A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires

A novel glucose biosensor was developed, based on the immobilization of glucose oxidase (GOD) with cross-linking in the matrix of bovine serum albumin (BSA) on a Pt electrode, which was modified with gold nanoparticles decorated Pb nanowires (GNPs-Pb NWs). Pb nanowires (Pb NWs) were synthesized by an l-cysteine-assisted self-assembly route, and then gold nanoparticles (GNPs) were attached onto the nanowire surface through –SH–Au specific interaction. The morphological characterization of GNPs-Pb NWs was examined by transmission electron microscopy (TEM). Cyclic voltammetry and chronoamperometry were used to study and to optimize the electrochemical performance of the resulting biosensor. The synergistic effect of Pb NWs and GNPs made the biosensor exhibit excellent electrocatalytic activity and good response performance to glucose. The effects of pH and applied potential on the amperometric response of the biosensor have been systemically studied. In pH 7.0, the biosensor showed the sensitivity of 135.5 μA mM⁻¹ cm⁻², the detection limit of 2 μM (S/N = 3), and the response time <5 s with a linear range of 5–2200 μM. Furthermore, the biosensor exhibits good reproducibility, long-term stability and relative good anti-interference.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

NF-κB inhibitory activity of cyclitols isolated from Hancornia speciosa

Hancornia speciosa Gomes (Apocynaceae) is a Brazilian plant traditionally employed to treat inflammatory conditions, among other uses. The chemopreventive effect of an ethanol extract from H. speciosa leaves (EHS) was evaluated in a battery of in vitro tests [inhibition of aromatase, NF-κB and ornithine decarboxylase (ODC), antioxidant response elements (ARE) induction and cell proliferation assays]. Bioassay-directed fractionation of EHS following by inhibition of 12-O-tetradecanoyl-13-acetate (TPA)-mediated NF-kB activation led to the isolation of the cyclitols quinic acid (1) (85.0±12.3 μM) and l-(+)-bornesitol (2) (IC₅₀=27.5±3.8 μM), along with rutin (26.8±6.3 μM). Based on these lead compounds, the cyclitols per-O-acetyl-1l-(+)-bornesitol (3) (IC₅₀=38.4±6.2 μM), myo-inositol (4) (>180.2 μM), scyllo-inositol (5) (83.0±13.7 μM) and β-d-galactoside-myo-inositol (6) (52.4±8.4 μM) were evaluated in the assay, but found to be somewhat less active than 1 and 2. None of the compounds was active in the ARE, aromatase or ODC assays and did not inhibit proliferation of MCF-7, LNCaP, HepG2 or LU-1 cell lines at a final concentration of 20 μg/ml (equivalent to 104.07–32.76 μM).This work identifies l-(+)-bornesitol, quinic acid and rutin as NF-κB inhibitors of H. speciosa and suggests cyclitols, in addition to myo-inositol, are potentially useful as chemopreventive agents.     

Angiotensin I-converting enzyme inhibitory peptides in red-mold rice made by Monascus purpureus

The ACE inhibitory activity in red-mold rice extracts, prepared from 24 strains of the genus Monascus, was measured. The most effective strain for ACE inhibition was Monascus purpureus IFO 4489 (IC₅₀ = 0.71 mg/ml). Although the antihypertensive substance γ-amino butyric acid was detected in the red-mold rice (85.2 mg/kg), it did not contribute to ACE inhibition. Four ACE inhibitory peptides were isolated from the extract and identified as Ile-Tyr (IC₅₀ = 4.0 μM), Val-Val-Tyr (22.0 μM), Val-Phe (49.7 μM) and Val-Trp (3.1 μM) by protein sequencing. The ACE inhibitory activity of these peptides was almost completely preserved after successive in vitro digestion by pepsin, chymotrypsin and trypsin. These results suggest that red-mold rice made by M. purpureus could be useful in alleviating hypertension.     

