergistic effects of electrostatic interactions, hydrophobic forces, and the comparatively high intrinsic quantum yield of SAs. Interestingly, PF-DBT-Im itself could not differentiate SAs from other proteins, demonstrating the superiority of the PF-DBT-Im/SDS self-assembly over PF-DBT-Im. Finally, an affordable smartphone-integrated point-of-care (PoC) device is also fabricated as a proof-of-concept for the on-site and rapid monitoring of SAs, validating the potential of the system in long-term clinical applications.

3-O-Sulfation induces sequence-specific compact topologies in heparan sulfate that encode a dynamic sulfation code

Heparan sulfate (HS) is arguably the most diverse linear biopolymer that is known to modulate hundreds of proteins. Whereas the configurational and conformational diversity of HS is well established in terms of varying sulfation patterns and iduronic acid (IdoA) puckers, a linear helical topology resembling a cylindrical rod is the only topology thought to be occupied by the biopolymer. We reasoned that 3-O-sulfation, a rare modification in natural HS, may induce novel topologies that contribute to selective recognition of proteins. In this work, we studied a library of 24 distinct HS hexasaccharides using molecular dynamics (MD). We discovered novel compact (C) topologies that are populated significantly by a unique group of 3-O-sulfated sequences containing IdoA residues. 3-O-sulfated sequences containing glucuronic acid (GlcA) residue and sequences devoid of 3-O-sulfate groups did not exhibit high levels of the C topology and primarily exhibited only the canonical linear (L) form. The C topology arises under dynamical conditions due to rotation around an IdoA → GlcN glycosidic linkage, especially in psi (Ψ) torsion. At an atomistic level, the L → C transformation is a multi-factorial phenomenon engineered to reduce like-charge repulsion, release one or more HS-bound water molecules, and organize a bi-dentate “IdoA-cation-IdoA” interaction. These forces also drive an L → C transformation in a 3-O-sulfated octasaccharide, which has shown evidence of the unique C topology in the co-crystallized state. The 3-O-sulfate-based generation of unique, sequence-specific, compact topologies indicate that natural HS encodes a dynamic sulfation code that could be exploited for selective recognition of target proteins.

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein.

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (KD) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein.

Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for β-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.

Ultrasensitive and Selective Protein Recognition with Nanobody‐Functionalized Synthetic Nanopores

The development of flexible and reconfigurable sensors that can be readily tailored toward different molecular analytes constitutes a key goal and formidable challenge in biosensing. In this regard, synthetic nanopores have emerged as potent physical transducers to convert molecular interactions into electrical signals. Yet, systematic strategies to functionalize their surfaces with receptor proteins for the selective detection of molecular analytes remain scarce. Addressing these limitations, a general strategy is presented to immobilize nanobodies in a directional fashion onto the surface of track-etched nanopores exploiting copper-free click reactions and site-specific protein conjugation systems. The functional immobilization of three different nanobodies is demonstrated in ligand binding experiments with green fluorescent protein, mCherry, and α-amylase (α-Amy) serving as molecular analytes. Ligand binding is resolved using a combination of optical and electrical recordings displaying quantitative dose-response curves. Furthermore, a change in surface charge density is identified as the predominant molecular factor that underlies quantitative dose-responses for the three different protein analytes in nanoconfined geometries. The devised strategy should pave the way for the systematic functionalization of nanopore surfaces with biological receptors and their ability to detect a variety of analytes for diagnostic purposes.

Protein-imprinted polymer films prepared via cavity-selective multi-step post-imprinting modifications for highly selective protein recognition.

