Nanostructured cantilevers as nanomechanical immunosensors for cytokine detection

The use of microfabricated cantilevers as immunosensors for the detection of human interleukin-1 beta (HIL1-β), one of the proinflammatory cytokines, is demonstrated. Cytokines are important biomarkers to monitor the effect of drugs influencing the immune system or inflammation. Nanostructured microcantilevers (MCs) functionalized with anti-human interleukin-1 beta (anti HIL1-β) antibody (Ab) are used for achieving immunonanome-chanical responses. Antibody-antigen binding interaction induces an apparent surface stress, thereby causing static bending of the MC that is detected in this work by an optical beam bending technique. Specific binding of HIL1-β to surface-bound Ab produces substantial compressive surface stress, which enables its detection in presence of other interfering proteins. Combination of biospecificity of antibodies with the sensitivity of nanomechanical surface stress detection using MCs with one side nanostructured allows detection of cytokine HIL1-β in the ppb range. The nanostructured immuno-MCs, when compared to the smooth surface MC, exhibit higher responses and yet better reversibility. The detection of HIL1-β shows good measurement reproducibility (CV=10%) in the same day tested via three replicate consecutive measurements of a solution of 0.5 ppm of HIL1-β. Also, a reasonable stability of the Ab functionalized MC within 7 d of functionalization is observed. It can be envisioned that differentially functionalized MC arrays can be made in a similar fashion.     

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

Small-scale upstream bioprocess development often occurs in flasks and multi-well plates. These culturing platforms are often not equipped to accurately monitor and control critical process parameters; thus they may not yield conditions representative of manufacturing. In response, we and others have developed optical sensors that enable small-scale process monitoring. Here we have compared two parameters critical to control in industrial cell culture, pH and dissolved oxygen (DO), measured with our optical sensors versus industrially accepted electrochemical probes. For both optical sensors, agreement with the corresponding electrochemical probe was excellent. The Pearson Correlations between the optical sensors and electrochemical probes were 98.7% and 99.7%, for DO and pH, respectively. Also, we have compared optical pH sensor performance in regular (320 mOsm/kg) and high-osmolality (450 mOsm/kg) cell culture media to simulate the increase in osmolality in pH-controlled cultures. Over a pH range of 6.38-7.98 the average difference in pH readings in the two media was 0.04 pH units. In summary, we have demonstrated that these optical sensors agree well with standard electrochemical probes. The accuracy of the optical probes demonstrates their ability to detect potential parameter drift that could have significant impact on growth, production kinetics, and protein product quality. We have also shown that an increase in osmolality that could result from controlling pH or operating the reactor in fed-batch mode has an insignificant impact on the functionality of the pH patches.     

High-pressure liquid chromatographic-reductive electrochemical detection analysis of serum trans-phylloquinone

A sensitive method for the determination of human serum trans-phylloquinone levels has been developed. Serum samples were extracted with hexane and subjected to preliminary separation on a silica semipreparative HPLC column with an 80% recovery as determined by the addition of [³H]phylloquinone. The portion of the eluate containing trans-phylloquinone was dried and injected into a μBondapak C-18 analytical HPLC column, and the concentration of the vitamin was determined by reductive electrochemical detection utilizing a glassy carbon electrode and and $\text{Ag}\text{AgCl}$ reference electrode. The presumed vitamin peak was confirmed as trans-phylloquinone by rechromatography at different HPLC conditions, hydrodynamic voltamography, and photodegradation. As little as 300 pg/ml rans-phylloquinone could be detected, and normal human serum concentrations were found to be in the range of 1 ng/ml.     

