Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

A Zanamivir-protein conjugate mimicking mucin for trapping influenza virion particles and inhibiting neuraminidase activity.

Influenza viruses contribute significantly to the global health burden, necessitating the development of strategies against transmission as well as effective antiviral treatments. The present study reports a biomimetic strategy inspired by the natural antiviral properties of mucins. A bovine serum albumin (BSA) conjugate decorated with the multivalent neuraminidase inhibitor Zanamivir (ZA-BSA) was synthesized using copper-free click chemistry. This synthetic pseudo-mucin exhibited potent neuraminidase inhibitory activity against several influenza strains. Virus capture and growth inhibition assays demonstrated its effective absorption of virion particles and ability to prevent viral infection in nanomolar concentrations. Investigation of the underlying antiviral mechanism of ZA-BSA revealed a dual mode of action, involving disruption of the initial stages of host-cell binding and fusion by inducing viral aggregation, followed by blocking the release of newly assembled virions by targeting neuraminidase activity. Notably, the conjugate also exhibited potent inhibitory activity against Oseltamivir-resistant neuraminidase variant comparable to the monomeric Zanamivir. These findings highlight the application of multivalent drug presentation on protein scaffold to mimic mucin adsorption of viruses, together with counteracting drug resistance. This innovative approach has potential for the creation of antiviral agents against influenza and other viral infections.

Chemoenzymatic synthesis of sialyl-α2,3-lactoside-functionalized BSA conjugate inhibits influenza infection.

Influenza remains a global public health threat, and the development of new antivirals is crucial to combat emerging drug-resistant influenza strains. In this study, we report the synthesis and evaluation of a sialyl lactosyl (TS)-bovine serum albumin (BSA) conjugate as a potential multivalent inhibitor of the influenza virus. The key trisaccharide component, TS, was efficiently prepared via a chemoenzymatic approach, followed by conjugation to dibenzocyclooctyne-modified BSA via a strain-promoted azide-alkyne cycloaddition reaction. Biophysical and biochemical assays, including surface plasmon resonance, isothermal titration calorimetry, hemagglutination inhibition, and neuraminidase inhibition, demonstrated the strong binding affinity of TS-BSA to the hemagglutinin (HA) and neuraminidase (NA) proteins of the influenza virus as well as intact virion particles. Notably, TS-BSA exhibited potent inhibitory activity against viral entry and release, preventing cytopathic effects in cell culture. This multivalent presentation strategy highlights the potential of glycocluster-based antivirals for combating influenza and other drug-resistant viral strains.

Peptide-Carrier Conjugation.

To produce antibodies against synthetic peptides, it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined.

Freezing Diluted Bovine Serum Albumin Standards Does Not Significantly Affect Standard Curves.

Total protein isolation followed by quantitation is a common protocol in many laboratories. Quantitation is often done using a colorimetric assay such as the bicinchoninic acid (BCA) assay in which a change in the color of the BCA reagent is related to protein concentration. Extracted protein samples are compared to a standard curve made with dilutions of a protein standard such as bovine serum albumin (BSA) to determine their concentrations. A series of experiments was designed to determine the most reproducible and accurate method for quantifying protein concentrations of samples in an experimental series over time. The effect of freezing on diluted standards was investigated. Standards were frozen at -20°C or -80°C and serially thawed and refrozen up to three times prior to their use in a BCA assay. Thawing and refreezing the standards had no significant effect on protein concentration and the resulting standard curves. Inter-person and intra-person variability in the preparation of standards was also investigated. Protein concentration differences due to inter-person and intra-person variability were greater than protein concentration variability resulting from freezing and thawing, regardless of the freezing temperature. The most reproducible and accurate method for determining the protein concentration of extracted samples in an experimental series over time is diluting a large batch of BSA standards and freezing them at either -20°C or -80°C. Reproducibility was maintained with up to three freeze-thaws.

Enantioseparation of six profenoid drugs by capillary electrophoresis with bovine serum albumin-modified gold nanoparticles as quasi-stationary phases.

