Novel composite nanomaterials based on magnetic molecularly imprinted polymers for selective extraction and determination of rutin in fruit juice.

In this work, with the attempt to further improve the selectivity, magnetism and loading proportion of existing adsorbents, a novel composite (MGO/MHNTs@MIPs) was synthesized by electrostatically combining molecularly imprinted polymer based on the surface of magnetic halloysite nanotubes (MHNTs@MIPs) with magnetic graphene oxide (MGO). Then some characterizations were done to prove its successful synthesis. Besides, the bonding experiment showed that it possessed a loading capacity of up to 132 mg·g-1, and the adsorption behavior of MGO/MHNTs@MIPs was elucidated by Langmuir isotherm model and Pseudo-second order model. By comparing its adsorption capacity to analogues, we concluded that the MGO/MHNTs@MIPs with the MHNTs@MIPs as basic elements exhibited higher selectivity (imprinting factor = 2.25) than that of MGO/MHNTs@NIPs based on MHNTs@NIPs for template rutin. Furthermore, a series of solid phase extraction conditions were optimized, and then the materials were used for the extraction and detection of rutin in fruit juice under the optimal conditions.

Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

Invader Assisted Enzyme-Linked Immunosorbent Assay for Colorimetric Detection of Disease Biomarkers Using Oligonucleotide Probe-Modified Gold Nanoparticles.

We successfully developed an invader assisted ELISA assay (iaELISA) for sensitive detection of disease biomarkers. The method includes three key steps as follows; biotinylated detection antibody was at first used to capture targeted antigen by sandwich ELISA. The biotinylated oligonucleotide was then attached to detection antibody via streptavidin. Finally, the cascade invader reactions were employed to amplify the biotinylated oligonucleotide specific to the antigen so that detection of the antigen was transformed into signal amplification of the antigen-specific DNA. To achieve colorimetric detection, oligonucleotide probe and modified gold nanoparticles (AuNPs) were coupled with the invader assay. Utilization of the hairpin probes in the invader reaction brought about free AuNPs, resulting in the positive read-out (red color). On the other hand, aggregation of the AuNPs occurred when the hairpin probes were not utilized in the reaction. This method was successfully used to detect as low as 2.4 x 10(-11) g/mL of HBsAg by both naked eye and spectrophotometer. This sensitivity was about 100 times higher than that of conventional ELISA method. The method was also used to assay 16 serum specimens from HBV-infected patients and 8 serum specimens from HBV-negative donors and results were in good agreement with those obtained from the conventional ELISA. As the invader assay is sensitive to one base sequence, a good specificity was also obtained by detecting other antigens like hepatitis A virus (HAV) and BSA. The method has therefore much potential for ultrasensitive and cost-effective detection of targeted proteins that have clinical importance.

A Pharmacometabonomic Approach To Predicting Metabolic Phenotypes and Pharmacokinetic Parameters of Atorvastatin in Healthy Volunteers.

Genetic polymorphism and environment each influence individual variability in drug metabolism and disposition. It is preferable to predict such variability, which may affect drug efficacy and toxicity, before drug administration. We examined individual differences in the pharmacokinetics of atorvastatin by applying gas chromatography-mass spectrometry-based metabolic profiling to predose plasma samples from 48 healthy volunteers. We determined the level of atorvastatin in plasma using liquid chromatography-tandem mass spectrometry. With the endogenous molecules, which showed a good correlation with pharmacokinetic parameters, a refined partial least-squares model was calculated based on predose data from a training set of 36 individuals and exhibited good predictive capability for the other 12 individuals in the prediction set. In addition, the model was successfully used to predictively classify individual pharmacokinetic responses into subgroups. Metabolites such as tryptophan, alanine, arachidonic acid, 2-hydroxybutyric acid, cholesterol, and isoleucine were indicated as candidate markers for predicting by showing better predictive capability for explaining individual differences than a conventional physiological index. These results suggest that a pharmacometabonomic approach offers the potential to predict individual differences in pharmacokinetics and therefore to facilitate individualized drug therapy.

Colorimetric detection of DNA sequences using an organic solvent to induce the aggregation of label-free gold nanoparticles.

A novel assay based on a solvent for inducing the aggregation of AuNPs colloid was proposed to discriminate ssDNA from dsDNA. Eleven organic solvents with different polarity were investigated, and it was found that DMSO was possible to aggregate AuNPs at the amount of only 0.4 microL in a 50-microL detection system. Further research showed that 0.8 microL of DMSO could discriminate the ssDNA from dsDNA. Colorimetric detection with various conditions, including the ratio of the target to the probe, and the concentration of AuNPs and DNA, was investigated. The proposed method was successfully used for SNP typing, and unambiguous discrimination of a wild type from a mutant was obtained for the templates with the mismatched base at the 5'-end or in the middle of the target sequence. As no requirement of gold modification and detection instrument, we believe that this method will be much low in the cost for DNA detection.

Pyrosequencing on templates generated by asymmetric nucleic acid sequence-based amplification (asymmetric-NASBA).

Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.