Analysis of Strychnos alkaloids in traditional Chinese medicines with improved sensitivity by sweeping micellar electrokinetic chromatography

A new amperometric method was developed for rapid detection of Escherichia coli (E. coli) density using a bienzyme biosensor. The bienzyme biosensor was fabricated based on the covalent immobilization of laccase and horseradish peroxidase (HRP) at indium tin oxide (ITO) electrode by (3-aminopropyl) triethoxysilane (APTES) monolayer. The bienzyme biosensor showed a high sensitivity in determination of the polyphenolic compounds, which was microbially generated from the salicylic acid (SA) added into the culture medium during the course of E. coli metabolism. Since the amount of polyphenolic compounds depends on E. coli density, the bienzyme biosensor was applied for the rapid and high sensitive detection of E. coli density after the E. coli solution was incubated in culture medium with salicylic acid for 2.5 h at 37 °C. By chronoamperometry, the amplified response current was obtained at the bienzyme biosensor, due to the substrate recycling of the polyphenolic compounds driven by bienzyme-catalyzed oxidation and electrochemical reduction. The amplified response current at the biosensor was linear with the E. coli density ranging from 1.6 × 10³ to 1.0 × 10⁷ cells/mL. The bienzyme biosensor could detect the E. coli density with a detection limit of 9.7 × 10² cells/mL within 3 h.     

Use of bioluminescent bacterial sensors as an alternative method for measuring heavy metals in soil extracts

The luminescence based bacterial sensor strains Pseudomonas fluorescens OS8 (pTPT11) for mercury detection and Pseudomonas fluorescens OS8 (pTPT31) for arsenite detection were used in testing their application in detecting heavy metals in soil extracts. Three different soil types (humus, mineral and clay) were spiked with 1, 100 or 500 μg g⁻¹ Hg²⁺ or As³⁺. Samples were taken 1, 14 and 30 days and extracted with water, ammonium acetate, hydrogen peroxide and nitric acid to represent water soluble, bioavailable, organic matter bound and residual fractions, respectively. The lowest mercury-concentration measured using biosensor (0.003 μg kg⁻¹) was considerably lower than by chemical method (0.05 μg kg⁻¹). The sensor strain with pTPT31 appeared to have a useful detection range similar to that of chemical methods. Concentration results with chemical and biosensor analysis were very similar in the case of mercury-spiked samples. Although some of the arsenite samples showed higher variation between methods, it is concluded that the bacteria can be used as an alternative traditional methods for different types of samples.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring

A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM⁻¹ (RSD = 6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02–0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7 h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4 °C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.     

A nanosized Ag-silica hybrid complex prepared by γ-irradiation activates the defense response in Arabidopsis

Silver nanoparticles have antimicrobial activity against many pathogenic microbes. Here, the preparation of a nanosized Ag-silica hybrid complex (NSS) prepared by γ-irradiation is described. The effects of both NSS and reduced Ag nanoparticles (Ag⁰) on the growth of the model plant Arabidopsis thaliana were tested. The application of 1-10 ppm NSS complex improved Arabidopsis growth in soil, whereas 100 ppm NSS resulted in weakly curled leaves. In addition, supplementation of Murashige and Skoog (MS) growth medium with 1 ppm NSS promoted the root growth of Arabidopsis seedlings, but root growth was inhibited by supplementation with 10 ppm NSS. To investigate whether the NSS complex could induce plant defense responses, the expression of pathogenesis-related (PR) genes that are implicated in systemic acquired resistance (SAR) in Arabidopsis plants was examined. PR1, PR2 and PR5 were significantly up-regulated by each application of 10 ppm NSS complex or Ag⁰ to the rosette leaves. Furthermore, pretreatment with the NSS complex induced more pathogen resistance to the virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) compared to water treatment in Arabidopsis plants.     

