Target induced aggregation of Ce(III)-based coordination polymer nanoparticles for fluorimetric detection of As(III).

As(III) is the most harmful substance of all over 20 kinds of arsenic compounds. In addition, the trivalent oxidation state of arsenic is not stable, which can be oxidized to pentavalent arsenic. Thus, it is attractive and challenging to sensitively and selectively monitor As(III) concentration, rather than As(V) concentration, in water. However, most of detection techniques suffer from the inability to distinguish As(III) and As(V), or even need specialized personnel and additional equipment. Herein, novel luminescent Ce(III)-based coordination polymer nanoparticles (Ce-CPNs) have been proposed for selective detection of As(III). The Ce-CPNs are dispersive and show a fluorescence peak at 353 nm under excitation at 280 nm. With the presence of As(III), aggregation of Ce-CPNs occurs, resulting in quenching of the fluorescent Ce-CPNs due to the aggregation-caused π-π stacked layers of Ce-CPNs. Under optimal conditions, the detection limit for As(III) is down to 0.7 ppb. In addition, the Ce-CPNs are selective for As(III) over other ions and has been successfully applied for fluorescence sensing of As(III) in environmental water samples.

Luminescence determination of microRNAs based on the use of terbium(III) sensitized with an enzyme-activated guanine-rich nucleotide.

A method is reported for the fluorometric quantitation of microRNA. It is making use of a luminescent probe deribed from terbium(III) ion whose fluorescence is sensitized with a guanine-rich (G-rich) nucleotide. The probe has a large Stokes' shift and strong and sharp emission bands. The assay relies on the wide substrate specificity of terminal deoxynucleotidyl transferase (TdTase), which catalyzes the formation of long G-rich nucleotides when using microRNA primer as a trigger to start the polymerization. The addition of Tb(III) induces the formation of a G-quadruplex from the G-rich nucleotide, and this strongly enhances the green fluorescence of Tb(III) (peaking at 545 nm upon photoexcitation at 290 nm). Specifically, microRNA-21 was chosen as the analyte. The fluorescence intensity of Tb(III) increases linearly in the 1 pM to 1 nM microRNA concentration range, and the detection limit is as low as 0.11 pM. The method can distinguish between family members of microRNA and performs excellently even when applied to extracts of cancer cells. Graphical abstract A fluorometric technique is reported for the determination of microRNA. It is based on signal enhancement based on the sensitization of terbium(III) via a guanine-rich nucleotide sequence. Klenow Fragment exo- (KFexo-) generates DNA sequence at the 3'-OH of microRNA, and terminal deoxynucleotidyl transferase (TdTase) catalyzes the formation of long G-rich nucleotides.

Electrochemical sensor for arsenite detection using graphene oxide assisted generation of prussian blue nanoparticles as enhanced signal label.

An electrochemical sensor was fabricated for arsenite detection using graphene oxide-assisted generation of prussian blue nanoparticles as enhanced redox signal label. The 5'-thiolate-labeled (GT)21-ssDNA was first self-assembled on a gold electrode surface via Au-S bond. Graphene oxide can interact with ssDNA through π-π stacking interaction and facilitate the generation of prussian blue nanoparticles on its surface as an electrochemically active indicator. In the absence of arsenite, plenty of graphene oxide/prussian blue nanoparticles can be adsorbed on the electrode surface to produce a stronger redox signal of prussian blue nanoparticles. While in the presence of arsenite, (GT)21-ssDNA can recognize and combine with arsenite via hydrogen bonds to form (GT)21-ssDNA/arsenite complex with a frizzy structure. The conformational change of (GT)21-ssDNA led to less adsorption of graphene oxide/prussian blue nanoparticles on the electrode surface, resulting in a reduced redox response. The arsenite-induced (GT)21-ssDNA structure switching can be used for sensitive detection of arsenite with a linear range from 0.2 to 500 ppb and a detection limit down to 0.058 ppb. Benefiting from (GT)21-ssDNA containing arsenite recognition sequence, the proposed sensor exhibited excellent specificity against other heavy metal ions. The applicability of the electrochemical biosensor for arsenite assay in real water samples demonstrated the great potential of this strategy for trace arsenite detection in environment.

Ratiometric electrochemical assay for sensitive detecting microRNA based on dual-amplification mechanism of duplex-specific nuclease and hybridization chain reaction.

