Electrogenerated chemiluminescence aptamer-based method for the determination of thrombin incorporating quenching of tris(2,2-bipyridine)ruthenium by ferrocene

A novel electrogenerated chemiluminescence (ECL) aptamer-based biosensing method for the determination of thrombin was developed on basis of a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA capture probe, composing of a ss-DNA sequence to adopt two distinct structures-a DNA double strand with a complementary DNA sequence tagged with a ECL signal producer tris(2,2^′-bipyridyl)ruthenium derivative and a DNA duplex with a thrombin-binding aptamer tagged with a ECL-quencher ferrocene (FcDNA), was self-assembled onto surface of a gold electrode. In the presence of thrombin, the aptamer sequence prefers to form the aptamer-thrombin complex and the switch of the binding partners occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA. The integrated ECL intensity versus the concentration of thrombin was linear in the range from 2.0 × 10⁻¹⁰ M to 2.0 × 10⁻⁷ M. The detection limit was 6 × 10⁻¹¹ M. The relative standard derivation for 2.0 × 10⁻⁹ M was 5.7% (n = 7). This work demonstrates that the sensitivity and specificity of ECL aptamer-based method for proteins can be greatly improved using quenching ECL signal producer by a chemical quencher such as ferrocene.     

Electrogenerated chemiluminescence biosensor based on Ru(bpy)3(2+) and dehydrogenase immobilized in sol-gel/chitosan/poly(sodium 4-styrene sulfonate) composite material

A new electrogenerated chemiluminescence biosensor was fabricated by immobilizing ECL reagent Ru(bpy)₃²⁺ and alcohol dehydrogenase in sol-gel/chitosan/poly(sodium 4-styrene sulfonate) (PSS) organically modified composite material. The component PSS was used to immobilize ECL reagent Ru(bpy)₃²⁺ by ion-exchange, while the addition of chitosan was to prevent the cracking of conventional sol-gel-derived glasses and provide biocompatible microenvironment for alcohol dehydrogenase. Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection for quantification of enzyme substrate and it was much simpler than previous double-layer design. The detection limit was 9.3 × 10⁻⁶ M for alcohol (S/N = 3) with a linear range from 2.79 × 10⁻⁵ to 5.78 × 10⁻² M. With ECL detection, the biosensor exhibited wide linear range, high sensitivity and good stability.     

Determination of itopride hydrochloride by high-performance liquid chromatography with Ru(bpy)3 electrogenerated chemiluminescence detection

In this work, a stable electrogenerated chemiluminescence (ECL) detector was developed. The detector was prepared by packing cation-exchanged resin particles in a glass tube, followed by inserting Pt wires (working electrode) in this tube and sealing. The leakage of Ru(bpy)₃²⁺ can be compensated by adding a small amount of Ru(bpy)₃²⁺ into solution phase. Coupled with high-performance liquid chromatography separation, the detector has been used for determination of itopride hydrochloride in human serum. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of itopride hydrochloride in the range of 1.0 × 10⁻⁸ g mL⁻¹ to 1.0 × 10⁻⁶ g mL⁻¹ and the detection limit was 3 × 10⁻⁹ g mL⁻¹ (S/N = 3). The as-prepared ECL detector displayed good sensitivity and stability.     

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO₂NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)₃]^2+/3+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)₃]^2+/3+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10⁻¹⁵ to 1 × 10⁻⁸ mol L⁻¹. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL⁻¹ with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)₃]^2+/3+ interaction with ssDNA before and after hybridization.     

Intact and permeabilized cells of the yeast Hansenula polymorpha as bioselective elements for amperometric assay of formaldehyde

Intact and permeabilized yeast cells were tested as the biorecognition elements for amperometric assay of formaldehyde (FA). For this aim, the mutant C-105 (gcr1 catX) of the methylotrophic yeast Hansenula polymorpha with a high activity of AOX was chosen. Different approaches were used for monitoring FA-dependent cell response including analysis of their oxygen consumption rate by the use of a Clark electrode, as well as assay of oxidation of redox mediator at a screen-printed platinum electrode covered by cells entrapped in Ca-alginate gel. It was shown that oxygen consumption rate of permeabilized cells reached its saturation at 4 mM of FA (23 °C). The detection limit was found to be 0.27 mM. In the presence of redox mediator 2,6-dichlorophenolindophenol (DCIP), the screen-printed platinum band electrode covered by permeabilized cells did not show any current output to FA. In contrast, well-pronounced amperometric response to FA was observed in the case of intact yeast cells in the presence of DCIP. It was shown that current output reached its maximum at 7 mM concentration of FA. The detection limit was found to be 0.74 mM. Obviously, it is necessary to perform a directed genetic engineering of the yeast cells to improve their bioanalytical characteristics in the corresponding biosensors.     

