Concentrations Of Con Research Articles

PIM1 inhibitor SMI-4a attenuated concanavalin A-induced acute hepatitis through suppressing inflammatory responses.

Serine/threonine kinase 1 (PIM1) plays a crucial role in cell growth, differentiation, and apoptosis. However, its role in the pathogenesis of concanavalin A (ConA)-induced acute hepatitis is not well understood. PIM1 kinase inhibitor can reduce the expression of PIM1. This study aims to investigate the effects of PIM1 kinase inhibitor and its protective mechanism in ConA-induced acute hepatitis. C57/BL six mice were injected with ConA (20, 15, and 12 mg/kg) to induce acute hepatitis, and PIM1 kinase inhibitor SMI-4a (60 mg/kg) was administered orally 24 h before ConA injection. The survival rate of the mice was observed after ConA injection. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was performed on liver tissue collected at different time points. The major cytokines expression in liver tissue was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The number of macrophages, T-cell and neutrophils in liver tissue were detected by flow cytometry (FCM). PIM1 in liver tissue was detected by western blot (WB) and qRT-PCR. SMI-4a (80 µM) was pretreated for 24 h and ConA (400 µg/mL) was stimulated for 12 h in RAW264.7 cell model. Phosphorylated p65 (p-p65) and cleaved caspase-3 (c-caspase-3) in liver tissue and macrophages were detected by WB. Different concentrations of ConA caused different acute hepatitis mortality, 12 mg/kg concentration within 24 h of the mortality showed a gradient increase. The levels of AST and ALT increased significantly at 12 h after ConA injection. PIM1 expression was upregulated at 12 h. SMI-4a can suppress the PIM1 expression. SMI-4a suppressed cytokines production, AST, and ALT in ConA-treated serum. SMI-4a suppressed the major cytokines in liver tissue. Tests in liver tissue showed that SMI-4a reduced the number of T cells, neutrophils, and macrophages. SMI-4a inhibited the inflammatory response by downregulating the expression of p-p65. Meanwhile, apoptosis was decreased by decreasing the expression of c-caspase-3. In conclusion, the protective effect of SMI-4a against acute hepatitis is by reducing the inflammatory response and apoptosis. These findings suggest that SMI-4a may have therapeutic potential in the treatment of autoimmune hepatitis.

Leptin-mediated ERK Signaling Pathway Promotes the Transformation of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust

Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust. Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R₂₀₀ cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method. Both the cells in the R group and R₂₀₀ group express leptin receptor OB-R. Compared to the R₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R₂₀₀ group (R₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R₂₀₀ (R₂₀₀LU) group. Additionally, the cells in the R₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R₂₀₀LU group and R₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R₂₀₀L group. However, when the cells in the R₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased. Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.

Increasing buffering capacity enhances rumen fermentation characteristics and alters rumen microbiota composition of high-concentrate fed Hanwoo steers

The buffering capacity of buffer agents and their effects on in vitro and in vivo rumen fermentation characteristics, and bacterial composition of a high-concentrate fed Hanwoo steers were investigated in this study. Treatments were comprised of CON (no buffer added), BC0.3% (low buffering capacity, 0.3% buffer), BC0.5% (medium buffering capacity, 0.5% buffer), and BC0.9% (high buffering capacity, 0.9% buffer). Four Hanwoo steers in a 4 × 4 Latin square design were used for the in vivo trial to assess the effect of treatments. Results on in vitro experiment showed that buffering capacity, pH, and ammonia–nitrogen concentration (NH3-N) were significantly higher in BC0.9% and BC0.5% than the other treatments after 24 h incubation. Individual and total volatile fatty acids (VFA) concentration of CON were lowest compared to treatment groups. Meanwhile, in vivo experiment revealed that Bacteroidetes were dominant for all treatments followed by Firmicutes and Proteobacteria. The abundances of Barnesiella intestinihominis, Treponema porcinum, and Vibrio marisflavi were relatively highest under BC0.9%,Ruminoccocus bromii and Succiniclasticum ruminis under BC0.5%, and Bacteroides massiliensis under BC0.3%. The normalized data of relative abundance of observed OTUs’ representative families have grouped the CON with BC0.3% in the same cluster, whereas BC0.5% and BC0.9% were clustered separately which indicates the effect of varying buffering capacity of buffer agents. Principal coordinate analysis (PCoA) on unweighted UniFrac distances revealed close similarity of bacterial community structures within and between treatments and control, in which BC0.9% and BC0.3% groups showed dispersed community distribution. Overall, increasing the buffering capacity by supplementation of BC0.5% and and BC0.9% buffer agents enhanced rumen fermentation characteristics and altered the rumen bacterial community, which could help prevent ruminal acidosis during a high-concentrate diet.