We propose a ratiometric electrochemical assay for detecting microRNA (miRNA) on the basis of dual-amplification mechanism by using distinguishable electrochemical signals from thionine (Thi) and ferrocene (Fc). The thiol-modified and ferrocene-labeled hairpin capture probes (CP) are first immobilized on an Au electrode via Au-S reaction. The target miRNA hybridizes with CP and unfolding the hairpin structure of CP to form miRNA-DNA duplexes. Then, kamchatka crab duplex specific nuclease (DSN) specifically cleaves the DNA in miRNA-DNA duplexes, leading to the release of miRNA and another cleaves cycle, meanwhile, numerous Fc leaves away from the electrode surface and leads to the signal-off of Fc. The residual fragment on electrode surface acts as a HCR primer to form dsDNA polymers through in situ HCR with the presence of the primer and two probes (HDNA and HDNA'), resulting in the capture of numerous DNA/Au NPs/Thi and the signal-on of Thi. The dual-amplification mechanism significantly amplifies the decrease of Fc signal and the increase of Thi signal for ratiometric readout (IThi/IFc), thus providing a sensitive method for the selective detection of miR-141 with a detection limit down to 11aM. The dual-signal ratiometric outputs have an intrinsic self-calibration to the effects from system, which is promising to be applied in biosensing and clinical diagnosis.

Simultaneously electrochemical detection of microRNAs based on multifunctional magnetic nanoparticles probe coupling with hybridization chain reaction.

We report a sensor combining two distinguishable magnetic nanoprobes (DNA1/Fe3O4 NPs/Thi and DNA2/Fe3O4 NPs/Fc) with target-triggered hybridization chain reaction (HCR) strategy for the simultaneous detection of microRNA-141 (miR-141) and microRNA-21 (miR-21). In the presence of targets, the thiol-modified hairpin capture probes (HCP1 and HCP2) specifically hybridize with miR-141 and miR-21 on a gold electrode, leading to the conformation change of HCP1 and HCP2, respectively. The conformation change subsequently triggers HCR to generate plentiful bonding sequences of magnetic nanoprobes. Thus, numerous thionine (Thi) modified DNA1/Fe3O4 NPs/Thi and ferrocene carboxaldehyde (Fc-CHO) modified DNA2/Fe3O4 NPs/Fc are captured by the well-designed HCR, via DNA hybridization respectively, giving rise to the dual magnified response of currents. The increase in the electrochemical currents at different potentials of the two magnetic nanoprobes enables us to simultaneously and quantitatively detect miR-141 and miR-21. Target-triggered HCR increases the amount of captured nanoprobes due to the increasing number of bonding sequences, greatly amplifying the currents of the two magnetic nanoprobes in the presence of targets, and ultimately realizing the dual signal amplification with increased sensitivity. The sensor can be applied for detecting miRNAs in cell lysates, thus, promising to be a clinic diagnosis of cancers by means of simultaneous detection of a variety of miRNA biomarkers.

Direct fluorescence detection of microRNA based on enzymatically engineered primer extension poly-thymine (EPEPT) reaction using copper nanoparticles as nano-dye.

A new strategy based on enzymatically engineered primer extension poly-thymine (EPEPT) and nanomaterials in situ generation technology is reported for direct detection of microRNA (miRNA) in a fluorescence turn-on format using the sequential and complementary reactions catalyzed by Klenow Fragment exo- (KFexo-) and terminal deoxynucleotidyl transferase (TdTase). The short miRNA can be efficiently converted into long poly-thymine (polyT) sequences, which function as template for in situ formation of fluorescence copper nanoparticles (CuNPs) as nano-dye for detecting miRNA. The polyT-CuNPs can effectively form and emit intense red fluorescence under the 340nm excitation. For the proof of concept, microRNA-21 (miR-21) was selected as the model target to testify this strategy as a versatile assay platform. By directly using miR-21 as the primer, the simple, rapid and sensitive miRNA detection was successfully achieved with a good linearity between 1pM and 1nM and a detection limit of 100fM. Thus, the EPEPT strategy holds great potential in biochemical sensing research as an efficient and universal platform.

[The role of amino acid sequence between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the regulation of the VWF binding to GP I b alpha].

OBJECTIVE: To explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha. METHODS: The VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope. RESULTS: The VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips. CONCLUSIONS: The amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.

[Establishment of Chinese hamster ovary cell line expressing recombinant GPIb-IX complex].

The aim of this study was to construct Chinese Hamster Ovary (CHO) cell models expressing recombinant wild-type GPIb-IX and mutant GPIb-IX complex, so as to provide the platform to study the related physiologic functions of GPIb-IX. The plasmids were extracted from E.coli expressing wild-type or deletion mutant GPIbalpha and were identified by digestion with EcoR I. Three plasmids containing GPIbalpha, GPIbbeta, and GPIX genes were co-transfected into CHO cells, and then the expression of GPIb-IX complex was detected by immune coprecipitation, Western blot and flow cytometry. The results showed that the expression of GPIb-IX complex could be detected in the lysate and on the surface of CHO cells at 48 hours after transfection. In conclusion, CHO cell models expressing recombinant wild-type or mutation GPIb-IX complex has been successfully constructed.

[Preparation and preliminary application of rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha].