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.     

A novel aptamer functionalized CuInS2 quantum dots probe for daunorubicin sensing and near infrared imaging of prostate cancer cells

In this paper, a novel daunorubicin (DNR)-loaded MUC1 aptamer-near infrared (NIR) CuInS₂ quantum dot (DNR–MUC1–QDs) conjugates were developed, which can be used as a targeted cancer imaging and sensing system. After the NIR CuInS₂ QDs conjugated with the MUC1 aptamer–(CGA)₇, DNR can intercalate into the double-stranded CG sequence of the MUC1–QDs. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of DNR on these conjugates. DNR–MUC1–QDs can be used to target prostate cancer cells. We evaluated the capacity of MUC1–CuInS₂ QDs for delivering DNR to cancer cells in vitro, and its binding affinity to MUC1-positive and MUC1-negative cells. This novel aptamer functionalized QDs bio-nano-system can not only deliver DNR to the targeted prostate cancer cells, but also can sense DNR by the change of photoluminescence intensity of CuInS₂ QDs, which concurrently images the cancer cells. The quenched fluorescence intensity of MUC1–QDs was proportional to the concentration of DNR in the concentration ranges of 33–88 nmol L⁻¹. The detection limit (LOD) for DNR was 19 nmol L⁻¹. We demonstrate the specificity and sensitivity of this DNR–MUC1–QDs probe as a cancer cell imaging, therapy and sensing system in vitro.     

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY_0.78F₄:Yb_0.2,Tm_0.02 UCNPs modified aptamer 1 and NaY_0.28F₄:Yb_0.70,Er_0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9964), and the signal was in the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL⁻¹ for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF₄ UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.     

One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

Herein, high quality DNA-CuInS₂ QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS₂ QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS₂ QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS₂ QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I₀ (I₀ is the original fluorescence intensity of DNA-CuInS₂ QDs, and I is the fluorescence intensity of DNA-CuInS₂ QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L⁻¹ with a detection limit of 0.013 nmol L⁻¹. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS₂ QDs also hold potential applications in bioimaging.     

Diterpenes and sesquiterpenes with anti-Coxsackie virus B3 activity from the stems of Illicium jiadifengpi

Seven new diterpenes, jiadifenoic acids J–P (1–7), two new sesquicarane sesquiterpenes, sesquicaranoic acids A and B (8 and 9), and four known compounds, were isolated from the stems of Illicium jiadifengpi. The trans-fused A/B ring junction in diterpenes was deduced from NMR data analysis and confirmed by single-crystal X-ray crystallography of 3. The absolute configurations of compounds 1 and 5 were determined using the Mo₂(OAc)₄-induced circular dichroism (ICD) method. Compounds 8 and 9 are a pair of C-10 epimers, and their absolute configurations were confirmed by analyzing their CD and ICD data. All of the isolated compounds were evaluated in vitro for their antiviral activities against Coxsackie virus B3 (CVB3). Among the isolated compounds, 4, 5, 7, 10, and 11 exhibited reasonable activity against CVB3, with IC₅₀ values of 7.0–22.2 μmol/mL and selective index values (SI=TC₅₀/IC₅₀) of 49.3, 37.1, 31.3, 45.6 and 40.9, respectively.     

Quick identification of apoptosis inducer from Isodon eriocalyx by a drug discovery platform composed of analytical high-speed counter-current chromatography and the fluorescence-based caspase-3 biosensor detection

