Rectangular coordination polymer nanoplates: large-scale, rapid synthesis and their application as a fluorescent sensing platform for DNA detection

In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs) assembled from Cu(II) and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1) RCPN binds dye-labeled single-stranded DNA (ssDNA) probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2) Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA) which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully.     

Layer-by-layer self-assembly of functionalized graphene nanoplates for glucose sensing in vivo integrated with on-line microdialysis system

In this work, a novel amperometric biosensor for hydrogen peroxide was fabricated through the layer-by-layer (LBL) self-assembling of amine-terminated ionic liquid (IL-NH(2)), and sulfonic acid (SO(3)(-)) functionalized graphene by covalent bonding. The modification of the two functionalities introduced positive and negative charge onto the surface of graphene respectively, thus facilitating the formation of a multilayer film denoted with {IL-RGO/S-RGO}(n) through electrostatic interaction and further immobilization of glucose oxidase (GOx). The resulting {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor displayed an excellent response to glucose at a potential of -200 mV. Combined with on-line microdialysis system, the glucose biosensor in the on-line system showed good linear range from 10 μM to 500 μM with the detection limit of 3.33 μM (S/N=3). Consequently, the basal level of glucose in the striatum of anesthetic rats was calculated to be 0.376 ± 0.028 mM (mean ± s.d., n=3). The {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor was further applied for in vivo sensing of the glucose level in the striatum when rats received intraperitoneal (i.p.) injection of 30 μL insulin, which resulted in an obvious decrease in the extracellular concentration of glucose within 30 min. The method was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.     

Biosynthesis of flower-shaped Au nanoclusters with EGCG and their application for drug delivery

Background

In the last decade, the biosynthesis of metal nanoparticles using organisms have received more and more considerations. However, the complex composition of organisms adds up to a great barrier for the characterization of biomolecules involved in the synthesis process and their biological mechanisms.

Results

In this research, we biosynthesized a kind of flower-shaped Au nanoclusters (Au NCs) using one definite component—epigallocatechin gallate (EGCG), which was the main biomolecules of green tea polyphenols. Possessing good stability for 6 weeks and a size of 50 nm, the Au NCs might be a successful candidate for drug delivery. Hence, both methotrexate (MTX) and doxorubicin (DOX) were conjugated to the Au NCs through a bridge of cysteine (Cys). The introduction of MTX provided good targeting property for the Au NCs, and the conjugation of DOX provided good synergistic effect. Then, a novel kind of dual-drug loaded, tumor-targeted and highly efficient drug delivery system (Au-Cys-MTX/DOX NCs) for combination therapy was successfully prepared. The TEM of HeLa cells incubated with Au-Cys-MTX/DOX NCs indicated that the Au-Cys-MTX/DOX NCs could indeed enter and kill cancer cells. The Au-Cys-MTX/DOX NCs also possessed good targeting effect to the FA-receptors-overpressed cancer cells both in vitro and in vivo. Importantly, the Au-Cys-MTX/DOX NCs resulted in an excellent anticancer activity in vivo with negligible side effects.

Conclusions

These results suggest that the biosynthesized Au-Cys-MTX/DOX NCs could be a potential carrier with highly efficient anticancer properties for tumor-targeted drug delivery.

     

Reproducible fabrication of miniaturized glucose sensors: preparation of sensing membranes for continuous monitoring.

The immobilization process of glucose oxidase(GOx) in the poly(1,3-diaminobenzene) (poly(1,3-DAB)) network was closely investigated in situ using an electrochemical quartz crystal microbalance(EQCM). GOx captured in approximately 50 nm thick poly-1,3-DAB layer causes a 514 Hz frequency increase, corresponding to 541 ng, and distributes mostly in the outer part of the polymer film. The presence of poly-L-lysine and glutaraldehyde during electropolymerization of poly(1,3-DAB) improves sensitivity by raising the amount of GOx immobilized. Adding a protective membrane on to the enzyme layer from poly(tetrafluoroethylene) (PTFE) dispersed in aqueous media lets the entire fabrication procedure finish perfectly without nonaqueous solvent. The finalized needle-type glucose sensors show competent functions in sensitivity, stability, biocompatibility, lifetime, interference and reproducibility.     