Electrochemical profiles of Herba Saussureae Involucratae by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection method has been developed for the determination of the pharmacologically active ingredients, acacetin, rutin, umbelliferone, kaempferol, apigenin, luteolin and quercetin, in Herba Saussureae Involucratae. Under optimum conditions, the seven analytes could be completely separated within 19 min in a 75 cm length capillary at a separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2). A 300 microm diameter carbon disk electrode, positioned opposite the outlet of the capillary in a wall-jet configuration at a potential of +950 mV (vs a saturated calomel electrode) was used as the working electrode. A good linear relationship was established between peak current and concentration of the analytes over two orders of magnitude with detection limits (signal-to-noise ratio = 3) ranging from 1.2 x 10(-7) to 4.1 x 10(-8) g/mL for all analytes. The proposed method has been successfully applied to the analyses of bio-active components of Herba Saussureae Involucratae samples after a relatively simple extraction procedure. The assay results show that the resultant electrochemical profiles are indicative of the content diversity of each electrochemically active ingredient in the various samples, and may also offer some evidence for phytotaxonomy.     

电化学生物传感器快速检测DNA研究进展

本简要地介绍了DNA电化学生物传感器研究的最新进展,重点讨论了改善生物传感器选择性和灵敏度的技术和方法。     

Gastric juice,gastric tissue and blood antibiotic concentrations following omeprazole,amoxicillin and clarithromycin triple therapy

Background. Amoxicillin and clarithromycin are key antibiotics in proton pump inhibitor‐based Helicobacter pylori eradication therapies. Aims. To study gastric mucus and tissue concentrations and collect basic data about optimal antibacterial doses. Methods. Plasma, gastric mucosa and gastric juice antibiotic concentrations were measured following either low‐ or high‐dose amoxicillin (750 or 1000 mg bid) and clarithromycin (400 or 500 mg bid) given in combination with omeprazole 20 mg bid to 12 male volunteers in an open crossover design. Gastric juice and mucosal biopsy collection was performed either 2 (n = 6) or 6 hours (n = 6) after dosing. Results. Amoxicillin concentrations 2 hours after high dosage were gastric juice > gastric body > antral mucosa > plasma. At 6 hours, plasma and gastric juice concentrations were still above the MIC for amoxicillin‐susceptible bacteria but no antibiotic was detectable in mucosa samples. Clarithromycin concentrations after high dosage were gastric juice > mucosa > serum; all above the MIC for clarithromycin‐susceptible bacteria at both 2 and 6 hours. Conclusions. Both dosage regimens provided effective antibiotic concentrations in gastric juice at 2 hours. After dosing, both antibiotics demonstrated high gastric tissue concentrations via local diffusion while clarithromycin also provided sustained delivery (6 hours) via gastric mucosa penetration.     

Antimicrobial screening involving Helicobacter pylori of nano-therapeutic compounds based on the amoxicillin antibiotic drug

Nano-structure Cu(II) complex [Cu(AMAB)₂]Cl₂ with Schiff base (AMAB) derived from the condensation between 4-(dimethylamino)benzaldehyde and amoxicillin trihydrate was prepared. (AMAB) Schiff base and its Cu(II) complex were identified and confirmed by different physicochemical techniques. The Schiff base (AMAB) was coordinated to copper ion through carbonyl oxygen and imine nitrogen donor sites. X-ray powder diffraction shows a cubic crystal system of the Cu(II) complex. The density functional theory was used to optimize the structure geometries of the investigated compounds. The molecular docking of the active amino acids of the investigated proteins' interactions with the tested compounds was evaluated. The bactericidal or bacteriostatic effect of the compounds was screened against some bacterial strains. The activity of Cu-chelate against Gram-negative bacteria was mainly more effective than its (AMAB) ligand and vice versa in the case of Gram-positive bacteria. The biological activity of the prepared compounds with biomolecules calf thymus DNA (CT-DNA) was determined by electronic absorption spectra and DNA gel electrophoresis technique. All studies revealed that the Cu-chelate derivative exhibited better binding affinity to both CT-DNA than the AMAB and amoxicillin itself. The anti-inflammatory effect of the designed compounds was determined by testing their protein denaturation inhibitory activity spectrophotometrically. All obtained data supported that the designed nano-Cu(II) complex with Schiff base (AMAB) is a potent bactericide against H. pylori, and exhibits anti-inflammatory activity. The dual inhibition effects of the designed compound represent a modern therapeutic approach with extended spectrum of action. Therefore, it can act as good drug target in antimicrobial and anti-inflammtory therapies. Finally, H. pylori resistance to amoxicillin is absent or rare in many countries, thus amoxicillin nanoparticles may be beneficial for countries where amoxicillin resistance is reported.     

