Supercharged Green Fluorescent Protein And Nano-materials Based Biosensors And Nano-materials Toxicity Analysis

Green fluorescence protein (GFP) is encoded and expressed by organism itself, whichis one of the most widely studied and exploited proteins in biochemistry and medicalscience. Recently discovered green fluorescent protein with a+36nettheoretical charge(supercharged GFP) is highly aggregation-resistant, quite stable in different pH, and coudcarry nucleic acid into mammanlian cells. The interaction between scGFP andnano-materials and further related biosensing application have never been exploited.Compared to the rapid development of the synthesis technology of nanomaterials and theirbroad application, the risk estimation of nano-materials to human health and environmenthas not yet been fully established.This work reported and analyzed the interaction between scGFP and graphene oxide(GO), and a novel lable-free fluorescentmethod for myoglobin detection was developedbased on this interaction. In addition, the toxic effect and the primary toxic mechanism ofcopper nanoparticles (CuNPs) were investigated using whole-cell bacterial biosensor. Thespecific works are summarized as follows:(1) The interaction among scGFP, DNA, and GO was firstly explored. It was foundthat scGFP could bind onto GO surface through electrostatic interaction and hydrophobicdomain interaction, according to the results of atomic force microscope, fluorescencespectrophotometry, and circular dichroism spectroscopy. Due to the attraction of scGFP,monolayer GO aggregated and formed multilayer-graphene/scGFP aggregates. In theseaggregates, scGFP was very close to GO, fluorescence resonance energy transfer (FRET)occured, and the fluorescence of scGFP was quenched by GO. Our previous work showedthat DNA and scGFP mixture can aggregate into big particles due to the electrostaticinteraction. Further study showed that the scGFP/DNA mixture particles were adsorbed onthe edge of GO, and there were no obvious GO aggregation and fluorescence quenching ofscGFP. We suspected that in scGFP/DNA particles, the hydrophobic domains and the mostcharges of scGFP were packaging inside, and only the positive charges on the suface of theparticles can interact with the hydroxy and carboxyl groups at the edge of GO. Because ofsteric hindrance, most scGFPs in the particles are too far away from GO to have FRET, andtheir fluorescence retain..(2) Based on the interaction between scGFP and GO, a novel immune-independent andlabel-free fluorescent aptasensor for effective sensing of myoglobin was developed. The strong interaction between scGFP and GO led to the fluorescence quenching of scGFP. Inthe presence of myoglobin aptamer (MA), scGFP and the aptamer formed big particles dueto the electrostatic interaction, which can protect scGFP from fluorescence quenching byGO. When there was myoglobin, MA specificly bounded with myoglobin and left scGFPalone. In this way, scGFP would bind to GO again, followed by the fluorescence quenching.The change of fluorescent intensity responded to the concentration of myoglobin very well.Such method has good selectivity, low detection limit (0.16μg/mL), and wide linear range(0.3μg/mL-10μg/mL). This method has been successfully applied for the myoglobindetection in the spiked serum sample, and satisfactory recoveries were obtained (between87.7and107.6%).Because scGFP and GO have good biological compatibility, this methodhas potential application in biological samples. Furthermore, when substituting themyoglobin aptamer with other aptamers, this strategy could be conveniently extended fordetection of other proteins and cancer cells.(3) The effect of morphology on toxicity of CuNPs are investigated based on astress-responsive whole-cell bacterial biosensor. Four kinds of copper nanomaterials,commercialized CuNPs, synthesized octahedral cage CuNPs, nanoflowers CuNPs, and10nm CuNPs were used. It was found that the particles showed different toxicity, and thetoxicity of the octahedral cage CuNPs was much higher than others.The production of Cu (I)from CuNPs wasfurther checked by2,9-dimethyl-1,10-phenanthroline (neocuproine, Nc)and it was found that the toxic effect of CuNPs with different morphologies were notrelated to Cu (I), but may depend on reactive oxygen species generation from CuNPs.