Canavalia ensiformis lectin induced oxidative stress mediate both toxicity and genotoxicity in Drosophila melanogaster

Mannose/glucose-binding lectin from Canavalia ensiformis seeds (Concanavalin A - ConA) has several biological applications, such as mitogenic and antitumor activity. However, most of the mechanisms involved in the in vivo toxicity of ConA are not well known. In this study, the Drosophila melanogaster model was used to assess the toxicity and genotoxicity of different concentrations of native ConA (4.4, 17.5 and 70 μg/mL) in inhibited and denatured forms of ConA. The data show that native ConA affected: the survival, in the order of 30.6 %, and the locomotor performance of the flies; reduced cell viability to levels below 50 % (4.4 and 17.5 μg/mL); reduced nitric oxide levels; caused lipid peroxidation and increased protein and non-protein thiol content. In the Comet assay, native ConA (17.5 e 70 μg/mL) caused DNA damage higher than 50 %. In contrast, treatments with inhibited and denatured ConA did not affect oxidative stress markers and did not cause DNA damage. We believe that protein-carbohydrate interactions between ConA and carbohydrates of the plasma membrane are probably the major events involved in these activities, suggesting that native ConA activates mechanisms that induce oxidative stress and consequently DNA damage.

Sandwich photoelectrochemical biosensing of concanavalin A based on CdS/AuNPs/NiO Z-scheme heterojunction and lectin-sugar binding

A CdS/AuNPs/NiO Z-scheme heterojunction was prepared on a fluorine-doped tin oxide (FTO) electrode by hydrothermal synthesis of NiO on FTO, electrodeposition of AuNPs on NiO/FTO electrode and then cast-coating of CdS quantum dots. The CdS/AuNPs/NiO/FTO electrode gave a notably increased photocurrent versus NiO/FTO, CdS/FTO, AuNPs/NiO/FTO, CdS/AuNPs/FTO and CdS/NiO/FTO electrodes. The CdS/AuNPs/NiO/FTO electrode was further cast-coated with chitosan to immobilize d-mannose by Schiff base reaction, and concanavalin A (ConA) and then horseradish peroxidase (HRP) were captured on the electrode surface by lectin-sugar binding. 4-Chloro-1-naphthol (4-CN) was oxidized to form an insoluble precipitate catalyzed by HRP in the presence of H2O2, and the presence of precipitate on the photoelectrode inhibited the photocurrent in the presence of holes scavenger ascorbic acid. The relevant electrodes were characterized by electrochemistry, quartz crystal microbalance (QCM), UV–vis spectrophotometry, scanning electron microscopy/energy dispersive spectroscopy, and transmission electron microscopy. The QCM revealed that the collection efficiency (η) of the 4-CN-electrooxidation precipitate on the electrode can be as high as 91.8%. Under the optimal conditions, the decline of photocurrent responded linearly to the common logarithm of ConA concentration from 50 pM to 500 nM, with a limit of detection of 17 pM (S/N = 3). Satisfactory results were obtained in the detection of real soybean samples.

An electrochemiluminescence biosensor based on boron nitride quantum dots as novel coreactant for quantitative determination of concanavalin A.

