| Dopamine (DA) is one of the most important neurotransmitters in the central nervous system and also is a precursor of norepinephrine in the biological pathway. It plays an important role in cardiovascular and central nervous system as well as endocrine system. Shortage of DA is correlated with kinds of neurological disorders, such as Alzheimer, schizophrenia and Parkinson’s disease. On the other hand, excess of DA can cause a state of excitement or anxiety, which may relate with anxiety disorder and attention-deficit hyperactivity disorder. The level of DA in biological samples is of great significance for the diagnosis and pathological mechanism research of neurological disorders in clinical practice. Due to the low concentration level of DA existing in complicated biological samples, it is urgent to establish a highly sensitive and selective method for the determination of DA.Carbon dots (CDs), namely fluorescent carbon nanoparticles, are a kind of quasispherical nanocarbons with sizes below 10 nm. CDs not only exhibit the excellent optical properties of traditional semiconductor quantum dots (such as broad excitation and narrow emission spectrum, resistance to photo-bleaching, high photoluminescence quantum yield), but also possess the property of excitation wavelength dependence, good stability, low-toxicity and excellent biocompatibility. Till now, fluorescence quenching principle is widely adopted in the biosensor based on CDs fluorescent probes. However, this kind of biosensor has a great disadvantage that detecting the degree of fluorescence quenching will influence the sensitivity of the biosensor constructed in the context of strong fluorescence. In order to improve the sensitivity of the determination, more and more researchers have started to focus on the establishment of on-off-on biosensors.In this disseration, a novel on-off-on biosensor based on DA aptamer labeled CDs (receptor unit) and NG (quencher) was synthesized and applied successfully in the determination of trace DA in biological samples. The process of the study was categorized as following:1. The synthesis of Aptamer-CDs/NG biosensorThe carboxy groups on the surface of CDs interacted with the amino groups of DA aptamer after activation, thus DA aptamer was successfully modified on the surface of CDs. After the introduction of NG, the fluorescence of Aptamer-CDs was quenched. In the absence of DA, Aptamer-CDs were adsorbed on the surface of NG through π-π stacking and hydrophobic interaction, which led to the fluorescence quenching of Aptamer-CDs. When it was in presence, DA combined with its aptamer anchored on the surface of CDs to form specific globular chain structure which would detach from the surface of NG and thus the fluorescence intensity of Aptamer-CDs was recovered. According to the linear relation between the degree of fluorescence restoration and DA concentration, the level of DA can be quantified. Hereon, related experimental conditions such as the amounts and ratio of reactants, reaction time and temperature were optimized. Then, fourier transform infrared spectra and the high-resolution transmission electron microscopic images were used to confirm the successful synthesis of Aptamer-CDs/NG biosensor.2. The application of Aptamer-CDs/NG biosensorAn analytical method for the determination of DA in urine samples was established by a fluorescent biosensor based on Aptamer-CDs and NG. The interference study demonstrated that the Aptamer-CDs/NG biosensor possessed excellent selectivity. And under the optimal conditions, the relative fluorescence intensity versus the natural logarithm of the concentration of DA showed linear relationship. The calibration curve could be expressed by the equation (F-F0)/F0=0.0638×ln[DA]+0.2192 with the correlation coefficient of 0.9995 (where, F and Fo represented the fluorescence intensities of the biosensor in presence and absence of DA, respectively). The limit of detection (LOD) was evaluated to be 0.055 nM, which was defined as 3σ/k (where, a is the standard deviation of blank measurements (n=10), and k is the slope of calibration graph. The proposed method was applied to analyze DA in human urine. The amounts of DA in two healthy volunteers from laboratory after 60-fold-dilution were 0.75 nM and 0.80 nM, respectively. The recoveries of DA in spiked urine samples at four different levels were in the range of 96.0%-105.2%, and the relative standard deviations (RSDs) were within 18.7%(n=3). It showed that the analytical method could meet the needs of analysis of real bio-samples.The establishment of the proposed fluorescent biosensor is green and convenient, and it can be applied to the rapid determination of DA in complex bio-samples just after dilution. |
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