Sialic acid (SA), a glycoprotein associated with many pernicious diseases, has been applied to quantify cancer cells. In this paper, a novel electrochemical cytosensor with three-dimensional (3D) micro/nanostructured sensing interface, which can provide a better platform for cell adhesion, was utilized to detect the expression of SA from the cell surface. The hollow horn-like PPy (hPPy) film and chitosan–Au nanoparticles (CS–Au NPs) were electrodeposited on stainless steel and then, combined with a targeting lectin molecule of Sambucus nigra agglutinin (SNA), for sensing A549 human lung cancer cells based on the molecular recognition between SNA and SA. The morphologies, wettability and cytotoxicity of CS–Au/hPPy were investigated by scanning electron microscope (SEM), energy dispersive spectrum (EDS), water contact angle test and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays. Furthermore, the electrochemical performances of this cytosensor with a 3D micro/nanostructured CS–Au/hPPy sensing interface were investigated. Under optimal conditions, the proposed cytosensor exhibited a good linear relationship, wide linear range of the cell concentration from 10 to 1.0 × 107 cells per mL and a detection limit as low as 2 cells per mL (S/N = 3). Moreover, the cytosensor also had good stability and specificity to analyze the over-expressed SA on living cells, implying that the new sensing interface we proposed may have a huge potential application in the study of tumor cells and greatly promote the cancer diagnosis and treatment in early stages.

Ultrafiltration and HPLC were employed to assess binding rates between rat plasma protein and two active compounds with lipid-regulating properties (alisol B 23-acetate and alisol A 24-acetate) from Alismaorientale rhizomes (Alismatis Rhizoma), a traditional Chinese medicine. SDS–PAGE was used for the evaluation of the binding between the alisol acetates and Hb in plasma. The fluorescence spectroscopy and circular dichroism spectroscopy were also combined with molecular modeling to explore binding mechanisms between Hb and the alisol acetates under imitative physiological condition. The ultrafiltration results show that alisol B 23-acetate bound more strongly than alisol A 24-acetate to plasma protein. SDS–PAGE results may suggest that alisols bind to Hb in plasma. The spectroscopy results are consisting with the molecular modeling results, and they indicate that the differences in plasma protein binding strength between the two compounds may be related to their side chains. A folded side chain/parent ring bound more strongly to Hb than an open side chain/parent ring.The side chain of alisol acetates may be the key group that determines the mode of interaction with the macromolecule, and therefore it plays a decisive role in interactions between alisol acetates and macromolecules.

Understanding conformation transitions of proteins in the presence of a chemical denaturant is a topic of great interest because the rich information contained in chemical unfolding is of fundamental importance for proteomic and pharmaceutical research. In this work, the conformational structure changes of glucose oxidase (GOx) induced by guanidinium ions (Gdm+) were studied in detail by a combination of electrochemical methods, various spectroscopic techniques including ultraviolet–visible (UV–vis) absorption, fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations with the purpose of revealing the mechanism of chemical unfolding of proteins. The results indicated that GOx underwent substantial conformational changes both at the secondary and tertiary structure levels after interacting with Gdm+ ions. The interaction of GOx with the chemical denaturant resulted in a disturbance of the structure of the flavin prosthetic group (FAD moiety) that induced the moiety to become less exposed to solvent than that in the native protein molecule. The calculation from quantitative second-derivative infrared and CD spectra showed that Gdm+ ions induced the conversion of α-helix to β-sheet structures. MD simulations and DFT calculations revealed that Gdm+ ions could enter the active pocket of the GOx molecule and interact with the FAD group, leading to a significant alteration in the structural characteristics and hydrogen bond networks formed between FAD and the surrounding amino acid residues. These alterations in the conformational structure of GOx resulted in a significant decrease in the catalytic activity of the enzyme to glucose oxidation. The study essentially provides an effective way for investigating the mechanism of chemical denaturant-induced protein unfolding, and this approach can be used for assessing the effect of drug molecules on proteins.

