||In this study, we used the advantages of good light stability, broad absorption band, high energy transfer and low biotoxicity, easy surface modification, based fluorescent semiconductor polymer nanoparticles (Pdots) to design the detection probe. Using the FRET (Förster resonance energy transfer) energy transfer mechanism, combined with Heptamethine cyanine as the radiation near infrared light sensor, developed to the ratiometric method to quantify pH values and quantification of glutathione.|
(i) Heptamethine cyanine Based Semiconducting Polymer Dots for Detection of Cellular pH.
The intracellular pH is very important for cell metabolism, and almost all proteins are based on pH values to maintain their structure and functionality. In addition, pH also plays a very important role in cell proliferation, apoptosis, drug resistance, phagocytosis, endocytosis and signaling.
In addition, it has recently been shown that the cytosolic acidity of cells creatine repair is also important. So the intracellular pH is very strict, as long as there is a little abnormal on behalf of some physical problems, such as cancer and Alzheimer's disease, the greater the deviation may even cause death. Therefore, if we can accurately understand and monitoring the entire cell pH in vivo is a very important breakthrough. However, the traditional pH quantitative methods such as electrochemical method because of poor spatial resolution, NMR is limited because of operational difficulties, So we have to develop a faster and more convenient way to detect. Which by fluorescence detection technology, with high resolution, non-destructive, able to design to achieve high selectivity, is currently used in cell research.
The ultimate goal of this fluorescent probe is to be able to achieve biological applications, so we use Heptamethine cyanine as a choice of dye, the use of its own structure with a long conjugate chain, modified with a electron-donating functional group based piperazine . The use of it in the acidic environment will be protonated, reducing the ability to donate electrons and extend the conjugate chain to absorb redshift phenomenon. Therefore, in this study, we use the advantage of Pdots easy - to - make the surface modification characteristics, in the polymer side we design of COOH functional groups, and in the near-infrared dye part is modified with NH2 functional groups. The FRET energy transfer mechanism was used to calibrate the pH value of the detective substance.
Keywords : Semiconducting polymer dots、Near-infrared dyes、pH sensing、FRET、Fluorescence ratiometric analysis
( ii )Heptamethine cyanine Based Semiconducting Polymer Dots for cellular Quantification of Glutathione
GSH is the most abundant bio-mercaptan in cells, the concentration range of 1~15 mmol / L. GSH is involved in many pathology and biology processes and plays an important role in maintaining the balance of intracellular redox states. ROS(reactive oxygen species), and different concentrations of GSH are also associated with a variety of cellular functions, including biological metabolism, gene regulation, intracellular signaling, stress response, immune response, and resistance to cancer、radiation treatment and chemotherapy. Therefore, in biochemical research and related diseases, so there is a great need to be able to know the cellular environment and the GSH concentration in vivo.
High performance liquid chromatography (HPLC), capillary electrophoresis, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LCMS, Liquid chromatography-mass spectrometry) Has been used in the detection of bio-mercaptan, but the above method is time-consuming and too expensive. In contrast, the appropriate probe combined with fluorescent technology is more dominant because of its non-destructive and high sensitivity, and can provide the location of the bio-mercaptan in the number and the location. Mostly based on 400nm-600nm of the radiation wavelength, such as coumarin, BODIPY, rhodamine, etc.. And fluorescent probe in vivo imaging wavelength range required in the NIR (650-900nm) is more appropriate and near infrared Light-emitting light can avoid spontaneous fluorescence of biological cells, in order to achieve a higher signal to noise ratio, and the advantages of small damage to biological samples. Therefore, in this study, we try to design a GSH detectable near-infrared light probe, modified on the Heptamethine cyanine with azobenzene functional group, as a light induced electron transfer (PET, photoinduced electron transfer) mechanism to dye quenching phenomenon. Using Semiconducting Polymer Dots to achieve the effect of signal amplification. In the presence of GSH environment, will replace the functional group, the original azobenzene cut off the dye and fluorescence recovery for quantitative analysis.