Profenoid drugs are a kind of common non-steroidal anti-inflammatory drugs and their chiral enantiomers often have huge differences in pharmacological activities. In this work, a novel chiral separation system by capillary electrophoresis (CE) was constructed using gold nanoparticles (AuNPs) functionalized with bovine serum albumin (BSA) as a quasi-stationary phase (QSP), and the enantioseparation of six profenoid drugs was efficiently accomplished. Under optimal chromatographic conditions, the enantioseparation performance of the AuNP@BSA-based chiral separation system was greatly improved compared with that of free BSA (Resolutions, Ibuprofen: 0.89 â†’ 8.15; Ketoprofen: 0 â†’ 10.02; Flurbiprofen:0.56 â†’ 9.83; Indoprofen: 0.88 â†’ 13.83; Fenoprofen: 0 â†’ 15.21; Pyranoprofen: 0.59 â†’ 5.34). Such high Rs are exciting and satisfying and it is in the leading position in the reported papers. Finally, through molecular docking, it was also found that the difference in binding energy between BSA and enantiomers was closely related to the resolutions of CE systems, revealing the chiral selection mechanism of BSA. This work significantly improves the CE chiral separation performance through a simple strategy, providing a simple and efficient idea for the chiral separation method.

Binding and displacement study of gentamicin, 5-fluorouracil, oxytetracycline and rolitetracycline with (BSA: Drug2) complex using spectroscopic and calorimetric techniques: Biophysical approach.

Understanding of energetics of interactions between drug and protein is essential in pharmacokinetics and pharmacodynamics study. The binding affinity (K) helps in investigating how tightly or loosely drug is bound to protein. The binding, displacement, conformational change and stability study of drugs- gentamicin (GM), 5-fluorouracil (5FU), oxytetracycline (OTC) and rolitetracycline (RTC) with bovine serum albumin (BSA) has been carried out in presence of each other drug by fluorescence, UV-visible spectroscopy, molecular docking, circular dichroism techniques and thermal denaturation method. The site marker study and docking methods have confirmed that 5FU and GM are able to bind at site 1 and OTC and RTC at site II of BSA. The order of their binding affinities with BSA for the binary system were as GM <5FU < OTC < RTC with the order of 102 < 103 < 105 < 105-6 M-1. The displacement study has shown that higher affinity drug decreases the equilibrium constant of another drug already in bound state with BSA if both these drugs are having the same binding site. Therefore 5FU, GM (binding site 1) drugs were not able to displace OTC and RTC (binding site 2) and vice-versa as they are binding at two different sites. The binding constant values were found to be decreasing with increasing temperature for all the systems involved which suggests static or mixed type of quenching, however can only confirmed with the help of TCSPC technique. The ΔG0 (binding energy) obtained from docking method were in accordance with the ITC method. From molecular docking we have determined the amino acid residues involved in binding process for binary and ternary systems by considering first rank minimum binding energy confirmation. From CD it has been observed that RTC causes most conformational change in secondary and tertiary structure of BSA due to the presence of pyrrole ring. OTC-RTC with higher affinity showed highest melting temperature Tm values while low affinity drugs in (5FU-GM) combination showed lowest Tm value. 5FU showed large endothermic denaturation enthalpy ΔHd0 due to the presence of highly electronegative fluorine atom in the pyridine analogue.

Multi-spectral and docking assessments to explore the combination of an antiviral drug, entecavir with bovine serum albumin.

Molecular interaction of entecavir (ETV) with the transport protein, albumin from bovine serum (BSA) was explored through multispectral and molecular docking approaches. The BSA fluorescence was appreciably quenched upon ETV binding and the quenching nature was static. The ETV-BSA complexation and the static quenching process were further reiterated using UV-visible absorption spectra. The binding constant (Ka) values of the complex were found as 1.47 × 104-4.0 × 103 M-1, which depicting a modarate binding strength in the ETV-BSA complexation. The experimental outcomes verified that the stable complexation was primarily influenced by hydrophobic interactions, hydrogen bonds and van der Waals forces. Synchronous and 3-D fluorescence spectral results demonstrated that ETV had significant impact on the hydrophobicity and polarity of the molecular environment near Tyr and Trp residues. Competitive site-markers displacement (with warfarin and ketoprofen) results discovered the suitable binding locus of ETV at site I in BSA. The molecular docking assessments also revealed that ETV formed hydrogen bonds and hydrophobic interactions with BSA, predominantly binding to site I (sub-domain IIA) of BSA.

Interaction mechanism of oseltamivir phosphate with bovine serum albumin: multispectroscopic and molecular docking study.