Determination of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) activity by high-performance liquid chromatography and electrochemical detection

The tripeptide glutathione (gamma-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of gamma-glutamylcysteine (gamma-GC), the product of GCL activity. This method allows for the direct detection of gamma-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 +/- 1.7 nmol gamma-GC synthesized/min/mg protein.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Determination of cholecystokinin tetrapeptide and cholecystokinin octapeptide sulfate in different rat brain regions by high-pressure liquid chromatography with electrochemical detection

A method for the determination of cholecystokinins in biological material, based on high-pressure liquid chromatography with direct electrochemical detection (HPLC-EC), is described. Using this method, the levels of cholecystokinin tetrapeptide and octapeptide sulfate in rat brain cortex, hippocampus, striatum, and brain stem were measured and found to be comparable to those reported using radioimmunoassay methods. We show that HPLC-EC is sensitive enough to accurately determine neuropeptides in brain tissue without prior derivatization and is therefore, due to its simplicity, an attractive alternative to existing methods.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Fluorescence‐based sensor for selective and sensitive detection of amoxicillin (Amox) in aqueous medium: Application to pharmaceutical and biomedical analysis

We here for the first time demonstrate an analytical approach for the highly selective and sensitive detection of amoxicillin (Amox) in aqueous medium based on the fluorescence quenching of quantum dots (QDs). The change in fluorescence intensity of mercaptopropionic acid‐capped cadmium sulphide (MPA‐CdS) QDs is attributed to the increasing concentration of Amox. The results show that the fluorescence quenching of QDs by Amox takes place through both static and dynamic types of quenching mechanism. The fluorescence quenching of QDs with increase in concentration of Amox shows the linear range between 5 μg ml^?1 and 30 μg ml^?1 and the limit of detection (LOD) is 5.19 μg ml^?1. There is no interference of excipients, which are commonly present in pharmaceutical formulation and urine samples. For the practical application approach, the developed method has been successfully applied for the determination of Amox in pharmaceutical formulations and urine samples with acceptable results.     

Detection and identification of optical activity using polarimetry – applications to biophotonics,biomedicine and biochemistry

Polarized light that is reflected or transmitted through chiral specimens can be used to detect and identify biological and chemical materials including human tissue. The determination of the silent footprints of the chiral properties of the biological materials on scattered polarized light is the basis for these investigations. It is of primary importance to identify which combinations of the elements of the Mueller matrix for reflected or for transmitted light can be used to determine the optical activity of the biochemical materials. The optical activity of chiral materials is characterized by optical rotation and circular dichroism. The explicit analytical dependence of these specific elements of the Mueller matrix, upon the angles of incidence and scatter, upon the wavelength and upon the type of chirality has the potential to provide experimentalists with guidance in determining the optimum use of optical polarimetric scatterometers. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Smart and intelligent optical materials for sensing applications

This special issue (SI) entitled ‘Smart and intelligent optical materials for sensing applications’, published by Luminescence, Wiley focuses on the recent advancement of smart and intelligent optical materials for the fabrication of sensor technology for use in numerous fields such as pharmaceutical, biomedical, and environmental. Also, detailed highlights of their prospects in the fields, for example, of personalized health care, wearable devices, and plant stress monitoring are given. This SI includes 46 peer-reviewed articles, of which 15 are reviews written by well established researchers with expertise in the field, and the remaining 31 are research articles from world-leading scientists.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

Determination of N7-(2-hydroxyethyl)guanine by HPLC with electrochemical detection