Analytical high-speed counter-current chromatography (HSCCC), a unique liquid-to-liquid separation technology, has an inherent capability to provide perfect fractionation for tracking active ingredients of medicinal herbs, in a quick, efficient, and high-recovery manner. A high throughput screening (HTS) method which utilizes a novel biosensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique, was newly established and proved to be very sensitive in detecting apoptosis induced by various known anticancer drugs. The first combination of both advanced techniques formed an efficient platform for drug discovery and succeeded in quickly identifying the most potent apoptotic constituent of a Chinese herb namely Isodon eriocalyx. The system of n-hexane/ethyl acetate/methanol/water was used as the separation solvent. The solvent ratio was first set at 3:5:3:5 to check the water-soluble part of the crude extract, and then 1:1:1:1 was used to isolate the target compounds. The active fraction was tracked and purified continuously using HSCCC which was guided by the apoptosis detection at gradually decreased drug concentrations. As a result, the most potent apoptosis inducer in this herb was discovered by analytical HSCCC equipped with a 16 ml mini-coil column, using less than 50 ml diphase solvent, from about 50 mg active fraction. It was identified as eriocalyxin B, a well-known antitumor natural product, by NMR analysis of the HSCCC purified fraction.     

Pheromone synthesis. Part 244: Synthesis of the racemate and enantiomers of (11Z,19Z)-CH503 (3-acetoxy-11,19-octacosadien-1-ol), a new sex pheromone of male Drosophila melanogaster to show its (S)-isomer and racemate as bioactive

The enantiomers of (11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol were synthesized from the enantiomers of 3,4-epoxy-1-butanol PMB ether. Its racemate was also synthesized. Its (S)-isomer and racemate were shown to possess the same pheromone activity as CH503, a long-lived inhibitor of male courtship in Drosophila melanogaster, although the racemate was less active.     

Structure and magnetic properties of the orthorhombic n=2 Ruddlesden-Popper phases Sr3Co2O5+δ (δ=0.91, 0.64 and 0.38)

The reduced Ruddlesden-Popper phases, Sr₃Co₂O_5+δ with δ=0.91, 0.64 and 0.38, have been prepared in a nitrogen atmosphere. The crystal structures were determined by powder neutron diffraction. Oxygen vacancies are found both in O(3) and O(4) sites but the majority are along one crystallographic axis in the CoO₂ plane, inducing an orthorhombic distortion of the normally tetragonal n=2 Ruddelsden-Popper structure. Superstructures due to oxygen ordering are observed by electron microscopy. The magnetic measurements reveal complex behavior with some ferromagnetic interactions present for Sr₃Co₂O_5.91 and Sr₃Co₂O_5.64.     

Pheromone synthesis. Part 243: Synthesis and biological evaluation of (3R,13R,1′S)-1′-ethyl-2′-methylpropyl 3,13-dimethylpentadecanoate, the major component of the sex pheromone of Paulownia bagworm, Clania variegata, and its stereoisomers

All of the four stereoisomers of (1′S)-1′-ethyl-2′-methylpropyl 3,13-dimethylpentadecanoate, the major component of the female sex pheromone of Clania variegata, were synthesized by employing olefin cross metathesis as the key reaction and starting from (R)- or (S)-2-methyl-1-butanol, (R)- or (S)-citronellal, and (S)-2-methyl-3-pentanol. Their bioassay revealed the (3R,13R,1′S)-isomer as the bioactive one, whose more efficient synthesis was achieved in two different ways by employing Wittig reaction as the key step.     

Effects of Pressure and Temperature on the Formation of Tungsten-Bronze-Related Phases, RExWO3+y, with RE=La and Nd

The influence of pressure (P) and temperature (T) on the formation of tungsten-bronze-related phases containing lanthanum and neodymium was investigated. A large number of samples with bulk compositions RE_xWO₃, prepared by solid-state reaction in the pressure and temperature regions P= 10-80 kbar and T= 1170-1620 K were examined by X-ray powder diffraction and electron microscopy, and a (P-T) diagram showing the phase relations was drawn. Three tungsten-bronze-related phases with perovskite (PTB)-, hexagonal (HTB)- and intergrowth (ITB)-type structures were identified. The PTB bronze RE_xWO₃ with x≈ 0.10 was formed at p≤50 kbar. The HTB-related phase with x≈ 0.10 was observed in samples prepared at P≥20 kbar, whereas phases of (n)-ITB-type were observed only in the 25-50 kbar region. In the latter pressure region, the PTB and ITB phases were only seen in samples prepared at T > 1520 K, while the HTB-related phase was found in almost all samples. The HTB- and ITB-related compounds are metastable, probably fully oxidized, high-pressure phases of composition RE_xWO_3+3x/2 with x≤0.13. They transform to a cubic PTB bronze during annealing in inert atmosphere under ambient pressure conditions. According to microanalysis studies of individual crystals, less than 40% of the hexagonal tunnel sites in the HTB and ITB structures are occupied by RE³⁺ ions. A superstructure of HTB-type with ≈60% occupancy of the hexagonal tunnel sites (x≈0.20) was observed in a few crystals from the samples prepared at P= 80 kbar. Ordered, defect and intergrowth structures are presented.     