Electrochemiluminescence biosensor for the assay of small molecule and protein based on bifunctional aptamer and chemiluminescent functionalized gold nanoparticles

An electrochemiluminescence (ECL) biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs) was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of N-(aminobutyl)-N-(ethylisoluminol) enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of 5.0 × 10⁻¹²–5.0 × 10⁻⁹ M with a detection limit of 2.2 × 10⁻¹² M. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer–target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from 5 × 10⁻¹⁴ M to 5 × 10⁻¹⁰ M with a detection limit of 1.2 × 10⁻¹⁴ M. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The sensitivity of the present biosensor is superior to most available aptasensors for adenosine and thrombin. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples.     

A reagentless DNA biosensor based on cathodic electrochemiluminescence at a C/CxO1−x electrode

A reagentless signal-on electrochemiluminescence (ECL) biosensor for DNA hybridization detection was developed based on the quenching effect of ferrocene (Fc) on intrinsic cathodic ECL at thin oxide covered glassy carbon (C/C_xO_1−x) electrodes. To construct the DNA biosensor, molecular beacon (MB) modified with ferrocene (3′-Fc) was attached to a C/C_xO_1−x electrode via the covalent bound between labeled amino (5′-NH₂) and surface functional groups. It was found that the immobilization of the probe on the electrode surface mainly depended on the fraction of surface carbonyl moiety. When a complementary target DNA (cDNA) was present, the stem-loop of MB on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the moving away of Fc from the electrode surface, and the restoring of the cathodic ECL signal. The restoration of the ECL intensity was linearly changed with the logarithm of cDNA concentration in the range of 1.0 × 10⁻¹¹ to 7.0 × 10⁻⁸ M, and the detection limit was ca. 5.0 pM (S/N = 3). Additionally, single-base mismatched DNA can be effectively discriminated from the cDNA. The great advantage of the biosensor lies in its simplicity and cost-effective with ECL generated from the electrode itself, and no adscititious luminophore is required.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

Lissoclibadin 1, a novel trimeric sulfur-bridged dopamine derivative, from the tropical ascidian Lissoclinum cf. badium

A novel tribenzotetrathiepin alkaloid, named lissoclibadin 1 (1), has been isolated from the ascidian Lissoclinum sp. (cf. L. badium Monniot and Monniot, 1996). The gross structure was assigned on the basis of the spectral data, and one of two possible isomers was selected by the computational modeling study. Lissoclibadin 1 inhibited the growth of the marine bacterium Ruegeria atlantica (15.2 mm at 20 μg/disk).     

A surface plasmon resonance biosensor for detecting Pseudomonas aeruginosa cells with self-assembled chitosan-alginate multilayers

A self-assembled multilayer (SAMu) including the alginate layer was prepared for detecting Pseudomonas aeruginosa cells in a solution and its potential was evaluated with a BIAcore system. After layer-by-layer formation, the refractive units (RU) values monitored with the biosensor increased by the interaction between the layers. The responses by the binding of P. aeruginosa cells to the alginate-immobilized SAMu were visualized immediately upon injection of the cell suspension. The RU values after injection of the cells were measured with approximately 1152, 656 and 173 for 1 × 10⁹, 1 × 10⁸ and 1 × 10⁷ CFU/ml. This result suggests that the alginate-immobilized SAMu will have useful application for detecting P. aeruginosa cells in a biosensor analysis.     

Antiplasmodial and antiviral cyclohexadepsipeptides from the endophytic fungus Pullularia sp. BCC 8613

Four new cyclohexadepsipeptides, pullularins A-D, were isolated from the endophytic fungus Pullularia sp. BCC 8613. Structures of these compounds were elucidated by interpretation of NMR spectroscopic and mass spectrometric data. The absolute configurations of amino acid and hydroxy acid residues were determined by HPLC analysis of depsipeptide acid hydrolyzates using a chiral column and Marfey's method. Pullularin A exhibited activities against the malarial parasite Plasmodium falciparum K1 (IC₅₀ 3.6 μg/mL) and herpes simplex virus type 1 (HSV-1; IC₅₀ 3.3 μg/mL), whereas it showed weak cytotoxicity to Vero cells (IC₅₀ 36 μg/mL).     