A novel biosensor based on l-homocysteine desulfhydrase enzyme immobilized in eggshell membrane

A novel biosensor for homocysteine determination has been developed. The biosensor was fabricated with l-homocysteine desulfhydrase immobilized on the ammonium selective electrode by means of eggshell membrane. The measurement principle is based on determination of ammonia due to the enzymatic reaction in the medium by ammonium selective electrode. The effects of enzyme loading, glutaraldehyde concentration, pH, buffer concentration, temperature, dithiotreitol (DTT) concentration and ionic strength adjustment buffer (ISA) on the biosensor response were investigated in detail. The linear detection range and limit of detection (LOD) for homocysteine were found to be 0.15–1.8 mM and 55 μM, respectively. Finally, the homocysteine biosensor has been applied to plasma samples for determination of total homocysteine contents.     

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC₅₀ = 3.3 μM, K_i = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.     

An amperometric biosensor fabricated from electro-co-deposition of sodium alginate and horseradish peroxidase

A convenient and effective strategy for fabrication of hydrogen peroxide biosensor based on sodium alginate (SA) and polyvinyl butyral (PVB) as matrices was reported in this paper. The horseradish peroxidase (HRP) and SA were electro-co-deposited onto the surface of gold electrode, and the HRP–SA/Au electrode was further coated with PVB. The interaction between HRP and SA was characterized by UV–vis absorption spectroscopy, and the fabricating process of biosensor was characterized by electrochemical impedance spectroscopy (EIS). The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Experimental conditions were investigated which influence the performance of the biosensor, such as pH, and applied potential. The biosensor showed a linear response to H₂O₂ over a concentration range from 7.0 × 10⁻⁶ to 4.1 × 10⁻³ M with a detection limit of 1.8 × 10⁻⁶ M based on a signal-to-noise ratio of 3 under optimum conditions. The value of HRP in the composite was evaluated to be 1.38 mM. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability.     

Development of a disposable glucose biosensor using electroless-plated Au/Ni/copper low electrical resistance electrodes

This paper presents a glucose biosensor, which was developed using a Au/Ni/copper electrode. Until now, research regarding the low electrical resistance and uniformity of this biosensor electrode has not been conducted. Glucose oxidase (GOD) immobilized on the electrode effectively plays the role of an electron shuttle, and allows glucose to be detected at 0.055 V with a dramatically reduced resistance to easily oxidizable constituents. The Au/Ni/copper electrode has a low electrical resistance, which is less than 0.01 Ω, and it may be possible to mass produce the biosensor electrode with a uniform electrical resistance. The low electrical resistance has the advantage in that the redox peak occurs at a low applied potential. Using a low operating potential (0.055 V), the GOD/Au/Ni/copper structure creates a good sensitivity to detect glucose, and efficiently excludes interferences from common coexisting substances. The GOD/Au/Ni/copper sensor exhibits a relatively short response time (about 3 s), and a sensitivity of 0.85 μA mM⁻¹ with a linear range of buffer to 33 mM of glucose. The sensor has excellent reproducibility with a correlation coefficient of 0.9989 (n = 100 times) and a total non-linearity error of 3.17%.     

Elderberry flavonoids bind to and prevent H1N1 infection in vitro

A ionization technique in mass spectrometry called Direct Analysis in Real Time Mass Spectrometry (DART TOF-MS) coupled with a Direct Binding Assay was used to identify and characterize anti-viral components of an elderberry fruit (Sambucus nigra L.) extract without either derivatization or separation by standard chromatographic techniques. The elderberry extract inhibited Human Influenza A (H1N1) infection in vitro with an IC₅₀ value of 252 ± 34 μg/mL. The Direct Binding Assay established that flavonoids from the elderberry extract bind to H1N1 virions and, when bound, block the ability of the viruses to infect host cells. Two compounds were identified, 5,7,3′,4′-tetra-O-methylquercetin (1) and 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexanecarboxylate (2), as H1N1-bound chemical species. Compound 1 and dihydromyricetin (3), the corresponding 3-hydroxyflavonone of 2, were synthesized and shown to inhibit H1N1 infection in vitro by binding to H1N1 virions, blocking host cell entry and/or recognition. Compound 1 gave an IC₅₀ of 0.13 μg/mL (0.36 μM) for H1N1 infection inhibition, while dihydromyricetin (3) achieved an IC₅₀ of 2.8 μg/mL (8.7 μM). The H1N1 inhibition activities of the elderberry flavonoids compare favorably to the known anti-influenza activities of Oseltamivir (Tamiflu^®; 0.32 μM) and Amantadine (27 μM).     