The use of molecularly imprinted polymers (MIPs) for achieving synthetic receptors capable of selective molecular recognition is promising; however, these polymers exhibit low selectivity derived from the heterogeneity of their created, imprinted cavities. To achieve highly selective protein recognition, we herein report the cavity-selective, multi-step, post-imprinting modification of MIPs. An MIP film for lysozyme was prepared by the copolymerization of {[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy acetic acid, a functional monomer possessing a modifiable disulfide bond, with acrylamide and N,N'-methylenebisacrylamide in the presence of lysozyme. After the removal of lysozyme, the disulfide bonds were cleaved by treatment with a reductant. A low concentration of lysozyme was then added to occupy the high-affinity cavities of the polymer and sterically protect the thiol groups within them. A poly(ethylene glycol)-based capping agent was reacted with the thiol groups residing in low-affinity cavities to hinder them. After the regeneration of the high-affinity cavities by washing out the bound lysozyme, the remaining thiol groups were reacted with 3-(2-pyridyldithio)propionic acid to introduce interacting groups, which produced capped MIPs. Comparing the capped and uncapped MIPs revealed that off-target protein binding was effectively suppressed by the capping treatment without any reduction in binding affinity (1.1 × 109M-1). Further investigation revealed that the lysozyme concentration during the capping process is critical for the selectivity of the capped MIP. In this case, highly selective MIPs were achieved when the lowest lysozyme concentration (100nM) was used. This facile process for creating highly selective, synthetic polymer receptors is a powerful approach for achieving plastic antibodies.

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes.

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

Aptamer-Based Western Blot for Selective Protein Recognition.

Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. Successful detection of the target proteins in these methods relies on the specific interaction of the antibodies, which often bring a high production cost and require a long incubation time. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition, and replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP, and His-tag. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis.

Polymeric nanoparticles for selective protein recognition by using thiol-ene miniemulsion photopolymerization

The fabrication of molecularly imprinted nanoparticles (MIP-NPs) specific for myoglobin by using thiol-ene photopolymerization in miniemulsion was described. Allyl derivatives of phenylalanine as a functional monomer was synthesized and copolymerized with acrylic monomers via miniemulsion polymerization to produce NIP-NPs with approximately 74 nm number average particle diameter. FTIR and 1H-NMR analysis confirmed the synthesis of functional monomer. MIP-NPs were prepared in the existence of myoglobin as a template protein. Morphological investigations exhibited that the particle size of the MIP-NPs, increased compared to the corresponding NIPs and the mean particle diameter by number was measured as 141 nm with narrow distribution. NIP-NPs that were polymerized without myoglobin were found to have less affinity to the target protein. In addition, the rebinding ability of MIP-NPs was much bigger than that of the corresponding NIPs. ELISA results showed that MIPs interact particularly with the myoglobin and show little affinity for BSA in competitive binding experiments.HighlightsAllyl N,N-diallyl phenylalaninate was synthesized as a functional monomer.Imprinted nanoparticles were prepared by using thiol-ene photopolymerization in miniemulsion.The nanoparticles were 141 nm with narrow size distribution.The imprinted nanoparticles showed selectivity toward myoglobin.

Advances on structural mechanisms of ubiquitinated nucleosome complexes

The ubiquitination of nucleosome histones (H2A, H2B, H3, H4) mediates a series of selective interactions between effector proteins and nucleosomes, and regulates the epigenetics of cells. In recent years, thanks to the development of new chemical and biological methods, sample acquisition technologies such as chemical modification of proteins, and structure observation technologies such as cryo-electron microscopy (Cryo-EM) have been well utilized. Some complex structures of ubiquitinated nucleosomes and effector proteins have been resolved, which revealed molecular mechanisms of ubiquitin regulated epigenetic process. This article summarizes the six complexes between ubiquitinated nucleosomes and effector proteins reported recently, comparatively analyzes the selective recognition and activation mechanism of effector proteins by ubiquitinated nucleosomes, and describes the key interaction sites on ubiquitinated nucleosomes.

Preparation of bovine hemoglobin surface molecularly imprinted cotton for selective protein recognition

Abstract We have developed a bovine hemoglobin (BHb) surface molecularly imprinted cotton based on degreasing cotton via surface imprinting technique for the efficient selective adsorption of BHb. The morphological structure of the samples was characterized by Scanning Electron Microscopy (SEM), and the chemical modification steps were characterized by Fourier Transform Infrared Spectroscopy (FTIR). The maximum adsorption capacity of the molecularly imprinted cotton (MIC) and non-imprinted cotton (NIC) for BHb was 62.95 mg/g and 8.32 mg/g, respectively, at the optimum pH value of 6.2. The kinetics studies demonstrated that the adsorption follows a pseudo-first-order kinetic model. The adsorption isotherm analysis indicated that the Langmuir adsorption isotherm fits well with the adsorption equilibrium data. Also, the selective adsorption shows the MIC has a good selectivity for BHb. In addition, the assessment of the reusability of the MIC was tested for five successive cycles revealed no significant decrease of the adsorption capacity. Electrophoretic analysis suggests the MIC were successfully applied to capture template proteins from the bovine blood sample.

