Investigation of 3,3 ',5,5 '-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme

in this Work, the suitability of 3,3',5,5'-tetramethylbenzidine sulfate (TMB) as the substrate of a DNAzyme catalytic system composed of a guanine-quadruplex DNA molecule and hemin was investigated. In the presence of H2O2, the hemin-DNA complex catalyzes the oxidation of TMB to produce two colored products, much like a peroxidase. The color-generating activity of this system could be influenced by several factors such as buffer type, pH value, DNA sequence, reaction time, and concentrations of both the hemin and H2O2. To illustrate the utility of this catalytic system, we designed a colorimetric assay, in which a synthetic oligonucleotide with a sequence complementary to the G-quadruplex DNA was used as the target. A detection limit of 1.86 nM was obtained. Our data have shown that TMB was an excellent colorimetric indicator that reported the peoxidase activities of the widely studied hemin-G-quadruplex DNAzyme system.

G-Quadruplex Aptamers with Peroxidase-Like DNAzyme Functions: Which Is the Best and How Does it Work?

Some G-quadruplex DNA aptamers have been found to strongly bind hemin to form DNAzymes with peroxidase-like activity. To help determine the most suitable DNAzymes and to understand how they work, five previously reported G-quadruplex aptamers were compared for their binding affinity and then the potential catalytic mechanism of their corresponding hemin-G-quadruplex DNAzymes was explored. Among these aptamers, a G-quadruplex named AGRO100 was shown to possess the highest hemin-binding affinity and the best DNAzyme function. This means that AGRO100 is the most ideal candidate for DNAzyme-based analysis. Furthermore, we found the peroxidase-like activity of DNAzyme to be primarily dependent on the concentration of H2O2 and independent of that of the peroxidase substrate (that is, 2,2-azino-bis(3-ethytbenzothiazoline-6-sulfonic acid)diammonium salt). Accordingly, a reaction mechanism for DNAzyme-catalyzed peroxidation is proposed. This study provides new insights into the G-quadruplex-based DNAzymes and will help us to further extend their applications in the analytical field.

Sensitive, selective and label-free protein detection using a smart polymeric transducer and aptamer/ligand system

We have demonstrated a smart polymeric transducer and aptamer/intercalating dye system that allows the label-free detection of protein with high sensitivity and selectivity.

Sensitive detection of protein by an aptamer-based label-free fluorescing molecular switch

We report an aptamer-based method for the sensitive detection of proteins by a label-free fluorescing molecular switch (ethidium bromide), which shows promising potential in making protein assay simple and economical.

Simple and sensitive aptamer-based colorimetric sensing of protein using unmodified gold nanoparticle probes

We describe herein simple and sensitive aptamer-based colorimetric sensing of protein (alpha-thrombin in this work) using unmodified gold nanoparticle probes.

SERS opens a new way in aptasensor for protein recognition with high sensitivity and selectivity

SERS aptasensors for protein recognition based on Au nanoparticles labeled with aptamers and Raman reporters have been developed, which opens a new way for protein recognition of high sensitivity and selectivity.

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T-m = 50.2 +/- 0.2 degrees C and a folding enthalpy Delta H degrees(fold) = -49.0 +/- 2.1 kcal mol(-1). These values agree with values of T-m = 49.6 degrees C and Delta H degrees(fold) = -51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy Delta G degrees(bind) = -5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with Delta H degrees(bind) = -8.7 kcal mol(-1). Combination of enthalpy and free energy produce ail unfavorable entropy of -T Delta S degrees = +3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K-1 was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures.

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillorum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3 h post-challenge. After a decrease at 6 h, the expression Level increased again and reached 207.8-fold at 12 h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCf KZSPI-1 2) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (K-i) of rCfKZSPI-12 to trypsin was 173 nmol L-1. These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response. (c) 2008 Elsevier Ltd. All rights reserved.