Microalgae fiber optic biosensors for herbicide monitoring using sol–gel technology

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L⁻¹) and the broadest dynamic calibration range (19–860 μg L⁻¹) with IC₅₀ 125 ± 14 μg L⁻¹. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

A genetically engineered whole-cell pigment-based bacterial biosensing system for quantification of N-butyryl homoserine lactone quorum sensing signal

N-Acyl homoserine lactone (AHL) is a widely conserved quorum sensing (QS) signal of Gram-negative bacteria and has received attention in fighting against human diseases and environmental pollution. However, a method for quantifying AHL is lacking although it is urgently required for diagnosis and bioprocess manipulation. This work screened out an aromatics degrader Pseudomonas aeruginosa for biosensing system development, which produced a blue–green pigment regulated by the RhlI–RhlR QS system. By taking advantage of the recognition of N-butyryl homoserine lactone (BHL, the signal molecule of RhlI–RhlR QS system and an AHL) by the product of rhlR, a new whole-cell biosensor P. aeruginosa ΔrhlIR/pYC-rhlR (rhlI⁻rhlR⁺⁺) was developed. It was constructed through abolishing its BHL production by in-frame deletion of rhlIR and over-expressing rhlR by introducing a multi-copy plasmid pYC-rhlR into ΔrhlIR. By using the pigment production which responded to exogenous BHL as biosensor output, BHL quantification in samples was simply done spectrophotometrically. Under optimum conditions, the calibration curve had the limit of detection (LOD), the 50% activation/effect concentration, the limit of quantification (LOQ), and the quantitative detection range of 1.3 nM, 2.77 ± 0.45 μM, 5.7 nM and 0.11–49.7 μM, respectively. The biosensor output was stable, culture samples could be stored 10 days under −20 °C, and this sensing system was resistant to interferences by toxic aromatic pollutants. It was successfully applied to environmental samples even without extraction. The new whole-cell biosensing system provided a simple, stable, toxic pollutants-tolerant, and cost-effective tool for quantitative investigation of the QS signals’ role in environmental processes.     

Graphitized macroporous carbon microarray with hierarchical mesopores as host for the fabrication of electrochemical biosensor

A novel graphitized ordered macroporous carbon (GMC, pore size 380 nm) with hierarchical mesopores (2–30 nm) and high graphitization degree was prepared by nickel-catalyzed graphitization of polystyrene arrays. The obtained GMC possessed high specific surface area, large pore volume, and good electrical conductivity, which was explored for the enzyme entrapment and biosensor fabrication by a facile method. With advantages of novel nanostructure and good electrical conductivity, direct electrochemistry of hemoglobin (a model protein) was observed on the GMC-based biocomposite with a formal potential of −0.36 V (vs. Ag/AgCl) and an apparent heterogeneous electron transfer rate constant (k_s) of 1.2 s⁻¹ in pH 7.0 buffer. Comparative studies revealed that GMC offered significant advantages over carbon nanotubes (CNTs) in facilitating direct electron transfer of entrapped Hb. The fabricated biosensor exhibited good sensitivity (101.6 mA cm⁻² M⁻¹) and reproducibility, wide linear range (1–267 μM), low detection limit (0.1 μM), and good long-term stability for H₂O₂ detection. GMC proved to be a promising matrix for enzyme entrapment and biosensor fabrication, and may find wide potential applications in biomedical detection and environmental analyses.     

Oleanane-type triterpene oligoglycosides with pancreatic lipase inhibitory activity from the pericarps of Sapindus rarak

The methanolic extract from the pericarps of Sapindus rarak DC. was found to show pancreatic lipase inhibitory activity (IC₅₀ = ca. 614 μg/mL). From the extract, oleanane-type triterpene oligoglycosides, rarasaponins I–III (1–3), and raraoside A (4), were isolated together with 13 known saponins and four known sesquiterpene glycosides. Among them, several saponin constituents including rarasaponins I (1, IC₅₀ = 131 μM) and II (2, 172 μM), and raraoside A (4, 151 μM) inhibited pancreatic lipase activity, which were stronger than that of theasaponin E₁ (270 μM).     