High dielectric constant PrYxOy sensing films electrolyte-insulator-semiconductor pH-sensor for the detection of urea

In this paper, we describe the structural and sensing properties of high-k PrY_xO_y sensing films deposited on Si substrates through reactive co-sputtering. Secondary ion mass spectrometry and atomic force microscopy were employed to analyze the compositional and morphological features of these films after annealing at various temperatures. The electrolyte-insulator-semiconductor (EIS) device incorporating a PrY_xO_y sensing membrane that had been annealed at 800 °C exhibited good sensing characteristics, including a high sensitivity (59.07 mV pH⁻¹ in solutions from pH 2 to 12), a low hysteresis voltage (2.4 mV in the pH loop 7 → 4 → 7 → 10 → 7), and a small drift rate (0.62 mV h⁻¹ in the buffer solution at pH 7). The PrY_xO_y EIS device also showed a high selective response towards H⁺. This improvement can be attributed to the small number of crystal defects and the large surface roughness. In addition, the enzymatic EIS-based urea biosensor incorporating a high-k PrY_xO_y sensing film annealed at 800 °C allowed the potentiometric analysis of urea, at concentrations ranging from 1 to 16 mM, with a sensitivity of 9.59 mV mM⁻¹.     

Belamide A, a new antimitotic tetrapeptide from a Panamanian marine cyanobacterium

The isolation and structure elucidation of belamide A from the marine cyanobacterium Symploca sp. is described. Belamide A is a highly methylated linear tetrapeptide with structural analogy to the important linear peptides dolastatins 10 and 15. Disruption of the microtubule network in A-10 cells was observed at 20 μM and displayed classic tubulin destabilizing antimitotic characteristics. The moderate cytotoxicity of belamide A (IC₅₀ 0.74 μM vs HCT-116 colon cancer line) provides new insights into structure-activity relationships for this drug class.     

Detection of P. aeruginosa using nano-structured electrode-separated piezoelectric DNA biosensor

A DNA biosensor for detection of Pseudomonas aeruginosa was set up based on the modification of two membranes (nano-TiO₂ and nano-TiO₂-polyethylene glycol hybrid membrane) to the ESPS surface. These two membrane materials were synthesized by sol-gel method. The detection was accomplished by modifying ss-DNA on the sensitive membrane and then hybridizing with their complementary strands from the P. aeruginosa in liquid phase. UV spectrum was used to identify the purity and concentration of extracted DNA; IR spectrum and SEM were used to characterize the properties of the membrane. The detection was highly improved by adoption of nanotechnology and hybrid membrane. Less than 3 h was sufficient. The detection linear range was from 10⁻¹ to 10⁻³ g l⁻¹ and the limit of detection was 10⁻⁴ g l⁻¹.     

Banchromene and other secondary metabolites from the endophytic fungus Fusarium sp. obtained from Piper guineense inhibit the motility of phytopathogenic Plasmopara viticola zoospores

A new chromene, (S)-banchromene (1), together with seven known compounds, ergosterol, beauvericin (2), fusaproliferin (3), radicinin (4), poly(3-hydroxybutyric acid) (PHB, 5), N-methylpyrrolidone and an inseparable mixture of isochromene derivatives 6a, 6b, were isolated from a culture of Fusarium sp. strain CAMKT24b1, an endophytic fungus from the leaves and twigs of Piper guineense (Piperaceae). The structures of these metabolites were elucidated on the basis of their spectroscopic data; the absolute configuration of 1 was determined by ab initio-calculation of the optical rotation. In tests with the zoospores of the grapevine downy mildew pathogen Plasmopara viticola, compounds 1–4 showed moderate to high levels of motility-impairing activity at concentrations as low as 2.5 μg/mL. Compound 2 was the most active, exhibiting both motility-halting and lytic activities. Furthermore, compounds 2 and 3 displayed significant cytotoxic activity against brine shrimp larvae (Artemia salina) at 10 μg/mL. This is the first report on motility inhibitory and lytic activities of metabolites from an endophytic Fusarium species against the zoospores of the downy mildew pathogen P. viticola.     