Study on extraction of agaropectin from Gelidium amansii and its anticoagulant activity

Gelidium amansii agar was fractionated on DEAE-cellulose and four fractions were obtained sequentially. The yields of 1.0 mol/L NaCl fraction and 2.5 mol/L NaCl fraction were 2.80% and 2.03%. They are highly sulfated agar, and named as agaropectin with sulfate content being 22.8% and 32.5%, respectively. The anticoagulant experiment results show that agaropectin could effectively prolong the coagulation time in a dose-dependent manner in vitro. Agaropection could be absorbed and effectively prolong the plasma coagulation time in vivo. After intragastric administration at the doses of 100, 200, and 400 mg/kg.d in rats for 15 days, TT (thrombin time), CT (coagulation time), PT (prothrombin time), and APTT (activated partial thromboplastin time) could be effectively prolonged and the plasma Fib level could be significantly lowered.

Purification and characterization of recombinant truncated human interleukin-11 expressed as fusion protein in Escherichia coli

Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.

A importância clínica da vitamina D

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Real Time Eye Tracking and Blink Detection with USB Cameras

A human-computer interface (HCI) system designed for use by people with severe disabilities is presented. People that are severely paralyzed or afflicted with diseases such as ALS (Lou Gehrig's disease) or multiple sclerosis are unable to move or control any parts of their bodies except for their eyes. The system presented here detects the user's eye blinks and analyzes the pattern and duration of the blinks, using them to provide input to the computer in the form of a mouse click. After the automatic initialization of the system occurs from the processing of the user's involuntary eye blinks in the first few seconds of use, the eye is tracked in real time using correlation with an online template. If the user's depth changes significantly or rapid head movement occurs, the system is automatically reinitialized. There are no lighting requirements nor offline templates needed for the proper functioning of the system. The system works with inexpensive USB cameras and runs at a frame rate of 30 frames per second. Extensive experiments were conducted to determine both the system's accuracy in classifying voluntary and involuntary blinks, as well as the system's fitness in varying environment conditions, such as alternative camera placements and different lighting conditions. These experiments on eight test subjects yielded an overall detection accuracy of 95.3%.

Prediction and prevention of venous thrombosis in pregnancy

Venous thromboembolism (VTE) remains the leading cause of maternal mortality. Reports identified further research is required in obese and women post caesarean section (CS). Risk factors for VTE during pregnancy are periodically absent indicating the need for a simple and effective screening tool for pregnancy. Perturbation of the uteroplacental haemostasis has been implicated in placenta mediated pregnancy complications. This thesis had 4 main aims: 1) To investigate anticoagulant effects following a fixed thromboprophylaxis dose in healthy women post elective CS. 2) To evaluate the calibrated automated thrombogram (CAT) assay as a potential predictive tool for thrombosis in pregnancy. 3) To compare the anticoagulant effects of fixed versus weight adjusted thromboprophylaxis dose in morbidly obese pregnant women. 4) To investigate the LMWH effects on human haemostatic gene and antigen expression in placentae and plasma from the uteroplacental , maternal and fetal circulation. Tissue factor pathway inhibitor (TFPI), thrombin antithrombin (TAT), CAT and anti-Xa levels were analysed. Real-time PCR and ELISA were used to quantify mRNA and protein expression of TFPI and TF in placental tissue. In women post CS, anti-Xa levels do not reflect the full anticoagulant effects of LMWH. LMWH thromboprophylaxis in this healthy cohort of patients appears to have a sustained effect in reducing excess thrombin production post elective CS. The results of this study suggest that predicting VTE in pregnant women using CAT assay is not possible at present time. The prothrombotic state in pregnant morbidly obese women was substantially attenuated by weight adjusted but not at fixed LMWH doses. LMWH may be effective in reducing in- vivo thrombin production in the uteroplacental circulation of thrombophilic women. All these results collectively suggest that at appropriate dosage, LMWH is effective in attenuating excess thrombin generation, in low risk pregnant women post caesarean section or moderate to high risk pregnant women who are morbidly obese or tested positive for thrombophilia. The results of the studies provided data to inform evidence-based practice to improve the outcome for pregnant women at risk of thrombosis.