Enzymatic preparation of chitooligosaccharides and their anti-obesity application

ABSTRACT

Chitooligosaccharides (COS) are derived from chitosan, which can be used as nutraceuticals and functional foods. Because of their various biological activities, COS are widely used in the food, medicine, agriculture, and other fields. COS were prepared by chitosanase from Pseudoalteromonas sp. SY39 and their anti-obesity activity was researched in mice in this study. The effects of hydrolysis time, temperature, the ratio of enzyme to chitosan, and pH on the productivity of COS were discussed. Preparation process of COS was established in a 5-L fermenter. COS were characterized and their anti-obesity activity was studied in animal experiments. The results showed that COS could effectively reduce serum lipid levels and obesity in mice, and have a good anti-obesity activity. The preparation technology and remarkable anti-obesity activity of COS further expand their applications in the food and pharmaceutical industries.     

Cyclen-based phenylboronate ligands and their Eu3+ complexes for sensing glucose by MRI

Novel cyclen-based phenylboronate ligands and their corresponding Eu(3+) complexes have been examined as glucose sensors using chemical exchange saturation transfer (CEST) MR imaging for detection. Two isomeric bis-phenylboronate complexes, Eu(4) and Eu(10), and a mono-phenylboronate complex, Eu(12), had been prepared and characterized by UV and circular dichroism spectroscopy, mass spectrometry, and CEST imaging. Both the free ligands and their Eu(3+) complexes bind to simple sugars, but their selectivity and binding affinities vary with sugar structure. Interestingly, the free ligands, 4 and 10, are selective for fructose over glucose, but this selectivity order switches in the respective Eu(3+) complexes. Of the complexes examined, Eu(4) shows the highest selectivity and binding affinity for glucose (2275 +/- 266 M(-)(1) at pH 10.2 and 339 +/- 29 M(-)(1) at pH 7). Glucose acts as a "capping"moiety in the Eu(4).glucose binary complex and modulates water exchange between a single Eu(3+)-bound water molecule and bulk water, an effect that can be detected by CEST imaging. Thus, Eu(4) represents a new class of metabolite-specific imaging agents that may allow mapping of metabolites by MRI of the bulk water signal.     

A new method for fish chromosome preparation

A new method for the preparation of fish chromosomes from abdominal cavity fluid has been developed. Cells were collected from fish abdominal cavity fluid after an in vivo PHA treatment, and cultured for a short time in medium with colchicine. After 30 min hypotonic treatment for marine fish and 35 min for freshwater fish, slides were prepared by the conventional air-drying method. The advantages ofthe method are: (1) it is technically simple; (2) it produces a reasonably high mitotic index; (3) chromosome spreading is good (4) there is very little cell breakage. Using this method, the chromosomes ofrainbow trout (2n=62); cod (2n=4546) and plaice (2n=46,47 and 48) were investigated.     

Concanavalin A for in vivo glucose sensing: a biotoxicity review

Over the last two decades there as has been surging scientific interest in employing the glucose- and mannose-specific lectin Concanavalin A (ConA) in affinity biosensors for in vivo glucose monitoring in diabetics. Numerous research groups have successfully shown in in vitro and in vivo studies that ConA-based affinity sensors can monitor glucose very accurately and reproducibly over many months, making ConA-based sensors an extremely interesting prospect for long-term implantation in humans. Despite this progress, there remains concern over the safety of ConA, which has widely been reported as a toxin in the literature. In this article, we review in vitro and in vivo studies related to ConA toxicity in order to assess the health risks posed by ConA in the context of an implantable biosensor. Based on the wealth of information available and on data from our own studies, we can conclude that the site of implantation (subcutaneous skin tissue) and the small amount of ConA (<10 microg/microl) being used in implantable glucose-sensitive detector devices like those proposed by various research groups would pose little or no health risk to its bearer even in the event of unexpected sensor rupture.     