An electrochemiluminescence (ECL) analytical platform is constructed based on boron nitride quantum dots (BNQDs) as a novel coreactant of luminol for quantitative assay of concanavalin A (Con A). Different from previous research that mainly focuses on its superior optical properties, BNQDs are used for the first timeas a coreactant for boosting ECL intensity of luminol, which has a 10-fold enhancement compared with individual poly(luminol/aniline) nanorods loaded on reduced graphene oxide (PLA-rGO) using GCE. On the basis that BNQDs contain an abundance of active amino, a possible mechanism of amino oxidation facilitating ECL emission is proposed. Firstly, luminol as light spices are oxidized to luminol•- and BNQDs generate an abundance of BNQDs-NH+• via electrochemical oxidization, producing reductive intermediates BNQDs-N• in alkaline conditions. Finally, BNQDs-N• react with luminol•- to obtain the excited species AP2-*, returning to ground state and emitting light. Due to the hindrance effect of Con A, the ECL intensity decreases gradually as various concentrations of Con A are modifying the electrodesurface. Therefore, a sensitive ECL biosensor for detecting Con A is constructed exhibiting awide linear range of1.0pg·mL-1 to 1.0μg·mL-1 and a low detection limit of 0.15pg·mL-1. Graphical abstract Schematic representation of an electrochemiluminescence (ECL) biosensor based on boron nitride quantum dots (BNQDs) as an efficient coreactant of reduced graphene oxide functionalized poly(luminol/aniline) (PLA-rGO) for quantitative assay of concanavalin A (Con A).

Revealing Lectin-Sugar Interactions with a Single Au@Ag Nanocube.

An individual nanoparticle-based plasmonic nanotechnology was used for real-time monitoring of lectin-sugar interactions, which could be designed as novel plasmonic nanobiosensors for the detection of trace concanavalin A (ConA) with high sensitivity and selectivity. The localized surface plasmon resonance (LSPR) spectra of Au@Ag nanocubes (NCs) are linearly shifted to a long wavelength with an increasing concentration of ConA. In fact, each Au@Ag NC can act as a nanobiosensor for the quantified detection of trace ConA, which enables the miniaturization of the biosensor system to nanoscale. Furthermore, the results demonstrated the perfect biosensing ability with the dual channel of dark-field microscopy images and LSPR spectra. We expect that this nanobiosensor system can provide an alternative important method for monitoring the specific binding of lectin-sugar at a single nanoparticle surface.

Concanavalin A- Sandwich-ELISA for the detection of antibodies against Newcastle disease virus (NDV) in chicken

Concanavalin A (Con A), a lectin interacts with carbohydrate moieties of viruses and provide stable and sensitive detection when used as a capture agent. Indirect ELISA methods need purified Newcastle disease virus (NDV) or recombinant antigens for adsorption, whereas use of Con A as capture agent will enable the use of non-purified and non-concentrated virus as antigen replacing costly and time-consuming virus purification step. Con A based sandwich ELISA with non-purified NDV whole virus antigen with single serum dilution format has been developed in this study. The optimum concentrations of the capture agent, Con A and non-purified antigen preparations were determined by checker-board titration. Briefly, microplates were coated with predetermined optimum concentration of ConA (0.5 mg/ml; 50µg per well) and incubated for 18h at 4°C. After washing, allantoic fluid with Newcastle disease virus (NDV) LaSota (HA titre, 210) at a constant predetermined dilution (1:1; 50µl) was coated and incubated for 45 min at 37°C, followed by blocking with 2 % bovine serum albumin for 45 min at 37°C. The antigen coated plates were used in the detection of antibody titre against NDV in serum samples at single serum dilution of 1: 500. Then, wells were added with goat anti-chicken IgG horseradish peroxidase conjugate and incubated for 1h at 37°C, followed by addition of TMB substrate and the plates were read spectrophotometrically at 450 nm. ELISA antibody titres were determined by standard serial dilution of positive sera and endpoints were calculated by a subtraction method. By using positive negative threshold curve (PNT), intercept and slope of the standard curve were calculated. Total of 271 random chicken serum samples were analyzed for antibodies against NDV by Haemagglutination inhibition assay (HI), indirect ELISA and compared with the Con A- S- ELISA developed in this study. The Con A-S-ELISA showed a high coefficient of correlation (r=0.85, n=271, P less than 0.01) and an agreement of ê=0.99 with the commercially available Indirect-ELISA. The relative sensitivity and specificity were 98% and 85% respectively in comparison to HI test. Hence, the Con A-S-ELISA is a simple, easy and effective for monitoring serum antibody levels against NDV.

