Preparation Of Functional Carbon Quantum Dots And Its Application In Biological Activity Detection

CQDs have excent dispersibility, chemical stability and light resistance, easy modification, multi-color excitation dependent emission, low toxicity and good biocompatibility, therefore they are expected to replace dye probes, toxicity semiconductor quantum dots and low fluorescent polymer, play an increasingly important role in the field of chemical sensors, biosensors, biological imaging, drug delivery, photovoltaic devices, optical catalysis.The CQDs were synthesized by chemical oxidation of activated carbon using concentrated sulfuric acid and nitric acid. Chemical oxidation was chosen as the preparation method in this work because it could introduce a great deal of oxygen-containing groups including hydroxyl and carboxyl, which endow as-prepared CQDs excellent aqueous solubility and functional groups on the surface. The as-prepared CQDs can exhibit strong yellowish green fluorescence, and are highly soluble in water and most polar organic solvents. Dopamine functionalized CQDs were synthesized using dopamine and CQDs through condensation reaction,a novel fluorescent nanoswitch for PPi on the basis of carbon quantum dots through integrating competitive interaction among CQDs, Ce (Ⅲ) and PPi was designed; making use of hydrolysis of ALP towards PPi and ATP,ACP towards PPi and the aggregation and the disaggregation mechanism,we realise the real time detection of ALP and ACP; A fluorometric assay for acetylcholinesterase activity and inhibitor screening with carbon quantum dots was realized by taking advantage of the competitive interaction among CQDs, Cu(Ⅱ) and thiol functionalized carbon quantum dots with dopamine for tyrosinase activity monitoring and inhibitor screening was realized basing on the PET process. The main contents are as follows:(1) A convenient and reversible nanoswitch with the assistance of cerium ions based on aggregation and disaggregation of carbon quantum dots was developed to achieve highly sensitive detection of pyrophosphate ions. Two states including aggregation state and disaggregation state corresponding to fluorescence off and on signaling can be readily switched in a reversible way. The aggregation state is resulted from large nanoassembly formed by carbon quantum dots and cerium ions due to their strong complexation affinity. The disaggregation state comes from competitive combination between cerium ion and pyrophosphate ion. The PPi ion severs as the external chemical stimulus to CQDs/Ce (Ⅲ) nanoassembly,and its quantity is responsive to fluorescence intensity of the sensing system. Quantitative evaluation of PPi in a broad range from 0.3 to 29.7μM with the detection limit of 0.1μM can be realized in this way.The PPi detection in human HeLa cells was also assessed, and fluorescence switching between on and off can be readily observed in response to presence and absence of PPi.(2) A convenient and real-time fluorometric assay based on aggregation and disaggregation of carbon quantum dots (CQDs) was developed to achieve highly sensitive detection of alkaline phosphatase activity.Method 1:CQDs and pyrophosphate anions (PPi) were used as the fluorescent indicator and substrate for ALP activity assessment respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by copper ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, PPi can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to copper ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by re-dispersion of CQDs in the presence of ALP and PPi.Quantitative evaluation of ALP activity in a broad range from 16.7 to 782.6 U/L with the detection limit of 1.1 U/L can be realized in this way。Method 2:CQDs and adenosine triphosphate(ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching.Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way。This two methods both have high enough sensitivity for practical detection in human serum. They broaden the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.(3) A reversible fluorescence nanoswitch by integrating carbon quantum dots nanoassembly and pyrophosphate ion is developed, and a reliable real-time fluorescent assay for acid phosphatase (ACP) activity is established on the basis of the fluorescence nanoswitch. Carbon quantum dots(CQDs) abundant in carboxyl groups on the surface, nickel(II) ion and pyrophosphate ion comprise the fluorescent nanoswitch, which operates in the following way:the nanoassembly consisting of CQDs and nickel ions can be triggered by pyrophosphate ion serving as an external stimulus. At the same time, the fluorescence nanoswitch switches between two fluorescence states(off and on) accompanying shifts in their physical states aggregation and disaggregation. Based on the nanoswitch, the introduction of ACP leads to breakdown of pyrophosphate ions into phosphate ions and resultant fluorescence quenching due to catalytic hydrolysis of ACP toward pyrophosphate ions (PPi). Quantitative evaluation of ACP activity in a broad range from 18.2 U/L to 1300 U/L, with a detection limit of 5.5 U/L, can be achieved in this way, which endows the assay with sufficiently high sensitivity for practical detection in human serum and seminal plasma.(4)We present a convenient and reliable fluorometric assay for acetylcholinesterase (AChE) activity and inhibitor screening by taking carbon quantum dots (CQDs) as the signal reporter. The detection mechanism is based on the following facts:the aggregation of CQDs by copper (Ⅱ) ions results in its fluorescence quenching due to strong coordination of copper ions to carboxyl groups of CQDs; AChE can catalyze the hydrolysis of acetylthiocholine into thiocholine which can induce fluorescence recovery owing to stronger affinity between thiocholine and copper ions; when its inhibitor tacrine is present,AChE loses its catalytic ability for hydrolysis of acetylthiocholine, and thus the fluorescence remains quenched. With this convenient method, the activity of AChE with a concentration as low as 4.25 U/L and a broad linear scope ranging from 14.2 to 121.8 U/L can be assayed, which possesses adequate sensitivity for practical samples of human serum and seminal plasma. The promising application of the proposed method in AChE inhibitor screening was demonstrated.(5) we develop a convenient and real-time assay with high sensitivity for TYR activity/level monitoring and its inhibitor screening based on biocompatible dopamine functionalized carbon quantum dots (Dopa-CQDs). Dopamine with redox property was functionalized on the surface of carbon quantum dots to construct a Dopa-CQDs conjugate with strong bluish green fluorescence. When the dopamine moiety in Dopa-CQDs conjugate was oxidized to a dopaquinone derivative under specific catalysis of TYR, an intraparticle photoinduced electron transfer (PET) process between CQDs and dopaquinone moiety took place, and then the fluorescence of the conjugate could be quenched simultaneously. Quantitative evaluation of TYR activity was established in terms of the relationship between fluorescence quenching efficiency and TYR activity. The assay covered a broad linear range of up to 800 U/L with a low detection limit of 7.0 U/L.In addtion,fluorescence quenching extent of the probe was reduced in the presence of arbutin. It is also demonstrated that Dopa-CQD conjugate possesses excellent biocompatibility, and can sensitively monitor intracellular tyrosinase level in melanoma cells and intracellular pH changes in living cells.