Synthesis and characterization of metal substituted AlxCr1−x(acetylacetonate)3 single-source precursors for their application to MOCVD of thin films

A detailed study, involving the synthesis of a single-source precursor containing two metal ions sharing the same crystallographic site, has been undertaken to elucidate the use of such a single-source precursor in a CVD process for growing thin films of oxides comprising these two metals, ensuring a uniform composition and distribution of metal ions. The substituted complexes Cr_1−xAl_x(acac)₃, where acac = acetylacetonate, have been prepared by a co-synthesis method, and characterized using UV–Vis spectroscopy, TGA/DTA measurements, and single crystal X-ray diffraction at low temperature. All the studied compositions crystallize in the monoclinic space group P2₁/c with Z = 4 in the unit cell. It was observed that the ratio (Al:Cr) of the site occupancy for the metal ions, obtained from single crystal refinement, is in agreement with the results obtained from complexometric titrations. All the solid state structures have the metal in an octahedral environment forming six-membered chelate rings. M–O acac bond lengths and disorder in the terminal carbon have been studied in detail for these substituted metal–organic complexes. One composition among these was chosen to evaluate their suitability as a single-source precursor in a LPMOCVD process (low-pressure metal–organic chemical vapour deposition) for the deposition of a substituted binary metal oxide thin film. The resulting thin films were characterized by X-ray diffraction, scanning electron microscopy, and infrared spectroscopy.     

Yb(OTf)3: an efficient catalyst for the synthesis of 3-arylbenzo [f]quinoline-1,2-dicarboxylate derivatives via imino-Diels-Alder reaction

A mild and facile method for the synthesis of 3-arylbenzo[f]quinoline-1,2-dicarboxylate derivatives is reported by an imino-Diels-Alder reaction in high yields (76-92%). This procedure includes a novel three-component reaction of aromatic aldehyde, naphthalen-2-amine, and but-2-ynedioate catalyzed by Yb(OTf)₃ in toluene. The results are obviously different from those of the simple substituted aniline-involved reactions catalyzed by ZnCl₂ or KOH in the literature.     

N-Chlorophthalimide as a mild and efficient chlorination reagent in the Gassman ortho alkylation of aromatic amines. Synthesis of 3-(methylthio)oxindoles

A practical modification of the Gassman 3-(methylthio)oxindole synthesis is reported. In our method, substituted anilines and 2-(methylthio)acetamide were reacted under mild reaction conditions, in the presence of N-chlorophthalimide as a chlorinating agent to give α-amidosulfides, which, in the next step of the process, were cyclized to give 3-(methylthio)oxindoles. The method was successfully applied for the synthesis of the key intermediate, 2-(2-amino-3-benzoylphenyl)-2-(methylthio)acetamide, in the process of the preparation of nepafenac, a commonly used ophthalmic drug.     

A simple and stereodivergent strategy for the synthesis of 3′-C-branched 2′,3′-dideoxynucleosides exploiting (Z)-but-2-en-1,4-diol and (R)-2,3-cyclohexylideneglyceraldehyde

Barbier type additions of allylic bromide 4, derived from (Z)-but-2-en-1,4-diol 2 to (R)-2,3-cyclohexylideneglyceraldehyde 1 were performed through mediation with Zn employing Luche’s procedure and also with low valent Cu, Co, and Fe which were produced via bimetal redox strategy in THF to afford 5c,d as the major products. From these, 5a,b were prepared following an oxidation-reduction protocol. Compound 5c was exploited as a representative starting material to develop a simple and inexpensive strategy toward the synthesis of 3′-C-branched 2′,3′-dideoxynucleosides having stereodiversity at 3′- and 4′-positions.     

FeCl3-SiO2 as a reusable heterogeneous catalyst for the synthesis of 5-substituted 1H-tetrazoles via [2+3] cycloaddition of nitriles and sodium azide

An efficient method for the preparation of 5-substituted 1H-tetrazole derivatives is reported using FeCl₃-SiO₂ as an effective heterogeneous catalyst. This method has the advantages of high yields, simple methodology, and easy work-up. The catalyst can be recovered by simple filtration and reused delivering good yields.