Novel Label-free Fluorescent Biosensing Methods Based On DNA-templated Copper Nanoparticles

With the development of the nanotechnology, nanomaterials have attracted domestic and foreign researchers’interests due to their special advantages, such as size effect, quantum tunneling effect and surface effect, and exhibit a wide range of applications in the fields of materials science, chemistry, physics and biology. In particular, the ultra-small metal nanoclusters/nanoparticles (such as Au nanoclusters, Ag nanoclusters, Pt nanoclusters and Cu nanoclusters/nanoparticles) caused widespread research interest. Compared with fluorescent dyes and fluorescent quantum dots, fluorescent metal nanoclusters/nanoparticles have low toxicity, high fluorescence quantum efficiency, strong anti-photobleaching performance, tunable fluorescence emission and so on. Among these fluorescent metal nanoparticles, DNA templated copper nanoparticles exhibit some advantages such as simple preparation, low cost and good biocompatibility. So, a series of simple, fast, sensitive and novel biosensing methods based on DNA templated copper nanoparticles were fabricated for the detection of small biomolecules (such as dopamine, L-histidine and glucose) and thiol-containing drug (such as N-acetylcysteine). Moreover, combined with small molecule DNA terminal protection technology, a simple, speed and interchangeable fluorescent sensing strategy was developed for the detection of protein. The main contents are as follows:(1) A novel label-free fluorescent sensing method based on double DNA (ds-DNA) templated copper nanoparticles was developed for the detection of dopamine. The fluorescent copper nanoparticles were synthesized by using 25bp random ds-DNA as template and ascorbic acid as reducing agent, and the obtained nanoparticles was characterized by transmission electron microscopy. In the prescence of dopamine, the fluorescent of copper naoparticles was effectively quenched though the photo-induced electrons transfer between dopamine and copper nanoclusters. Under the optimized conditions, the proposed method could be realized the ultrasensitive and high selective detection of dopamine and showed a linear range from 0.1 to 10 nmol/L with the detection limit of 20 pmol/L. The proposed method was applied in the detection of dopamine hydrochloride injection samples.(2) A novel label-free fluorescent sensing method was fabricated for the detection of glucose by using H2O2-mediated fluorescence quenching of ds-DNA templated copper nanoparticles. The copper nanoparticles was obtained though a simple method by using ds-DNA as template in the room temperature, and used as fluorescent probes. In this strategy, the fluorescence intensity of ds-DNA templated copper nanoparticles was quenched effectively in the presence of H2O2. Similarly, glucose could be monitored indirectly based on the enzymatic conversion of glucose by glucose oxidase to generate H2O2. The strategy exhibited sensitive detection of glucose in a linear range from 0.05 μmol/L to 100 μmol/L with a detection limit of 12 nmol/L. In addition, the proposed method was applied in the detection of glucose in human serum samples. The result showed that the method possessed some advantages such as simple operation and free label.(3) A new label-free sensing method based on inhibition of ds-DNA templated copper nanoparticles was constructed for the sensitive detection of L-histidine. In the presence of L-histidine, the strong chelation interaction between L-histidine and Cu2+ could result in the inhibition of copper nanoparticles, which leads to the low fluorescence intensity. Under the optimized conditions, the proposed method could realize the rapid and sensitive detection of L-histidine in a linear range from 0.2 μmol/L to 100 μmol/L with the detection limit of 20 nmol/L. Furthermore, the method was successfully applied in the detection of L-histidine in human urine samples.(4) A simple and fast fluorescent sensing method was developed for the detection of thiol-containing drug by using poly (thymine){poly (T)} templated copper nanoparticles as fluorescent probes. Cu nanoparticles were synthesized by using poly (T) ss-DNA as template and ascorbic acid as reducing agent, which was characterized by transmission electron microscopy. In the presence of N-acetylcysteine, a coordination complex was formed though Cu-S metal-ligand bond between N-acetylcysteine and copper nanoparticles, which resulted in the effectively quenching of fluorescent copper nanoparticles. Under the optimized conditions, the proposed method could realize the sensitive detection of L-histidine in a linear range from 0.2 μmol/L to 100 μmol/L with the detection limit of 50 nmol/L.(5) A novel label-free fluorescent sensing method based on poly (T) templated copper nanoparticles and small molecule DNA terminal protection strategy was constructed for the detection of the protein. In this method, the poly (T) ss-DNA was designed with biotin at its 3’terminus and could be as a template for the formation of fluorescent copper nanoparticles. In the absence of target protein, the poly (T) ss-DNA labeled with biotin was hydrolyzed to yield mononucleotides by Exo I, which destroyed the template for the synthesis of copper nanoparticles, resulting in the low fluorescence intensity. In the prescence of protein, the poly (T) ss-DNA labeled with biotin could specifically bond to streptavidin with strong affinity, and the steric hindrance of 3’terminus was increased, which protected the template of copper nanoparticles from being hydrolyzed by Exo I, exhibiting high fluorescence intensity. The proposed method could realize the sensitive detection of protein in a linear range from 0.5 nmol/L to 100 nmol/L with the detection limit of 0.1 nmol/L. The result showed that the proposed method possessed high sensitivity, good selective and was free of complex labeling.