Effects Of An Nanoparticles On The Specific Amplification Of PCR And Determination Of ACC By HPLC

Polymerase Chain Reaction (PCR) has become one of the most popular techniques in modern biological and medical sciences. Owing to the exponential amplification ability of PCR, this highly sensitive technique enables a broad spectrum of application, including DNA sequencing, molecular diagnosis and genetic analysis. Nevertheless, although it is highly sensitive, the current PCR technique is limited by its relative low level of specific amplification. Recently, people try to find ways of optimizing PCR from the rising subjects and technologies. Nanobiological technology coming from the combination of nanotechnology and bioscience technology has catched more and more people’s attention because of its special superiorities. Gold nanoparticles have been found broad and important applications in biology, because they are biocompatible nanomaterials and owing to the availability of versatile chemistry for functionalization at their surface as well as their unique optoelectronic properties. It has been discovered recently that the use of Au nanoparticles can reduce the level of nonspecific amplification and enhance the level of specific amplification, but the reports are about small DNA fragments (less than1000bp), there are no reports about long DNA fragments (more than1000bp) in PCR. So the effects of different concentrations of Au nanoparticles on the specific amplification of polymerase chain reaction (PCR) were studied.Ethylene is a plant hormone which regulates many aspects of plant growth, development and senescence. Many of these effects are of high commercial importance in agriculture.1-Aminocyclopropane-l-carboxylic acid (ACC) is the immediate precursor of the plant hormone ethylene. It is found to be directly correlated to its endogenous ACC content. An assay whereby the level of ACC in plant tissues and organs can be determined is essential to an understanding of ethylene-regulated physiological process. Gas chromatography in which ACC is oxidized to ethylene by NaClO has been a common and simple method for the determination of ACC. However, this classical method has shown some shortcomings, such as low and unstable conversion efficiency, high sample consumption. To the best of our knowledge, there is no report for the determination of ACC with HPLC coupled to a fluorescence detector using fluorescamine as a pre-column deriverization reagent. A new, sensitive and simple method was described for the determination of1-aminocyclopropane-l-carboxylic acid (ACC) in apple and peach extracts.The method was based on the derivatization of ACC with fluorescamine in borate buffer systems of pH8.0to yield a highly fluorescent product. The experimental parameters affecting the derivatization reaction efficiency were optimized by fluorimetric analysis. Under optimum derivatization conditions, the derivative product of ACC in apple and peach extracts without extra purification was successfully chromatographed on a C-18column by HPLC coupled to fluorescence detection. The derivative product of ACC with fluorescamine could be well separated from other concomitant substances or their derivatives that might interfere with the determination of ACC. The linearity of ACC was measured in the range of23.82-238.82μg·L-1with a good correlation coefficient of0.9997. Based on signal-to-noise ratio of3, a low detection limit of5.0μg·L-1could be reached. The proposed method was applied successfully to the determination of ACC in the crude apple and peach extracts without extra purification with low RSDs of0.19-1.9%and good recoveries of90.89-104.4%. The sensitive HPLC quantitative method is of great significance for the investigations of ACC metabolism in plants.