Protein conformational changes may be associated with particular properties such as its function, transportation, assembly, tendency to aggregate, and potential cytotoxicity. In this study, the mechanism of the effects of thermal unfolding of proteins on their catalytic activities and conformational structures were studied by utilizing glucose oxidase (GOx) as a model protein. The characteristic kinetic constants for the enzymatic reaction were evaluated by the use of an electrochemical approach under a substrate-saturated condition. A combination of quantitative second-derivative infrared analysis, two-dimensional infrared correlation spectroscopy (2D IR), and theoretical calculation was used to elucidate the conformational structures that were responsible for inactivation and denaturation of GOx induced by heat. The IR analysis demonstrated that the conformational structures of GOx, especially the α-helix and unordered structures, were greatly dependent on the system temperature. Thermal treatment resulted in the increase of the unordered structure accompanied by the loss of the α-helical structure in GOx conformation. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations revealed that thermal treatment could significantly alter the electronic characteristics and the intramolecular electron transfer ability of FAD (flavin adenine dinucleotide), and hydrogen bond networks formed between FAD and the amino acid residues around the cofactor, leading to the change of the secondary structure and the catalytic activity of GOx. The study essentially paves an effective approach to investigation of the mechanism of protein unfolding.

Crystal structure of the imiquimod has been determined by single crystal X-ray analysis, imiquimod crystallizes in orthorhombic space group P212121 and the molecules are linked along the c axis by the strong N–H⋯N hydrogen bonds. A density functional theory (DFT) study on the electronic properties of imiquimod and its synthetic intermediates has been performed at B3LYP/6-31G∗ level, while taking solvent effects into account. Both the single configuration interaction (CIS) method and the time-dependent DFT (TDDFT) approaches have been used to calculate the electronic absorption spectra, and there is a good agreement between the calculated and experimental UV–visible absorption spectra. The fluorescence emission spectra of these three compounds in solution have also been measured, the relatively low fluorescence intensity is attributed to a chlorine-modulated heavy atom effect that enhances intersystem crossing between excited singlet and triplet states, and the relatively high fluorescence intensity of imiquimod results from an extended π-conjugated system which enhances S1 → S0 radiant transition.Fluorescence emission and UV–visible spectra, crystal structure, and DFT study of imiquimods a (R = H), b (R = Cl), and c (R = NH2) were carried out in this work.

Understanding conformation transitions of proteins in the presence of a chemical denaturant is a topic of great interest because the rich information contained in chemical unfolding is of fundamental importance for proteomic and pharmaceutical research. In this work, the conformational structure changes of glucose oxidase (GOx) induced by guanidinium ions (Gdm+) were studied in detail by a combination of electrochemical methods, various spectroscopic techniques including ultraviolet–visible (UV–vis) absorption, fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations with the purpose of revealing the mechanism of chemical unfolding of proteins. The results indicated that GOx underwent substantial conformational changes both at the secondary and tertiary structure levels after interacting with Gdm+ ions. The interaction of GOx with the chemical denaturant resulted in a disturbance of the structure of the flavin prosthetic group (FAD moiety) that induced the moiety to become less exposed to solvent than that in the native protein molecule. The calculation from quantitative second-derivative infrared and CD spectra showed that Gdm+ ions induced the conversion of α-helix to β-sheet structures. MD simulations and DFT calculations revealed that Gdm+ ions could enter the active pocket of the GOx molecule and interact with the FAD group, leading to a significant alteration in the structural characteristics and hydrogen bond networks formed between FAD and the surrounding amino acid residues. These alterations in the conformational structure of GOx resulted in a significant decrease in the catalytic activity of the enzyme to glucose oxidation. The study essentially provides an effective way for investigating the mechanism of chemical denaturant-induced protein unfolding, and this approach can be used for assessing the effect of drug molecules on proteins.