Acrolein induces a cellular stress response and triggers mitochondrial apoptosis in A549 cells

Acrolein is a highly reactive, α,β-unsaturated aldehyde that is an omnipresent environmental pollutant. Humans are exposed to acrolein in food, vapors of overheated cooking oil, cigarette smoke and by combustion of organic products. Acrolein is a toxic by-product of lipid peroxidation resulting from oxidative stress, which is implicated in pulmonary, cardiac and neurodegenerative diseases. Low dose exposure to toxic compounds often leads to adaptive responses. If the adaptive response does not counteract the adverse exposure, death processes such as apoptosis will eliminate the cell. This study investigates the activation of antiapoptosis survival factors in relation to the induction of cell death by apoptosis, following exposure to low doses of acrolein, in A549 human lung cells. Exposure to acrolein (<15 μM, 30 min) activated the survival factor AKT, which led to phosphorylation of Bad and induction of antiapoptosis proteins cIAP1/2. Acrolein (10–50 μM, 30–60 min) increased reactive oxygen species and caused mitochondrial membrane hyperpolarisation. Inhibition by the antioxidants catalase, polyethylene glycol-catalase, sodium pyruvate and MnTBAP showed that acrolein-induced reactive oxygen species were responsible for mitochondrial membrane hyperpolarisation. Acrolein (3–27 μM, 30–60 min) activated early stage processes in the mitochondrial pathway of apoptosis, such as Bax translocation to mitochondria, cytochrome c release, caspase-9 activation, and translocation of apoptosis-inducing factor to the nucleus. Acrolein (10–50 μM) triggered later stage processes such as activation of caspases-3, -7 and -6, phosphatidylserine externalization and cleavage of poly(ADP)ribose polymerase after longer times (2 h). These events were inhibited by polyethylene glycol-catalase, showing that apoptosis was mediated by overproduction of reactive oxygen species by acrolein. The novel findings show that antiapoptosis processes dominate at low dose (<15 μM)/shorter exposure times to acrolein, whereas proapoptotic processes dominate at higher dose (10–50 μM)/longer exposure times. Acrolein induced apoptosis through the mitochondrial pathway that was mediated by reactive oxygen species.     

Development of an interference-free biosensor for l-glutamate using a bienzyme salicylate hydroxylase/l-glutamate dehydrogenase system

An amperometric biosensor was developed for the interference-free determination of l-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l-glutamate consuming NAD⁺. The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of l-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate with a detection limit of 3.0 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and electroactive substances.     

A Highly Sensitive and Selective Hydrogen Peroxide Biosensor Based on Gold Nanoparticles and Three-Dimensional Porous Carbonized Chicken Eggshell Membrane

A sensitive and noble amperometric horseradish peroxidase (HRP) biosensor is fabricated via the deposition of gold nanoparticles (AuNPs) onto a three-dimensional (3D) porous carbonized chicken eggshell membrane (CESM). Due to the synergistic effects of the unique porous carbon architecture and well-distributed AuNPs, the enzyme-modified electrode shows an excellent electrochemical redox behavior. Compared with bare glass carbon electrode (GCE), the cathodic peak current of the enzymatic electrode increases 12.6 times at a formal potential of −100mV (vs. SCE) and charge-transfer resistance decreases 62.8%. Additionally, the AuNPs-CESM electrode exhibits a good biocompatibility, which effectively retains its bioactivity with a surface coverage of HRP 6.39×10⁻⁹ mol cm⁻² (752 times higher than the theoretical monolayer coverage of HRP). Furthermore, the HRP-AuNPs-CESM-GCE electrode, as a biosensor for H₂O₂ detection, has a good accuracy and high sensitivity with the linear range of 0.01–2.7 mM H₂O₂ and the detection limit of 3μM H₂O₂ (S/N = 3).