Hydroxylation of ionized aromatics including carboxylic acid or amine using recombinant Streptomyces lividans cells expressing modified biphenyl dioxygenase genes

The bphA1(2072)A2A3A4 gene cluster codes for a shuffled biphenyl dioxygenase holoenzyme with broad substrate specificity. These bphA1(2072)A2A3A4 genes were expressed in the actinomycetes Streptomyces lividans using a thiostrepton-inducible promoter P_tipA. Biotransformation experiments of various aromatics including carboxylic acid or amine in their molecular structure, such as 1-naphthoic acid, 2-(1-naphthyl)acetic acid, diphenylamine, and 1-benzyl-4-piperidone, were performed using the recombinant S. lividans cells. These ionized aromatics were converted to the corresponding 1,2-dihydrodiol, mono- or tri-hydroxy forms in 48 h. The structure of the converted products was determined by their EI-MS, ¹H- and ¹³C NMR analysis, and several products were found to be novel compounds.     

An amperometric biosensor based on peroxidases from Brassica napus for the determination of the total polyphenolic content in wine and tea samples

An amperometric biosensor based on peroxidases from Brassica napus hairy roots (PBHR) used to determine the total polyphenolic content in wine and tea samples is proposed by the first time. The method employs carbon paste (CP) electrodes filled up with PBHR, ferrocene (Fc), and multi-walled carbon nanotubes embedded in a mineral oil (MWCNT + MO) at a given composition (PBHR-Fc-MWCNT + MO). The biosensor was covered externally with a dialysis membrane, which was fixed at the electrode body side part with a Teflon laboratory film and an O-ring. Calibration curves obtained from steady-state currents as a function of the concentration of a polyphenolic standard reference compound such as t-resveratrol (t-Res) or caffeic acid (CA) were then used to estimate the total polyphenolic content in real samples. The reproducibility and the repeatability were of 7.0% and 4.1% for t-Res (8.4% and 5.2% for CA), respectively, showing a good biosensor performance. The calibration curves were linear in a concentration range from 0.05 to 52 mg L⁻¹ and 0.06 to 69 mg L⁻¹ for t-Res and CA, respectively. The lowest polyphenolic compound concentration values measured experimentally for a signal to noise ratio of 3:1 were 0.023 mg L⁻¹ and 0.020 mg L⁻¹ for t-Res and CA, respectively.     

Surface plasmon resonance detection of oligonucleotide sequences of the rpoB genes of Mycobacterium tuberculosis

Oligonucleotide sequences related to the normal and mutated rpoB genes of Mycobacterium tuberculosis are detected using a surface plasmon resonance (SPR) biosensor system. A bioselective element was prepared by immobilizing the thiol-modified oligonucleotides of the selected sequence (the capture probe P2) that contains the mutated TCG → TTG codon 531 (evoking drug resistance) of the rpoB gene of M. tuberculosis on a gold sensor surface. Specific hybridization between immobilized probe P2 and complementary target T2 gave the highest sensor response, single-base mismatched oligonucleotide TN (corresponding to the normal gene sequence) produced somewhat smaller response and no response was observed at injection of noncomplementary oligonucleotide TC. The P2-T2 hybridization efficiency is calculated ca. 30% (5 × 10¹² molecules cm⁻²), and the lowest detection limit of T2 was 10 nM. An extended T2E oligonucleotide sequence consisting of T2 sequence and additional 24 nucleotides was shown to cause more pronounced sensor response (at least 5 nM T2E was easily detected). Injection into the sensor cell of the oligonucleotides complementary to the free additional part of T2E after P2-T2E hybridization gave a significant additional SPR response, thus showing that the sandwich hybridization format further improves the sensor sensitivity and decreases the lowest detection limit. The experimental results on surface hybridization between the studied oligonucleotides were in good agreement with thermodynamic parameters of the hybridization calculated for solution conditions. The described approach could be proposed as a basis for creating a biosensor for real-time and label-free diagnostics of drug resistant tuberculosis.     

An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18

In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 × 10⁴ cells and 9.86 × 10⁴ cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.     

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10⁻⁷ M), short response time (within 10 s), high sensitivity (88.6 μA mM⁻¹ cm⁻²) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H₂O₂ by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083 ± 0.001) ×10⁻³ M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.