Synthesis of new head-activator analogues and their application for improved radioimmunoassays

New analogues of head activator were produced for receptor and radioimmunoassay studies. The precursor molecules [(4'-I)Phe11] head activator and [Tyr11] head activator were synthesised for catalytic tritiation and iodination, respectively. With the tracer [(3,5-125 I2)Tyr11] head activator the sensitivity range of the radioimmunoassay was 5-100 fmol.     

一种新的制备多克隆抗体的方法

用亲和色谱纯化的rhEPO作为抗原免疫KM小鼠和Bal b/c小鼠,然后腹腔注射S180细胞或SP2/0细胞。S180细胞可刺激KM小鼠产生腹水,腹水量大,抗体效价高。而直接注射SP2/0细胞既未能使KM小鼠产生腹水,也未能使Bal b/c小鼠产生腹水。实验提供了一种新的简便、快速、经济地制备高效价多克隆抗体的方法。     

A new reagent for the preparation of glycoconjugates

The thioglycoside derivative 2-carbazoylethyl 1-thio-beta-D-galactopyranoside hydrochloride was synthesized. Conversion of the carbazoyl group into the reactive azidocarbonyl function leads to a well suited reagent for the preparation of glycoconjugates via amidation of proteins or synthetic carriers in aqueous media.     

Assembly of dandelion-like Au/PANI nanocomposites and their application as SERS nanosensors

The monodisperse, uniform dandelion-like Au/polyaniline (PANI) composite nanospheres were synthesized by a simple one-step process without any additives or templates. The nanospheres are really composed of many short nanorods and the average diameter of whole nanospheres is about 180 nm. The morphology of Au/PANI composites could be controlled by adjusting the molar ratio of HAuCl(4) to aniline. The prepared nanocomposite is developed as a wonderful sensor for the detection of Hg(2+) ions, which is based upon the Raman intensity response of PANI to Hg(2+) ions. Results from the morphology-dependent sensitivity investigations show that the dandelion-like nanospheres have an ultra sensitive response (as low as 10(-11)M) compared with other morphologies. The nanosensor also exhibits good reproducibility and greater selectivity for Hg(2+) ions than the other heavy metal ions. And the mechanism was proposed. The proposed nanosensors can be applied for highly sensitive and selective chemical analysis in a variety of environmental detection.     

A new method for preparation of an antiserum to penicillin and its application for novel enzyme immunoassay of penicillin.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.     

A method for preparation of dry thrombin for topical application

Thrombin for topical hemostasis can be prepared from bovine or human blood plasma. The prothrombin is isolated by means of adsorption on DEAE-Sephadex A-50 and consecutively activated by CaCl₂ and thromboplastin. Thrombin is precipitated and purified by acetone. The specific activity of the thrombin preparation is 122 + 23 IU/mg protein while the yield is 36,360 ± 6623 IU/liter plasma.     

Zinc sensing for cellular application

Numerous tools for Zn2+ sensing in living cells have become available in the past three years. Among them, fluorescence imaging using fluorescent sensor molecules has been the most popular approach. Some of these sensor molecules can be used to visualize Zn2+ in living cells. Some of the biological functions of Zn2+ have been clarified using these sensor molecules, especially in neuronal cells, which contain a high concentration of free Zn2+.     

Glucoamylase and glucose oxidase preparations and their combined application for conversion of maltose to gluconic acid

The kinetic behavior of a system of multiple enzyme in solution has been studied in a variable volume batch reactor at pH 5, controlled dissolved oxygen concentration, and T = 30°C. The enzymes used were glucoamylase (R. delemar), glucose oxidase (A. niger), and gluconolactonase (A. niger), all of which are important commercial biocatalysts, and a disaccharide was employed as the starting substrate. This study includes the basic kinetic properties of individual enzymes and interactions between components of the reaction mixture. Classical Michaelis–Menten single substrate or two substrate kinetic with parameters based on initial rate data predict correctly the batch time course of the sequential reaction network.