OBJECTIVE: To prepare rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha. METHODS: Two peptides corresponding to human platelet GPIbalpha C-terminus were synthesized and purified by high-performance liquid chromatography (HPLC). The peptides were cross-linked with keyhole limpet hemocyanin (KLH). Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbalpha intracellular peptides phosphorylation was tested with these polyclonal antibodies by ELISA. RESULTS: The titers of the two polyclonal antibodies against the GPIbalpha C-terminus peptides were 1:32 000 and 1:64 000 respectively and both of these antibodies reacted with purified GPIbalpha. CONCLUSIONS: Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbalpha have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular peptide of human platelets.

Group-selective immunoassay for the detection of morphine in urine.

A new competitive inhibition immunoassay (group-selective immunoassay; GSI) has been developed to detect free morphine in urine with the Fab' fragments of monoclonal antibodies (MAbs) (1B(12)F(9)B(4), IgG(1), kappa, K(aff) = 9.66 x 10(10)M(-1)). At the first assay step, microtiter plates were coated with morphine-ovalbumin (M-6-S-OVA), in which free amino acids were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. At the second assay step, anti-morphine MAbs' Fab' fragments, in which free amino groups were biotinylated by N-hydrosuccinimide-biotin ester, were bound to chemically modified immunosorbent. The biotin residues were then detected by the streptavidin-peroxide conjugate. This method has a sensitivity of 3.50 x 10(-15) mol/L using very little volume of sample, covering up to almost 1.20 x 10(-11) mol/L of standard concentration of morphine with good reproducibility. Standard curve prepared in urine indicated a good correlation between the concentration of morphine and the value of OD (y = 1/ax + b; r = 0.99939257, S = 0.01138127). Coefficients of variation for this immunoassay were 1.41 approximately 6.61% within-a-day assay and 2.31 approximately 8.99% between days assay. The recoveries were 94 approximately 101.4% from negative urine and 95.2 approximately 107.5% from positive urine samples, respectively. This method has application as a specific screen for morphine in drug abusers, to study the metabolism of the drug in the body, or to screen the monoclonal antibodies (MAbs) against morphine.

A novel competitive ELISA for both free and protein-bound nitrotyrosine.

3-Nitro-L-tyrosine (nitrotyrosine) has recently been considered to be useful as a biomarker of endogenous production of several reactive nitrogen species including peroxynitrite. In the present study, nitrotyrosine was coupled to human serum albumin (HSA) using a two-step glutaraldehyde method and immunized mouse with multifocal intradermal injections. Using a conventional immunization protocol, 12 stable monoclonal antibodies (MAbs) producing cell lines recognizing nitrotyrosine were obtained. Six MAbs were selected for further characterization. A study of cross-reactions with nitrotyrosine-like compounds showed that the antibodies had a high specificity for nitrotyrosine, but no detectable reactivity with L-tyrosine, p-nitro-L-phenylalanine, o-phospho-L-tyrosine or 3-amino-L-tyrosine. Using these high titer and affinity antibodies, a competitive inhibition ELISA was developed with a lower detection limit of approximately 20 nmol/L to detect both free and protein-bound nitrotyrosine in biological systems.

[Effects of simulated weightlessness and mechanical loading on bone interstitial fluid flow in rats].

OBJECTIVE: To investigate the effects of simulated weightlessness and mechanical loading on bone interstitial fluid flow. METHOD: Thirty-six male Sprague-Dawley [correction of Spargue-Danley] rats were divided into 3 groups: the control group, the tail-suspension group, and the tail-suspension plus mechanical loading group, with four rats in each group. All the rats were injected via a lateral tail vein with horseradish peroxidase on the 21st day of the experiment. Tibial tissue specimens were explanted, fixed, decalcified and cut into 30 micrometers frozen sections 3 h after the intravenous injection. RESULT: There were less peroxidase reaction product in the bony matrix and bone lacunae in tail-suspended rats than the control and tail-suspended plus mechanical loading rats. CONCLUSION: Tail-suspension decreased fluid movement through the bone, while mechanical loading increased it.

[Space flight and peroxidative damage].

Space flight is associated with an increase of peroxidative damage after returning to 1 g. The effect is more pronounced after long-duration space flight and can even last for several weeks after landing. In humans there is increased lipid peroxidation in erythrocyte membranes, reduced blood antioxidants, and increased urinary excretion of 8-iso-prostaglandin F2alpha, and 8-oxo-7, 8 dihydro-2 deoxyguanosine. Isoprostane 8-iso-prostaglandin F2alpha and 8-oxo-7, 8 dihydro-2 deoxyguanosine are markers for oxidative damage to lipids and DNA, respectively. The changes are attributed to a combination of energy deficiency that occurs during flight and substrate competition for amino acids occurring between repleted muscle and other tissues during the recovery phase. The observations in humans have been complemented by studies in rodents, which showed increased production of lipid peroxidation products and decreased antioxidant enzyme activity afterflight. The changes in rodents were attributed to the stress associated with re-entry into Earth's gravity. Reducing the imbalance between the production of endogenous oxidant defenses and oxidant production by increasing the supply antioxidants in diet may lessen the severity of the postflight increase in oxidative stress.