Synthesis, Properties And Biosensing Study Of Novel Organic Fluorescent Probes

Fluorescent probes showed significant scientific interests in the field of medicaldiagnosis and disease surveillance. This thesis reported the synthesis, properties andbiosensing study of novel organic fluorescent probes.1. Aptasensor based on aggregation-induced emission-active9,10-distyrylanthracene fluorescent probeWe report here a fluorescent biosensor for highly sensitive determination ofsingle-stranded DNA (ssDNA) with remarkable fluorescence enhancement, andlabel-free sensing of S1nuclease activity and inhibition in real-time based onssDNA-controlled self-assembly of9,10-distyrylanthracene (DSA) probe (BTAESA)with the aggregation-induced emission (AIE) property, thereby avoiding sophisticatedfabrication process and aggregation-caused quenching (ACQ) effect. Firstly, wesynthesized small molecule BTAESA (probe1), showing good water-solubility and AIEproperty. When BTAESA was binded to the ssDNA by the surpmoleculareffect-electrostatic interaction and hydrophobic effect, the fluorescence of BTAESA andssDNA system will be enhanced because of restricted intramolecular rotation inBTAESA. So BTAESA can be made as the effect fluorescent probe (Probe1) foraptasing. Then we study the fluorescence of Probe1in the presence of ssDNA with diferent base lengths varying from6-to24-mer. We found that longer ssDNA results inmore signifcant fuorescence enhancement, and a linear range is observed from6mer to24mer (R=0.998). We studied the fluorescence of Probe1in the presence ofssDNA24mer with different concentration, A linear range is observed from0to200nM(R=0.998), the detection limit could reach as low as150pM. We also studied thefluorescence of the compound system of Probe1and ssDNA in the presence of S1nuclease, we observed a linear correlation (R=0.995) between reaction rate of S1nuclease and the concentration of S1nuclease over the range of6-32U mL1. Moreover,the fluorescent intensity with various concentrations of S1nuclease becomes highlydiscriminating after3–16min. We also studied S1nuclease inhibitor (Na2ATP) for S1nuclease based on the fluorescence of the compound system of Probe1and ssDNA. Thedigestion of ssDNA24mer by S1nuclease is effectively inhibited by Na2ATP at theconcentration of0.4mM. Na2ATP was found to inhibit S1nuclease with IC50values of0.076mM. The assay present here was simple in design and offered a convenientprotocol for homogeneous, real-time and label-free determination with good linearrelation and high sensitivity.Besides, we also developed a label-free fuorescence turn-on approach for theselective sensing of Pb2+based on AIE-active probe1through Pb2+-induced allostericG-quadruplex (G4). The method shows simplicity, easy operation, cost-effectiveness,good sensitivity and good selectivity.2. Fluorescent aptasensing based on AIE probe and graphene oxideRecently, a great variety of AIE-active molecules have been utilized to designbioprobes for label-free fluorescent turn-on aptasensing with high sensitivity. In thiswork, a9,10-distyrylanthracene with two ammonium groups (DSAI) is synthesized as anovel AIE probe, which possesses two positive charges, thus showing good watersolubility. Besides, DSAI possess a non-planar conformational DSA structure. It showsweak fluorescence in their solutions but intense emission in their aggregate states. TheDSA moiety is the key factor of the AIE property due to the restricted intramoleculartorsion between the9,10-anthylene core and the vinylene segment. Upon addition ofssDNA aptamer (P1), the aggregation complex of DSAI and P1aptamer (DSAI/P1complex) are formed due to the electrostatic interactions between the ammonium cation in DSAI and backbone phosphate anions in P1, and the hydrophobic interactionbetween aryl rings in DSAI and nucleosides in P1. Accordingly, the fluorescence of thecomplex would be expected to be enhanced obviously owing to the restriction ofintramolecular rotation of DSAI in the aggregated state. Herein, DSAI is an effect probe(Probe2) for fluorescent aptasing.However, due to nonspecific binding interaction between aptamer and AIE probe,these AIE-based aptasensors have nearly no selectivity, thereby significantly limitingthe development. We studied the fluorescent change of DSAI/P1complex with theaddition of two differecent aptamer: targeted complementary ssDNA(T1), interferentialmismatched ssDNA (M1). P1is hybridized to form a duplex dsDNA with the additionof T1other than T1. We found the fluorescence of DSAI/P1complex will be enhancedwith addition of T1other than T1, therefore the DSAI/P1complex cannot recognize thetarget DNA. Then we imported the graphene oxide (GO), which can adsorb ssDNA andorganic probes.when ssDNA was hybridized with its complementary strand and formeddouble-stranded DNA (dsDNA), or hybridized with its targeted protein and formedG-quadruplex DNA (G4), the formed dsDNA or G4was desorbed from the surface ofGO. Then we studied the fluorescent changes of DSAI/P1/GO complex system with theaddition of T1and M1, and found that the fluorescent intensity of DSAI/P1/GOcomplex system wound be enhanced with addition of T1other than M1. Herein, wecould recognize targeted ssDNA based on AIE aptasensor with help of GO. Besides, theselectivity of the sensor described herein has also been determined by examining thefluorescent intensity of P1-DSAI-GO complex toward target complementary T1and thesingle-base mismatch ssDNA (SM1), the fluorescent intensity of SM1-P1-DSAI-GOcomplex is about84%of fluorescent intensity of T1-P1-DSAI-GO complex, showingthat the AIE/GO system can be as the fluorescent indicator for sensing of single-base inDNA.We also studied TB-DSAI-GO complex for targeted protein-thrombin. Whenthrombin was added into DSAI/GO/TB complex system, TB aptamer would be formedquadruplex structure and released from GO, the DSAI probe gathered on thequadruplex-thrombin complex exhibit the obvious fluorescence. However,TB-DSAI-GO complex showed a much lower fluorescent response to these control proteins. Herein, this aptasensor could easily recognized targeted protein.This DSAI and GO-based fluorescent aptasensor was developed for selective andsensitive sensing of targeted DNA and thrombin protein. Given its AIE property, highselectivity and sensitivity, this aptasensor can be also exploited to detect other targets.3. Self-assembled graphene quantum dots induced by cytochrome c: A novelbiosensor for trypsin with remarkable fluorescence enhancementGraphene quantum dots (GQDs) possess the advantages of both graphene and QDs,such as stable photoluminescence, high fluorescent activity, low toxicity, excellentsolubility and biocompatibility, so can be used in the field of bioimaging. However, asthe fluorescent probes, GQDs are rarely used in the biosensor field. Herein, we developa label-free fluorescent turn-on biosensor for trypsin based on the self-assembled GQDs.In this work, GQDs are found to possess self-assembled aggregation andfluorescencequenching by Cytochrome c (Cyt c), and trypsin can disturb theinteractions of GQDs-Cyt c complex and results in restoration of GQDs-based FL due totrypsin-catalyzed hydrolysis of Cyt c. Besides, trypsin cleaves peptide bonds of Cyt c tolysine and arginine residues, both of which make GQDs chemical reduction intoreducing-GQDs (r-GQDs) and subsequent increase in the FL intensity. Such anunlabeled GQDs-Cyt c complex can serve as a novel eco-friendly FL turn-on biosensorfor trypsin with remarkable fluorescence enhancement, as well as high selectivity andsensitivity...