Oseltamivir phosphate (OP) is an antiviral drug with potential risks to human health due to overuse, leading to serious consequences such as gastrointestinal disturbances, abnormal neuropsychiatric symptoms, and sudden death. Therefore, gaining an in-depth understanding of its interaction with proteins is crucial. We investigated the interaction between OP and bovine serum albumin (BSA) utilizing multispectral methods (i.e., fluorescence, ultraviolet absorption, circular dichroism) combined with molecular docking techniques. Fluorescence spectroscopy indicated that OP quenched BSA fluorescence by forming the OP-BSA complex. The Stern-Volmer constants (KSV) between OP and BSA were determined to be 3.06 × 103 L/mol, 2.36 × 103 L/mol, and 1.86 × 103 L/mol at 293 K, 298 K, and 303 K, respectively. OP occupies exclusively one binding site on BSA, and the fluorescent probe displacement measurements revealed that this is BSA site I. Thermodynamic data (∆H, ∆S, and ∆G) obtained by fitting the van't Hoff equation were - 77.49 kJ/mol, -176.54 J/(mol∙K), and - 24.88 kJ/mol, respectively, suggesting that hydrogen bonding and van der Waals forces mainly participate in OP-BSA complex stabilization. Moreover, the reaction occurs spontaneously at room temperature. Synchronous fluorescence spectra indicated that OP interacts with tryptophan residue of BSA. The results of ultraviolet (UV) and 3D fluorescence spectroscopy indicated that the OP-BSA complex formation altered the microenvironment around amino acid residues. Circular dichroism spectra revealed that the addition of OP decreased the α-helix content of BSA by 7.13%. Docking analysis confirmed that OP binds to BSA site I through hydrogen bonding with amino acids VAL342, SER453, and ASP450. Finally, ADMET studies were conducted to explore the pharmacokinetics of OP as an antiviral drug.

Mathematical modelling of genipin-bovine serum albumin interaction using fluorescence intensity measurements.

The interaction between genipin and a model protein bovine serum albumin (BSA), with and without the addition of acetic acid, has been studied experimentally and by modelling. The number of amino groups available to react was determined to be 5.6 % of the total number of amino acid building blocks on BSA. Fluorescence intensity was used to record the progress of the reaction over the 24 h, while the modelling study focused on capturing the kinetic profiles of the reaction. The experiments revealed a slow start to the BSA and genipin interaction, that subsequently accelerated in an S-shaped curve which the modelling study linked with the existence of the feedback cycle for both reactive amino groups and genipin. At BSA concentrations ≥30 mg/mL the reaction was accelerated in the presence of acid, while below 30 mg/mL the acidified conditions delayed the onset of the reaction. Contrary to the reaction mechanisms previously proposed, a degree of breakdown of the fluorescent links in the products formed was denoted both experimentally and in a modelling study. This indicated the reversibility of the processes forming fluorescent product/s and suggested feasibility of the successful release of the protein following prospective encapsulation within the genipin-crosslinked hydrogel structure.

Fabrication and characterization of bovine serum albumin-phycocyanobilin conjugate: effect on antioxidant and ligand-binding properties.

BACKGROUND: Phycocyanobilin (PCB) is an open-chain blue tetrapyrrole chromophore of C-phycocyanin (C-PC), a major chromoprotein derived from the cyanobacterium Arthrospira platensis having numerous health-promoting effects. Relying on the ability of PCB to attach to the sulfhydryl group of proteins, we propose a new method for covalent attachment of PCB to bovine serum albumin (BSA) as a means of its functionalization. RESULTS: Traut's reagent (TR, 2-iminothiolane), modifying lysine residues, was used to optimize the introduction of sulfhydryl groups in BSA. A higher degree of BSA thiolation by TR induces more profound alterations of its structure, resulting in minor oligomerization and aggregation. A 50-fold molar excess of TR was found to be the optimal, balancing thiolation level and adverse effect on protein structure. PCB was covalently attached to newly introduced sulfhydryl groups at pH 9 at 20-fold PCB/BSA ratio. An increase in the TR/BSA molar ratio leads to increased efficiency of PCB conjugation with thiolated BSA. Compared to native BSA, BSA-PCB conjugate binds quercetin with similar affinity but has higher antioxidant activity and increased oxidative stability. CONCLUSIONS: PCB-modified BSA could serve as a stable, food-compatible carrier of bioactive PCB, but also bind other ligands that would be protected from oxidative damage due to the high antioxidant potential of covalently bound PCB. Thiolation by TR is, at the same time, a simple method for the covalent functionalization of virtually any protein by bioactive PCB or for obtaining PCB-based fluorescent probes. © 2024 Society of Chemical Industry.