Construction of a Concanavalin A electrochemical sensor base on a novel sandwich capture mode

Herein, a concanavalin A (ConA) electrochemical sensor was constructed via a sandwich mode. Firstly chitosan functionalized graphene oxide (CS/GO) composite was introduced on the of glassy carbon electrode (GCE) as the substrate. D–mannose (D-man) was immobilized on the surface of CS/GO via Schiff base reaction. Due to the specific affinity between mannose and ConA, the prepared D–man/CS/GO sensing interface could specifically capture ConA which could further react with mannose residues of HRP and form a sandwich configuration. Based on the catalytic effect of horseradish peroxidase (HRP) to H2O2, a ConA electrochemical sensor with enzyme catalytic amplification was constructed by taking hydroquinone as electrical mediator. The morphology of CS/GO was investigated by transmission electron microscope and Fourier transform infrared spectroscopy. Electrochemical techniques including electrochemical impedance spectroscopy and differential pulse voltammogram (DPV) were used for reveal the characteristics of the sensing platform. Benefit from the high surface area of GO and enzyme catalytic amplification of HRP, the peak current from DPV is linearly dependent on the ConA concentration ranging from 5×10−9 to 5×10−7mol/L with a detection limit of 1.24×10−9mol/L. The results demonstrated that the proposed sensor might exploit a new direction for determination of ConA.

Electrochemical impedance spectroscopy study of Concanavalin A binding to self-assembled monolayers of mannosides on gold wire electrodes

The interactions of the lectin Concanavalin A (Con A) with self-assembled monolayers (SAMs) of thiolated mono-, di-, and tri-mannosides were studied on the surface of gold wires using electrochemical impedance spectroscopy (EIS). The SAMs of mannosides were prepared either pure or along with thiolated triethylene glycol (TEG) at different molar ratios (1:1, 1:2, 1:4, 1:9, and 1:19) to better understand and optimize the interaction conditions. The charge-transfer resistance of the [Fe(CN)6]3−/4− redox probe was compared before and after the interaction at different concentrations of Con A to determine the equilibrium dissociation constant (Kd) and limit of detection (LOD). Values of Kd were found in the nanomolar range showing multivalent interactions between mannosides and Con A, and LOD was found ranging from 4 to 13nM depending on the type of mannoside SAM used. Analysis using the Hill equation suggests negative cooperativity in the binding behavior. Peanut agglutinin was used as a negative control, and cyclic voltammetry was used to further support the experiments. We have found that neither the pure nor the widely dispersed monolayers of mannosides provide the conditions for optimal binding of Con A. The binding of Con A to these SAMs is sensitive to the molar ratio of the mannoside used to prepare the SAM and to the structure of the mannoside. A simple cleaning method has also been shown to regenerate the used gold wire electrodes. The results from these experiments contribute to the development of simple, cheap, selective, and sensitive EIS-based bioassays, especially for lectin–carbohydrate interactions.

Temperature-stable quartz oscillator and its applications in pressure gauges, gas sensing, and gas concentration measurements