Self-organized nanocrystal rings formed by microemulsion for selective recognition of proteins and immunoassays.

A simple and cheap method to fabricate a nanocrystal ring pattern was developed by utilization of a microemulsion in this study. The mixture of polystyrene and stabilizer dichloromethane solution that contained nanocrystal aqueous solution, prepared through shaking, was applied to fabricate a reverse microemulsion. After spreading and evaporating the solvent of microemulsion on a glass slide, an ordered honeycomb film was produced, accompanied by the formation of a nanocrystal ring pattern. The nanocrystal pattern could be readily applied for immunoassays and recognition of proteins. The pattern with antibody marked by a green colored nanocrystal specifically bound with antigen labeled by a red colored nanocrystal, leading to the enhancement in red fluorescent ring pattern and decrease in green fluorescent pattern. When the unlabeled antigen was added, the green fluorescent pattern was recovered. In addition, the ring pattern with immunocomplex could selectively recognize antigen and transferrin proteins. This strategy reveals that these patterns have potential applications in biochips, biosensors, imaging analysis and so forth.

Molecularly imprinted polymers fabricated via Pickering emulsions stabilized solely by food-grade casein colloidal nanoparticles for selective protein recognition.

Novel molecularly imprinted polymers (MIPs) based on denatured casein nanoparticle (DCP)-stabilized Pickering emulsions were developed for the first time. Casein, a phosphoprotein, is the main protein in milk. In this work, DCPs were solely used as Pickering-type interfacial emulsifiers for fabrication of MIPs for the selective recognition of proteins for the first time. DCPs were prepared by acidification and heat denaturation (at 80 °C) of casein. Their dispersions have satisfactory colloidal stability over a wide pH range. The DCPs acted as natural, food-grade, and edible interfacial emulsifiers, and adsorbed at the oil-water interface to form Pickering emulsions. After the polymerization of monomers, the template protein was removed by elution. During the elution, the interfacial DCPs were also removed, allowing more imprinted cavities to become exposed. The interfacial imprinting technology causes nearly all the imprinted sites to locate on the surface of the polymeric material. Therefore, the MIPs obtained exhibit fast rebinding and excellent specific recognition ability toward the analytes. Overall, this work provides a promising method for designing and fabricating natural-protein-based structured emulsions to prepare MIPs and thus offers new insight into protein separation and purification. Graphical Abstract Pickering emulsions stabilized by denatured casein particles.

On-Chip FRET Graphene Aptasensor

We review the recent advances in the use of our originally developed on-chip graphene aptasensor to detect biologically important proteins, such as cancer markers. The detection mechanism, based on fluorescence resonance energy transfer (FRET), occurs at a graphene–biomolecule interface. In our system, the graphene surface is modified with a pyrene–aptamer–dye probe. Pyrene functions as a linker to the graphene surface, the aptamer as a probe for selective protein recognition, and the dye as a fluorescence detection tag. Here, graphene behaves simultaneously as both an efficient acceptor for FRET over the entire visible region and as a strong adsorbate for single-stranded DNA (ssDNA), such as aptamers, via π-π interactions in the sp2 domain. The system allows us to perform molecular detection on a solid surface, which is advantageous for realizing on-chip sensors. Such on-chip sensors allow parallel analysis systems, such as array sensors. This enables the quantitative comparison of different samples by forming a multichannel configuration and/or a micropattern with different probes. Moreover, detecting the target protein is possible simply by adding a sample of less than 1 μL to the on-chip sensor; detection is completed in approximately 1 min. Aptasensors can be used for the detection of many different targets simply by replacing the aptamers. The simultaneous detection of multiple target molecules on a single chip using a 2 × 3 linear-array aptasensor was demonstrated here. Improved sensitivity was observed when a DNA spacer was incorporated into the aptamer, demonstrating that the probe can be modified in interesting ways.

Novel application of amphiphilic block copolymers in Pickering emulsions and selective recognition of proteins

An amphiphilic block copolymer stabilized Pickering high internal phase emulsions and was mildly exfoliated to improve imprinting efficiency.