Study Of Novel Bio-analytical Methods Based On Biological And Chemical Reactions

Biochemical analysis is critical to human health and environment protection. There are many biochemical reactions which have direct or indirect influence on human beings in nature. How to develop novel bio-analytical methods by using these biochemical reactions has always received research interest. We have employed certain organic reactions and some specific reactions based on biological affinity for the development of new bio-analytical methods..1. Electrochemical detection of nitrite based on Griess reaction and functionalized platinum nanoparticlesNitrite can binds to hemoglobin to cause methemoglobinemia in infants, and it is also relevant to the formation of carcinogenic nitrosamines. While contamination of drinking water supplies with nitrite continues to be a big problem throughout the world, designing a new high sensitivity and simple method to detect nitrite is more and more important. In this work, a novel electrochemical method for nitrite detection by using functionalized platinum nanoparticles (PtNPs) is proposed. Firstly, a gold electrode is immobilized with 4-(2-aminoethyl)benzenamine. Then, PtNPs are modified with 5-[1,2]dithiolan-3-yl-pentanoic acid [2-(naphthalene-1-ylamino)-ethyl]amide (DPAN). Consequently, in the presence of nitrite ions, Griess reaction occurs between 4-(2-aminoethyl)benzenamine on the electrode and DPAN on PtNPs, thus PtNPs are localized onto the electrode surface. So, PtNPs-electrocatalyzed reduction of H2O2 can be achieved to correlate the electrochemical signal with the concentration of nitrite ions. The linear concentration range can be as wide as 10-1000μM, while the detection limit is as low as 5μM. The proposed method has been also successfully applied to the detection of nitrite with the local lake water, and the result is well consistent with that obtained by UV-visible spectrophotometric method. So this method has potential use for monitoring nitrite in drinking water supplies in the future.2. Combination of terminal protection and Nicking endonuclease assisted amplification for sensitive electrochemical detection of protein binding with small moleculesSmall organic compounds that bind to a particular protein with reasonable affinity and specificity offer invaluable probes to perturb the function of the proteins for chemical genetics studies, and track the location and concentration of the proteins for molecular diagnostics. So, novel techniques for detecting small molecule-protein interactions would be of greatly benefit to molecular diagnostics and therapeutics. In this work, we have developed a new electrochemical strategy for homogeneous assay of protein-small molecule interactions based on the combination of terminal protection and nicking endonuclease assisted amplification (NEA). Specifically, the terminal protection help us convert the small molecule-protein interaction assay into the detection of specific DNA, while the NEA process offers handling convenience and ultrahigh detection sensitivity and selectivity. Here, we take folate receptor-folate interaction as the model target. The terminal protection is ExoⅠfails to catalyze the stepwise hydrolysis of the small-molecule-linked DNA when folate receptor is bound to folate-ssDNA though the small-molecule moiety, leaving the ssDNA intact. Such terminal protection translates the binding of small molecules to proteins into the presence of specific DNA sequences. The NEA process is the nicking endonuclease recognizes specific nucleotide sequences in double-stranded DNA and cleaves only one of the strands, probe 2. Then the released stand, probe 1, can hybridize to another probe 2 modified on an electrode surface again, thus and initiate the second cycle of cleavage. Eventually, each probe 1 can go through many cycles, resulting in the cleavage of many probe 2. Consequently, the blocking effect of probe 2 against [Fe(CN)6]3-/4- becomes lower, leading to increased electrochemical signals. Compared with existing protein-binding assay technologies, our assay strategy is able to offer desirable sensitivity, high selectivity, increased throughput, low cost, and simplified operations.