Synthesis Of Peptide-programmed Quantum Dots For Biomedical Application

Background Colloidal semiconductor nanocrystals, known as quantum dots (QDs), have attracted significant attention due to their unique optical and electronic properties and many potential applications, including light-emitting devices, lasers, solar cells, and especially biological labeling and imaging. Several unequaled advantages over organic dyes such as size-tunable photoluminescence (PL) spectra with broad excitation spectra and narrow emission bandwidths, quantum dots may adjust the wavelength range of400nm-2000nm, coverage is very wide, from the ultraviolet to near-infrared region (NIR), Width of the absorption spectrum of the quantum dots and the continuous photoluminescence of the same wavelength can be simultaneously excited with the different size of the quantum dots to emit different colors of photoluminescence, it is possible to achieve the elementary excitations, polyhydric emission, applicable in the field of bio-imaging in the multicolor marked, high photoluminescent quantum yields (PLQYs), quantum dot molar absorption coefficient of up to106L/(mol-cm), which can produce strong fluorescence signal, excellent photostability, and superb resistance to photobleaching, quantum dots have a large Stokes shift and long fluorescence lifetime (20-50ns), background and other fluorophores can significantly distinguish by signal, so can be applied in the time-resolved optical imaging, make them an ideal candidate for biological labeling and imaging.Although the preparation of the highly fluorescent II-VI semiconductor QDs by the TOP-TOPO method has been well-developed. For biological applications, these hydrophobic QDs must be further modified by bifunctional thiol ligands, silica, or polymer coating to impart their water-solubility and biocompatibility. Unfortunately, these additional complicated procedures during modification resulted in a drastic increase of the dot size and a reduction in the QDs PLQYs, which is unfavorable for biological applications. To overcome these obstacles, direct water-based synthesis of QDs with thiols as capping ligands has been developed. After considerable effort, significant progress has been achieved and the qualities of QDs are comparable to those prepared by the organometallic route. CdTe QDs, some of the most extensively studied QDs, have been well studied and characterized and the fluorescence of CdTe QDs with different sizes can cover almost the whole visible spectral range due to strong quantum confinement effect. Despite the fact that CdTe QDs have found numerous applications ranging from electronics to sensors, their use for biological applications is still limited. This is because the release of toxic Cd2+ions from thiol-capped CdTe QDs in living cells can cause cytotoxicity. For bioimaging, the QDs emission wavelength should ideally be in near-infrared (NIR) region to improve tissue penetration depth and to reduce background fluorescence. Moreover, NIR fluorescent QDs for in vivo imaging not only need excellent optical performance, but also should be ultrasmall size, and free of extremely toxic elements, such as Cd, Pb and Hg. However, it is still challenging to prepare NIR fluorescent QDs with ultrasmall size and low toxicity for bioimaging application. Thus, it is imperative for the development of new, water-soluble, highly luminescent and low-Cd containing QDs with low cytotoxicity so as to realize their wide biomedical applications.The main subject of this study is the aqueous phase synthesis of quantum dots. Quantum dots were synthesized by changing the reaction conditions to optimize the conditions to find the best experimental conditions. Furthmore, we developed one step synthesis of peptide-programmed quantum dots. This peptide functionalized quantum dots can be applied directly on biological labeling and imaging. The synthesis method is simple, easy to operate, and safe, we test different choose four different types of peptides to demonstrate the versatility of the aqueous phase one-step synthesis of peptide-programmed quantum dots. We have compared absorption and emission of quantum dots with different reaction conditions, the fluorescence and colloidal stability of the quantum dots in different pH, salt concentration, cell imaging and cell toxicity tests to evaluate the quantum dots. Besides quantum dots which containing cadmium, non-toxic Zn-based and Ag-based quantum dots have been researched and compared with Cd containing QDs on the influence of cell activity, to be better in biological applications.Chapter1. Aqueous phase synthesis and properties of CdTe quantum dotsThis section mainly studied the aqueous phase synthesis of CdTe quantum dots. We carried out aqueous phase synthesis of CdTe quantum dots with hydrophilic coatings of1-thioglycerol, thioglycolic acid, mercaptopropionic acid, L-cysteine and acetyl cysteine, respectively. Influence of the Cd/Te mole, reaction time and different coatings on the optical properties of CdTe quantum dots were investigated. Through comparing the optical properties by absorption, emission wavelength, the fluorescence quantum yield and the full width half maximum, we found that QD with coatings of1-thioglycerol slowest growth and thioglycolic acid fastest-growing. CdTe quantum dots with coatings of1-thioglycerol have the narrowest FWHM and well monodisperse, higher quantum yield can also be seen when coatings of acetyl cysteine. Change the Cd, Te molar ratio, we found that Cd/Te ratio of1:1.23generated quantum dots FWHM is quite narrow and high quantum yield. Moreover, the fluorescent microscopy images showed the hydroxyl group on TG can be used to minimize nonspecific cellular binding of QDs.Chapter2. One-step synthesis of peptide-programmed QDs for biomedical applicationThis section on