A method has been developed for the determination of N7-(2-hydroxyethyl)guanine (N7-EtOHGua) via HPLC with electrochemical detection (EC). N7-EtOHGua is the major base adduct formed in DNA upon exposure to ethylene oxide. N7-EtOHGua, released from DNA, was separated from the unmodified nucleobases by chromatography on a reversed-phase column. For electrochemical detection, an amperometric detector cell was used with a glassy carbon working electrode, set at 1.35 V relative to an Ag/AgCl reference electrode. With purified N7-EtOHGua a linear dose-response relation was observed in the range between 0.11 and 13 pmol. The signal-to-noise ratio during analysis of 0.11 pmol N7-EtOHGua was about 8 to 1. Determination of adducts in a series of DNA samples treated with 0.16–10 mM ethylene oxide showed a linear dose-dependent increase in the level of N7-modifications. For DNA samples, the detection limit of this HPLC-EC analysis is 1 N7-EtOHGua per 6 × 10⁶ nucleotides.     

抗生素抗性基因在环境中的传播扩散及抗性研究方法

抗生素在医药、畜牧和水产养殖业的大量使用造成了环境中抗性耐药菌和抗性基因日益增加,抗生素抗性基因作为一种新型环境污染物引起人们的广泛关注.本文综述了近年来国内外有关抗生素抗性基因的研究进展,其在水、土壤、空气等环境介质中和动,植物体内的传播扩散,以及开展环境中抗生素抗性基因研究的必要性,重点介绍了有关抗生素抗性（包括抗性细菌和抗性基因）的研究方法,指出抗性基因研究中存在的问题,并对未来的相关研究进行了展望.     

Recent advances in nanomaterials-based optical sensors for detection of various biomarkers (inorganic species,organic and biomolecules)

This review briefly emphasizes the different detection approaches (electrochemical sensors, chemiluminescence, surface-enhanced Raman scattering), functional nanostructure materials (quantum dots, metal nanoparticles, metal nanoclusters, magnetic nanomaterials, metal oxide nanoparticles, polymer-based nanomaterials, and carbonaceous nanomaterials) and detection mechanisms. Furthermore, the emphasis of this review is on the integration of functional nanomaterials with optical spectroscopic techniques for the identification of various biomarkers (nucleic acids, glucose, uric acid, oxytocin, dopamine, ascorbic acid, bilirubin, spermine, serotonin, thiocyanate, Pb²⁺, Cu²⁺, Hg²⁺, F⁻, peptides), and cancer biomarkers (mucin 1, prostate specific antigen, carcinoembryonic antigen, CA15-3, human epidermal growth factor receptor 2, C-reactive protein, and interleukin-6). Analytical characteristics of nanomaterials-based optical sensors are summarized in the tables, providing the insights of nanomaterials-based optical sensors for biomarkers detection. Finally, the opportunities and challenges of nanomaterials-based optical analytical approaches for the detection of various biomarkers (inorganic, organic, biomolecules, peptides and proteins) are discussed.     

Tapered plastic optical fiber-based biosensor--tests and application

Cells detection is crucial in microbiological analysis of clinical, food, water or environmental samples. However, currently employed methods are time consuming. Plastic optical fiber (POF) biosensors consist in a viable alternative for rapid and inexpensive scheme for detection. In order to study the sensitivity of tapers for microbiological detection, geometric parameters are studied, such as the taper waist diameter since the formation of taper regions are the key sensing element in this particular type of sensors. In this study, a series of POF taper sensors were prepared using a specially developed tapering machine, and the dispersion of geometric dimensions is evaluated, aiming to achieve the best tapering characteristics which will provide a better sensitivity on the sensor response. The fiber tapers that presented the finest results were those constructed in U-shaped (bended) configurations, with taper waist diameters ranging from 0.40 mm up to 0.50 mm. These fiber tapers were used as the main section of the monitoring device, and when chemically treated as immunosensors for the detection of bacteria, yeast and erythrocytes.