Peculiarities of the Dynamical Hydration Shell of Native Conformation Protein Using a Bovine Serum Albumin Example.

This paper describes an approach based on the method of terahertz time-domain spectroscopy, which allows the analysis of dynamical hydration shells of proteins with a thickness of 1-2 nm. Using the example of bovine serum albumin in three conformations, it is shown that the hydration shells of the protein are characterized by increased binding of water molecules in the primary hydration layers, and in more distant areas of hydration, on the contrary, the water structure is somewhat destroyed. The fraction of free or weakly bound molecules, usually observed in the structure of liquid water in hydration shells, become more numerous but its average binding is greater than in undisturbed water. The energy distribution of hydrogen bonds in hydration shells is narrowed compared to undisturbed water. All these manifestations of hydration are most pronounced for the native conformation of the protein. Also, the hydration shells of the native protein are characterized by a smaller number of hydrogen bonds and a tendency to decrease their average energy compared to non-native conformations. The fact of a pronounced peculiarity of the hydration shells of the protein in the native conformation has been noted for different proteins before. However, the methodological approach used in this work for the first time allowed this peculiarity to be described by specific parameters of the intermolecular structure and dynamics of water.

Detection of Low-Concentration Biological Samples Based on a QBIC Terahertz Metamaterial Sensor.

Quasi-bound state in the continuum (QBIC) can effectively enhance the interaction of terahertz (THz) wave with matter due to the tunable high-Q property, which has a strong potential application in the detection of low-concentration biological samples in the THz band. In this paper, a novel THz metamaterial sensor with a double-chain-separated resonant cavity structure based on QBIC is designed and fabricated. The process of excitation of the QBIC mode is verified and the structural parameters are optimized after considering the ohmic loss by simulations. The simulated refractive index sensitivity of the sensor is up to 544 GHz/RIU, much higher than those of recently reported THz metamaterial sensors. The sensitivity of the proposed metamaterial sensor is confirmed in an experiment by detecting low-concentration lithium citrate (LC) and bovine serum albumin (BSA) solutions. The limits of detection (LoDs) are obtained to be 0.0025 mg/mL (12 µM) for LC and 0.03125 mg/mL (0.47 µM) for BSA, respectively, both of which excel over most of the reported results in previous studies. These results indicate that the proposed THz metamaterial sensor has excellent sensing performances and can well be applied to the detection of low-concentration biological samples.

Interactions of three berberine mid-chain fatty acid salts with bovine serum albumin (BSA): Spectroscopic analysis and molecular docking.

In this paper, the interaction of three berberine mid-chain fatty acid salts ([BBR][FAs]), viz. berberine caproate ([BBR][CAP]), berberine heptylate ([BBR][HEP]) and berberine octoate ([BBR][OCT]), with bovine serum albumin (BSA) was studied by means of UV-visible absorption spectroscopy, fluorescence spectroscopy, fourier transform infrared spectroscopy (FT-IR) and molecular docking techniques. Fluorescence experiments revealed that three berberine salts quench the fluorescence of BSA by static quenching mechanism resulted from a stable [BBR][FAs]-BSA complex formation. The stoichiometric numbers of [BBR][FAs]-BSA complexes were found to be 1:1. Synchronous and three-dimensional fluorescence spectra as well as FT-IR demonstrated that the binding of [BBR][FAs] altered the microenvironment and conformation of BSA. The binding average distance from [BBR][FAs] to BSA (3.2-3.5 nm) was determined according to Förster energy transfer theory. Site probe investigation showed that [BBR][FAs] bound to BSA active site I (sub-domain IIA). The binding promotes the esterase-like activity of BSA. The molecular docking results confirmed the fluorescence competition findings and provided the type of binding forces. Furthermore, the relationship between the anionic chain length of [BBR][FAs] and the interaction was explored, and the positive correlation was found.

Albumin-Based Hydrogel Films Covalently Cross-Linked with Oxidized Gellan with Encapsulated Curcumin for Biomedical Applications.