In this paper, a temperature-stable quartz oscillator (TSQO) is introduced that eliminates the influence of temperature on the output, which is the electrical impedance (Z)-converted voltage. Z depends on the pressure, viscosity, and density of the measured gas. Temperature stability was confirmed for temperatures varying from 30 to 300 °C at atmospheric pressure. As temperature was increased from 31 to 200 °C, fluctuations were decreased to below 0.30% of the average TSQO output. The TSQO exhibited an improved temperature stability compared with the output fluctuation of a conventional quartz oscillator, which is usually approximately 2.0% of the output voltage over the temperature range of 15–85 °C. The measured fluctuation in the TSQO output over the temperature range of 31–100 °C is 0.22%, which is acceptable for outdoor hydrogen sensing. This is because the fluctuations are smaller than the 0.33% change induced by hydrogen (H2) contamination at a concentration of 0.33 vol. %. The concentration of contaminated hydrogen is sufficiently less than the minimum detection level, which is one-fourth of the fluctuation introduced by low-level explosions of H2 in air (4%). The TSQO output could be implemented in various measurement applications. The dependence of the TSQO output on the total pressure in the range of 0.5–100 kPa indicates that it can function as a pressure gauge. The difference between the TSQO output measured for pure nitrogen (N2) and 0.5 and 1.0 vol. % H2 in N2 at atmospheric pressure indicates that the TSQO can operate as a hydrogen sensor. Moreover, depending on the TSQO output on hydrogen concentration at atmospheric pressure confirmed that the TSQO can be used to measure hydrogen concentration. The hydrogen sensing and concentration measurement applications stated above were possible up to 300 °C. Therefore, it can be concluded that the TSQO is practically useful for carrying out various measurements under the conditions of temperature fluctuations and exhibits an optimum performance in the temperature range of 31−300 °C.

A novel “on-off” electrochemiluminescence sensor for the detection of concanavalin A based on Ag-doped g-C3N4

In this work, a novel “on-off” electrochemiluminescence (ECL) biosensor was successfully constructed for ultrasensitive detection of concanavalin A (ConA). The “signal-on” state with strong initial ECL signal was obtained by Ag-doped graphitic carbon nitride nanosheet (Ag-g-C3N4), which was modified onto the glassy carbon electrode (GCE) for immobilizing phenoxy dextran (DexP) through π-π interaction. Then, the target ConA was anchored onto the DexP/Ag-g-C3N4 modified film via the specific carbohydrate-ConA interaction. Successively, polyaniline-3,4,9,10-perylenetetracarboxylic acid-DexP conjugate (denoted as PANI-PTCA-DexP), as the ECL signal quenching probe, was incubated onto the electrode through the carbohydrate-ConA interaction to achieve the “signal-off” state. Here, PTCA was used as a matrix for the high loading of PANI and DexP, PANI served as the quencher towards Ag-g-C3N4 system, and DexP as the recognition element for bounding ConA. With the formation of the sandwiched structure of DexP, ConA and PANI-PTCA-DexP, a desirable quenching ECL signal was measured with S2O82− as the co-reactant of Ag-g-C3N4. The quenching effect of PANI towards Ag-g-C3N4 is positively correlated with the concentration of ConA. With such an “on-off” system, the detection of ConA was achieved with a wide linear range from 0.001ng/mL to 50ng/mL as well as a low detection limit down to 0.0003ng/mL.

Synthesis and optical properties of a novel sugar coated poly(p-phenyleneethynylene) effectively quenched by concanavalin A

The synthesis of a novel sugar coated poly(p-phenyleneethynylene) (PPE) via Pd-catalyzed Sonogashira reaction and its subsequent interaction with concanavalin A (Con A) are described. UV–Vis and fluorescence spectra were employed to investigate the response of the polymer toward various concentrations of Con A. The polymer showed a green fluorescence which can be quenched by Con A due to complex formation at ground state. The protocols reported herein provide intermediate products that can be converted into useful sensing elements from cheap and commercially available starting materials, in a few number of steps.