the basis of the first portion under the optimal reaction conditions to be tested. Major introduce the method of one-step synthesis of peptide-programmed QDs in aqueous phase. A typical synthesis of CdTe QDs is described as follows:First, sodium hydrogen telluride (NaHTe) was freshly made before each synthesis by dissolving0.025g sodium borohydride (NaBH4) in1ml deionized water and then0.040g tellurium powder was added into the NaBH4solution. This reaction was conducted at room temperature overnight in an eppendorf tube. CdCl2.2.5H2O (14.5mg) and12μl1-thioglycerol (1.25g/ml) were dissolved in 50ml ultrapure water and stirred for10min. The solution was adjusted to pH11.0by dropwise addition of NaOH solution (0.1M). A solution containing fresh NaHTe (10ul) was added to the above-mentioned solution (1ml) in an Eppendorf tube (1.5mL), and then a solution containing peptides(1mg/ml) was added. The reaction was conducted at100℃for1h and then gradually cooled to room temperature. For synthesis of CdHgTe QDs, CdCl2.2.5H2O was replaced with a mixture of cadmium chloride and Mercuric chloride with different ratio.1-thioglycerol [1] and specific-sequence peptides were used as co-ligands to stabilize the QDs. The hydroxyl group on TG can be used to minimize nonspecific cellular binding of QDs. The peptide consisted of three domains:a His6-tag (which preferentially bind to the surface of QDs over metal-affinity coordination), a tetramer of PEG (which was introduced as a spacer to reduce nonspecific interactions and improve solubility) and a functional group or a peptidic sequence of biological interest (which can be grafted on the terminal of the PEG). Peptide-programmed quantum dots had good application prospects in biosensing detection, biological labeling and imaging. UV-Vis absorption spectrophotometer, fluorescent spectrometer, Zetasizer Nano ZS (Malvern Instruments), laser scanning confocal microscope, transmission electron microscopy (TEM), microplate reader on the characterization of quantum dots in the course of the study. To investigate the absorption, the emission wavelength, and particle size, potential, intracellular imaging, intracellular labeling, cytotoxicity, morphology and monodisperse of the quantum dots.In conclusion, we provided a convenient method to prepare ready-to-use peptide-functionalized QDs with a high colloidal stability, efficient optical properties, and a small size of about6nm. This method is simple and reliable, does not involve complex bioconjugate chemistry or additional reagents that can impair the QD stability and fluorescence and is of a potential interest for high-throughput, rapid and reproducible biosensing detection and biomedical imaging.Chapter3. Non-toxic quantum dot researchZn-base and Ag-base quantum dot as a new type of quantum dots that is green and low toxicity have been studied more recent years. We synthesized Ag:ZnSe, Ag2Se, Ag2S quantum dots based on other groups reported and researched the characterization and cytotoxicity assays. For bioimaging, the QDs emission wavelength should ideally be in near-infrared (NIR) region to improve tissue penetration depth and to reduce background fluorescence. Moreover, NIR fluorescent QDs for in vivo imaging not only need excellent optical performance, but also should be ultrasmall size, and free of extremely toxic elements, such as Cd, Pb and Hg. However, it is still challenging to prepare NIR fluorescent QDs with ultrasmall size and low toxicity for bioimaging application. Silver chalcogenides are ideal narrow-bandgap semiconductor materials for preparing low toxicity NIR fluorescent QDs. Nevertheless, the small solubility product constant (Ksp=6.3×2510-50) and the fast crystal growth of Ag2S make it difficult to obtain ultrasmall Ag2S QDs. Even so, great progress has been made for preparing Ag2S QDs recently.Base on the synthesis of ZnSe, we doped Ag+in ZnSe quantum dots for synthesis Ag:ZnSe. Influence of the Zn/Ag/Se ratio, reaction time and ligands on the photoluminescence (PL) were investigated. The results showed that either change the reaction time or Zn2+/Ag+/Se2-molar ratio the emission wavelength of the Ag:ZnSe quantum dot almost all the same, only changing the fluorescence intensity. We also found that the dithiophosphate lyxose alcohol (DTT) or alpha-lipoic acid (DHLA) as ligands of QDs precipitation and no fluorescence. And when1-thioglycerol as ligands of QDs has a longer reaction time and the fluorescence intensity is weak. Therefore, glutathione as a surface-modifying agent is more desirable.Ag2Se quantum dots no fluorescence detected by fluorescence spectrometer, so it is temporarily unable to applications in biology.Influence of the Ag+/MPA ratio and reaction time the photoluminescence (PL) were investigated. Study found that the Ag+/MPA ratio of1:12and reaction time in7minutes is the optimum conditions of the synthetic Ag2S quantum dots. Study found that the silver nitrate and mercaptopropionic acid (MPA), a ratio of1:12when the wavelength tuning range is relatively large, and the fluorescence intensity; reaction time in7minutes, the quantum yield of the Ag2S quantum dots, so the above ratio, and timesynthetic Ag2S quantum dots optimum conditions. Cytotoxicity test Ag2S and CdTe quantum dots, the experiment proved once again that even if Ag2S quantum dots in a lot of concentration on cell activity almost no effect, but CdTe quantum dots or, Ag2S quantum point for the study of low or no toxicityquantum dots in biology has laid a good foundation. Comparing the cytotoxicity of Ag2S and CdTe quantum dots, Growth inhibition tests showed that Ag2S QDs do not exhibit any cytotoxicity toward MCF-7cells up to a concentration of120mg/L, indicating that these nanocrystals can be considered as building blocks for the synthesis of bioprobes for cells and tissues imaging.