Bovine serum albumin (BSA) hydrogels are non-immunogenic, low-cost, biocompatible, and biodegradable. In order to avoid toxic cross-linking agents, gellan was oxidized with NaIO4 to obtain new functional groups like dialdehydes for protein-based hydrogel cross-linking. The formed dialdehyde groups were highlighted with FT-IR and NMR spectroscopy. This paper aims to investigate hydrogel films for biomedical applications obtained by cross-linking BSA with oxidized gellan (OxG) containing immobilized ß-cyclodextrin-curcumin inclusion complex (ß-CD-Curc) The ß-CD-Curc improved the bioavailability and solubility of Curc and was prepared at a molar ratio of 2:1. The film's structure and morphology were evaluated using FT-IR spectroscopy and SEM. The swelling degree (Q%) values of hydrogel films depend on hydrophilicity and pH, with higher values at pH = 7.4. Additionally, the conversion index of -NH2 groups into Schiff bases increases with an increase in OxG amount. The polymeric matrix provides protection for Curc, is non-cytotoxic, and enhances antioxidant activity. At pH = 5.5, the skin permeability and release efficiency of encapsulated curcumin were higher than at pH = 7.4 because of the interaction of free aldehyde and carboxylic groups from hydrogels with amine groups from proteins present in the skin membrane, resulting in a better film adhesion and more efficient curcumin release.

A Comparative Study on the Interaction Between Protein and PET Micro/Nanoplastics: Structural and Surface Characteristics of Particles and Impacts on Lung Carcinoma Cells (A549) and Staphylococcus aureus.

The interaction between particles and proteins is a key factor determining the toxicity responses of particles. Therefore, this study aimed to examine the interaction between the emerging pollutant polyethylene terephthalate micro/nanoplastics from water bottles with bovine serum albumin. The physicochemical characteristics of micro/nanoplastics were investigated using nuclear magnetic resonance, x-ray diffraction, Fourier transform infrared, dynamic light scattering, and x-ray energy dispersive spectroscopy after exposure to various concentrations and durations of protein. Furthermore, the impact of protein-treated micro/nanoplastics on biological activities was examined using the mitochondrial activity and membrane integrity of A549 cells and the activity and biofilm production of Staphylococcus aureus. The structural characteristics of micro/nanoplastics revealed an interaction with protein. For instance, the assignment of protein-related new proton signals (e.g., CH2, methylene protons of CH2O), changes in available protons s (e.g., CH and CH3), crystallinity, functional groups, elemental ratios, zeta potentials (-11.3 ± 1.3 to -12.4 ± 1.7 to 25.5 ± 2.3 mV), and particle size (395 ± 76 to 496 ± 60 to 866 ± 82 nm) of micro/nanoplastics were significantly observed after protein treatment. In addition, the loading (0.012-0.027 mM) and releasing (0.008-0.013 mM) of protein also showed similar responses with structural characteristics. Moreover, the cell-based responses were changed regarding the structural and surface characteristics of micro/nanoplastics and the loading efficiencies of protein. For example, insignificant mitochondrial activity (2%-10%) and significant membrane integrity (12%-28%) of A549 cells increased compared with control, and reductions in bacterial activity (5%-40%) in many cases and biofilm production specifically at low dose of all treatment stages (13%-46% reduction) were observed.

Synthesis of green nanosorbent from bovine serum albumin and curcumin for magnetic solid phase extraction of pesticides from food samples.

For the first time, a magnetic carbon nanocomposite was synthesized using one-step hydrothermal procedure, employing bovine serum albumin, curcumin, and ferric ammonium citrate. Additionally, the application of this novel composite as an adsorbent for magnetic dispersive solid phase extraction of fungicides and pesticides from water and food samples is a unique aspect of this study. Under optimum conditions (salt concentration: 5.0% w/v, pH: 7.0, desorption solvent: ethanol, sorbent amount: 20 mg, extraction time: 20 min, desorption time: 3 min, stirring rate: 500 rpm, sample volume: 30 mL, extraction temperature: room temperature, and desorption solvent volume: 150 µL) linearity (2.5 to 1400 ng mL-1), coefficients of determination (R2 ≥ 0.997), limits of detection (0.75 to 1.5 ng mL-1), and limits of quantification (2.5 to 5.0 ng mL-1) were achieved. The method validation results showed extraction recovery ranging from 71.2% to 93.4%, and preconcentration factors ranging from 142.5 to 186.1.

