[Identification of horn-derived animal medicines based on detection of keratin-derived characteristic peptides].

Due to the highly stable structure of keratin, the extraction and dissolution steps of animal medicines rich in keratin are complex, which seriously restricts the detection efficiency and flux. Therefore, this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-derived animal medicines. For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA), the ion pair conditions of the characteristic peptides were optimized, and the retention time, intensity and other data of the specific peptides were compared between the samples treated with/without IAA. Two pre-treatment methods, direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis, were used to prepare horn-derived animal medicine samples. The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn, water buffalo horn, goat horn, and yak horn were compared regarding the retention time of specific peptides and ion intensity. The results indicated that after direct enzymatic hydrolysis, the specific peptides in the samples without IAA treatment can be detected. Compared with the characteristic peptides in the samples treated with IAA, their retention time shifted back and the mass spectrometry response slightly decreased. The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines. Overall, the process of direct enzymatic hydrolysis can be used to treat horn samples, omitting the steps of protein extraction and dithiothreitol and IAA treatment, significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines. This study provides ideas for quality research and standard improvement of horn-derived animal medicines.

Telomerase-triggered DNAzyme spiders for exponential amplified assay of cancer cells.

A highly flexible electrochemical assay based on target-triggered DNAzyme spiders was proposed for the detection of telomerase. The DNAzyme-telomerase substrate primers (D-TSP) containing Cu2+-dependent DNAzymes serve as recognition elements, and primers of telomerase. Telomerase extracted from Hela cells recognize the D-TSP and elongated with DNA sequence repeats. A synthetic telomerase product hybridized with scaffold sequences of two DNAzyme-tethered probes on the basis of the mechanism of the proximity-ligation assay. The three-leg DNAzyme spiders has been assembled and initiated the autonomous hybridization/nicking/displacement cycles on substrate modified surface. The cleaved ferrocene-labeled fragements are adsorbed on gold surface leading to an increase in the electrochemical signal. As a result, the one input target, telomerase, release large amount of ferrocene-labeled DNA strands, achieving an exponential signal amplification and an excellent improvement in sensitivity over single molecule or two-component 'sandwich' binding complexes. Our proposed biosensor showed a nonlinear dependence with Hela cell numbers, ranging from 25 to 2000 with a detection limit of 10 cells. Telomerase activities from different cell lines were also successfully evaluated. Our electrochemical strategy based on target-triggered DNAzyme spiders was enzyme-free, PCR-free, simple in operation which indicated that it expected to expand the scope of DNA nanotechnology in the areas of clinical diagnosis.