Synthesis, characterization and detection of Concanavalin A based on a mannose-substituted conjugated polymer through aggregation-enhanced FRET

A conjugated polymer (P1) with pendant mannose linkages has been designed and synthesized, which is a highly efficient ratiometric probe for Concanavalin A (Con A) detection. P1 is doped with a low-bandgap fluorophore, porphyrin, which in conjunction with favorable spectral overlap between the poly(fluorene–phenylene) and porphyrin facilitates efficient FRET process in aqueous solutions in presence of Con A. UV/vis spectroscopy and dynamic light scattering studies indicate that P1 and Con A form into nanoparticles by carbohydrate–lectin interactions. Addition of Con A results in the quenching of the poly(fluorene–phenylene) emission at 456nm with simultaneous enhancement of porphyrin emission at 625nm, exhibiting visual emission color change from blue to purple. The intensity ratio of I625/I456 is enhanced 9.3-fold when the concentration of Con A reaches 10μmol/L. Nonspecific proteins including bovine serum albumin (BSA), pepsin (Pep), lysozyme (Lys) show poor fluorescence response, thus highlighting the selectivity and sensitivity of the FRET-based sensing of Con A by P1. Moreover, P1 shows low cytotoxicity by CCK-8 assay, which makes its potential application as fluorescent agent for cellular imaging.

Apoptosis induction by concanavalin A in gut cells of grain aphid

In this study, the toxicity and mechanism of action of concanavalin A (ConA) in the grain aphid (Sitobion avenae) were studied. Feeding assays with S. avenae on an artificial diet containing different concentrations of ConA demonstrated an inhibitory effect on fecundity as well as high mortality caused by this lectin. ConA also increased the pre-reproductive period and the development time and reduced the intrinsic rate of natural increase. Moreover, an extract of the gut of treated S. avenae demonstrated an increase in caspase-3 activity together with DNA fragmentation, suggesting that ConA can induce the apoptotic pathway. These results suggest that ConA may be detrimental in insect gut tissues and the interaction of ConA with epithelial cells may be responsible for the observed insecticidal effects.

An ultrasensitive electrochemiluminescent biosensor for the detection of concanavalin A based on poly(ethylenimine) reduced graphene oxide and hollow gold nanoparticles.

A highly sensitive electrochemiluminescent (ECL) biosensor was designed for the detection of concanavalin A (ConA) based on glucose oxidase (GOx) as a recognition element by carbohydrate-lectin biospecific interaction, and poly(ethylenimine) (PEI) reduced graphene and hollow gold nanoparticles (HAuNPs) as supporting matrix and signal amplifier. The modification process and detection principle of the biosensor are briefly described as follows. First, PEI reduced graphene oxide with abundant amino groups was cast onto the surface of glassy carbon electrode to adsorb HAuNPs for improving the signal intensity in luminol/H2O2 ECL system. Next, GOx was further assembled onto the electrode by the interaction between Au and -NH2. In the presence of glucose in the detection solution, GOx catalyzed glucose to generate H2O2 in situ, which served as a co-reactant of luminol to enhance ECL signal of luminol. Based on the fact that ConA could result in a decrease in ECL signal when immobilized on the electrode, an ECL biosensor was prepared for the determination of ConA. The ECL signal intensity was linear with the logarithm of ConA concentration and the linear range was from 1.0 to 20ng/mL with a low detection limit of 0.31ng/mL (signal to noise ratio =3). This strategy led to a nearly 1000-fold improvement in detection limit for ConA assays compared with previously reported method, thus exhibiting a great potential application in sensitive bioassays of ConA.

Double‐layer silver nanoparticle arrays‐based localised surface plasmon resonance optical sensor for sensitive detection of concanavalin A

A localised surface plasmon resonance (LSPR) optical sensor with double-layer silver nanoparticle (Ag NP) arrays has been developed for the sensitive detection of concanavalin A (ConA). Based on the principle of carbohydrate–protein interactions, double-layer dextran capped Ag NP arrays were initially constructed. The increase of adsorption apparent mass and LSPR coupling between the two Ag NP arrays can effectively enhance the LSPR spectral shift response. Compared with the single-layer Ag NP arrays configuration, the double-layer Ag NP arrays configuration improves the limitation of detection of ConA concentration from 3.42 to ∼1.14 µg/ml.