A Ratiometric Green Fluorescent Carbazole-Bis(hydrazinobenzothiazole) Probe for the Selective Detection of Toxic Hg2+ Ions in Real Water Samples.

A dyad Carbazolyl-bis(hydrazinobenzothiazole) was designed to form a symmetrical structure that containing two-arm active binding sites facilitates coordination with Hg2+ ion. This sensor has imparted a colorimetric and fluorometric changes in presence of Hg2+ ions. The ligand showed a selective blue shift in presence of Hg2+even in co-existence with heavy metal ions with luminescence change from colorless to blue and colorless to green under day light. Enhanced Intramolecular charge transfer process is responsible for fluorescence transformation when ligand interacts with Hg2+ ion. The emission spectra showed a ratiometric response to increasing addition of Hg2+ ions. The sensor is capable of detecting above the lower concentration of 6.8025×10-8 M. The fluorescence efficiency of CBT-2 with Hg2+ ion is quite stable under different co-metal ions and wide range of pH 6 to 9. The sensor CBT-2 forms a 1 : 1 stoichiometric complex with Hg2+ ions and the binding nature is confirmed from the 1H-NMR, FTIR, and mass spectroscopic studies. The sensor CBT-2 and its Hg2+ complex possess good binding nature to protein in Bovine Serum Albumin which could be good in biological applications. Additionally, wedevelop a practical application in real water sample analysis and electrochemical detection via oxidation potential discrimination.

Lyophilised protein formulations as a patient-centric dosage form: A contribution toward sustainability paradigm.

At present, society has embraced the fact apropos population aging and climate changes, that demand, amongst others, innovative pharmaceutical technologies, emphasising the development of patient-specific delivery systems and thus the provision of efficient and sustainable drugs. Protein drugs for subcutaneous administration, by allowing less frequent application, represent one of the most important parts of the pharmaceutical field, but their development is inevitably faced with obstacles in providing protein stability and suitable formulation viscosity. To gain further knowledge and fill the gaps in the already constructed data platform for the development of monoclonal antibody formulations, we designed a study that examines small model proteins, i.e., bovine serum albumin. The main aim of the presented work is to evaluate the effect of protein concentrations on critical quality attributes of both, pre-lyophilised liquid formulations, and lyophilised products. Through the study, the hypothesis that increasing protein concentration leads to higher viscosity and higher reconstitution time without affecting the stability of the protein was confirmed. The most important finding is that sucrose plays a key role in the lyophilisation of investigated protein, nevertheless, it can be predicted that, to ensure the beneficial effect of mannitol, its amount has to prevail over the amount of sucrose.

In Situ Preparation of Star-Shaped Protein-"Smart" Polymer Conjugates with pH and Thermo-Dual Responsibility for Bacterial Separation.

To achieve effective separation and enrichment of bacteria, a novel synthetic scheme was developed to synthesize star-style boronate-functionalized copolymers with excellent hydrophilicity and temperature and pH responsiveness. A hydrophilic copolymer brush was synthesized by combining surface-initiated atom-transfer radical polymerization with amide reaction using bovine serum albumin as the core. The copolymer brush was further modified by introducing and immobilizing fluorophenylboronic acids through an amide reaction, resulting in the formation of boronate affinity material BSA@poly(NIPAm-co-AGE)@DFFPBA. The morphology and organic content of BSA@poly(NIPAm-co-AGE)@DFFPBA were systematically characterized. The BSA-derived composites demonstrated a strong binding capacity to both Gram-positive and Gram-negative bacteria. The binding capabilities of the affinity composite to Staphylococcus aureus and Salmonella spp. were 195.8 × 1010 CFU/g and 79.2 × 1010 CFU/g, respectively, which indicates that the novel composite exhibits a high binding capability to bacteria and shows a particularly more significant binding capacity toward Gram-positive bacteria. The bacterial binding of BSA@poly(NIPAm-co-AGE)@DFFPBA can be effectively altered by adjusting the pH and temperature. This study demonstrated that the star-shaped affinity composite had the potential to serve as an affinity material for the rapid separation and enrichment of bacteria in complex samples.