Electrochemical displacement sensor based on ferrocene boronic acid tracer and immobilized glycan for saccharide binding proteins and E. coli

Pathogens such as viruses and bacteria use their envelope proteins and their adhesin lectins to recognize the glycan residues presented on the cell surface of the target tissues. This principle of recognition is used in a new electrochemical displacement sensor for the protein concanavalin A (ConA). A gold electrode was first modified with a self-assembled monolayer of a thiolated mannose/OEG conjugate and a ferrocene boroxol derivative was pre-assembled as reporter molecule onto the mannose surface. The novel tracer molecule based on a 2-hydroxymethyl phenyl boronic acid derivative binds even at neutral pH to the saccharides which could expand the application towards biological samples (i.e., urine and feces). Upon the binding of ConA, the tracer was displaced and washed away from the sensor surface leading to a decrease in the electrochemical signal. Using square wave voltammetry (SWV), the concentration of ConA in the sample solution could be determined in the dynamic concentration range established from 38nmolL−1 to 5.76µmolL−1 with a reproducible detection limit of 1µgmL−1 (38nmolL−1) based on the signal-to-noise ratio (S/N=3) with fast response of 15min. The new reporter molecule showed a reduced non-specific displacement by BSA and ribonuclease A. The sensor was also successfully transferred to the first proof of principle for the detection of Escherichia coli exhibiting a detection limit of approximately 6×102cells/mL. Specificity of the displacement by target protein ConA and E. coli was demonstrated since the control proteins (i.e., BSA and RNaseA) and the control E. coli strain, which lack of type 1 fimbriae, were ineffective.

Electrochemical sensing of concanavalin A using a non-ionic surfactant with a maltose moiety

To electrochemically detect concanavalin A (ConA), a new method was developed using mixed micelles between a non-ionic surfactant with a maltose moiety and electroactive daunomycin. The surfactants, in which the length of the alkyl chain was different, were n-decyl-β-d-maltoside, n-dodecyl-β-d-maltoside, and n-tetradecyl-β-d-maltoside. The measurement principle was due to the micelle breakdown caused by the binding between the ConA and maltose moieties. When ConA was combined with maltose moieties at a concentration of surfactant that was near the critical micelle concentration, the daunomycin that formed the micelles was moved to a solution from the micelles. As a result, the peak current of daunomycin increased as the concentration of ConA was increased. The mechanism was proposed using voltammetry, spectrometry, and gel filtration. The linear range using n-tetradecyl-β-d-maltoside was 2.0×10−9 to 8.0×10−8M of ConA, and it was the most sensitive in the presence of the three surfactants. To examine whether selective binding took place, measurements with several proteins were carried out. The electrode responses of daunomycin were not influenced by the presence of 5.0×10−6M protein. Furthermore, this method could be applied to the determination of ConA in a serum, and to the measurement of sugar chains that can be combined with ConA on the cell surface.

Flow cytometric evaluation of T cell activation markers after cardiopulmonary bypass.

Background. Cardiopulmonary bypass surgery (CPBS) is associated with an increased risk for infections or with subsequent organ dysfunction. As T cell activation is a central mechanism during inflammatory processes, we developed an assay to evaluate T cell activation pathways in patients undergoing CPBS. Methods. Blood was obtained from eleven patients undergoing CPBS preoperatively, on postoperative day (POD)-3, and on POD-7 and was stimulated with different concentrations of Concanavalin A (ConA). Cyclosporine and sirolimus, inhibiting different pathways of the T cell cycle, were added to blood ex vivo. Expression of T cell activation markers CD25 and CD95 was analyzed by flow cytometry. Results. In untreated blood, expression of CD25 and CD95 significantly increased with higher ConA concentrations (P < 0.05) and decreased for all ConA concentrations for both antigens over the study time (P < 0.05). Independently from the ConA concentration, inhibition of CD25 and CD95 expression was highest preoperatively for sirolimus and on POD-3 for cyclosporine. At all time points, inhibition of CD25 and CD95 expression was significantly higher after cyclosporine compared to sirolimus treatment (P < 0.001). Conclusion. Our results showed that different pathways of T cell activation are impaired after CPBS. Such knowledge may offer the opportunity to identify patients at risk for postoperative complications.