Synthesis of highly fluorescent GSH-CDTE nanoparticles (quantum dots)

Abstract

The invention relates to a method for the synthesis of glutathione-capped cadmium-telluride (GSH-CdTe) quantum dots in an aqueous medium, including the steps of: a) preparing a precursor solution of cadmium in a citrate buffer; b) adding glutathione (GSH) to the preceding mixture via strong agitation; c) adding a telluride (potassium or sodium telluride) oxyanion as a telluride donor to the preceding mixture; d) allowing the preceding mixture to react; and e) stopping the reaction by incubation at low temperature.

Claims

1. A synthesis method in aqueous medium of cadmium-tellurium quantum dots joined with glutathione (CdTe-GSH), CHARACTERIZED in that it comprises the steps of: a. preparing an aqueous soluble cadmium salt solution in a buffer, the aqueous soluble cadmium salt solution having a pH of 9-13, the buffer being selected from the group consisting of a citrate buffer, a phosphate buffer, a Tris-HCL buffer, a Luria Bertani (LB) bacterial culture media buffer, and a MP buffer; b. adding glutathione (GHS) to the cadmium salt solution while applying agitation to from an agitated solution; c. adding a tellurium oxyanion as a tellurium donor, to the agitated solution of step b, the tellurium oxyanion being selected from the group consisting of sodium tellurite and potassium tellurite to from a composition; d. heating the composition from step c from 37° C. to 130° C. to from a reaction to provide a reactant solution; e. stopping the reaction by cooling the reactant solution to 4° C. to from a cooled solution; and f. incubating the cooled solution for at least 30 minutes.

2. The method according to claim 1, CHARACTERIZED in that in said step a, the cadmium salt in the aqueous soluble cadmium salt solution is selected from the group consisting of CdCl.sub.2, CdSO.sub.4 and Cd(CH.sub.3CO.sub.2).sub.2.

3. The method according to claim 1, CHARACTERIZED in that the composition of step c comprises CdCl.sub.2:GSH:K.sub.2TeO.sub.3 at a ratio of 4:10:1.

4. The method according to claim 1, CHARACTERIZED in that in step d, the reaction time is not greater than 24 hours.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent of application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows the absorption spectrum of the QDs which have been generated by the method of the present application. A) Aliquots of the synthesis mixture of CdTe-GSH nanoparticles, taken at different times of reaction (0, 1, 3, 6, 12, 18 and 24 h of treatment at 60° C.). B) Absorption spectra of the aliquots which have been obtained after at 1, 6, 12, 18 and 24 hours.

(3) FIG. 2: Fluorescence spectrum of the nanoparticles present in aliquots obtained at different times.

(4) FIG. 3: Aliquots exposed to UV light (trans-illuminator).

(5) FIG. 4: SDS-PAGE of the anti S. Typhi antibody joined to CdTe-GSH revealed with Comassie blue or evaluated by fluorescence in an UV (A) transiluminator. A Dot-Blot was prepared with the Ab+NPs complex, and the microorganism were detected by forming the complex, and the fluorescence associated with it (B). As corresponds to the antigens to be detected (S. Typhi or S. Typhimurium), PBS is the saline phosphate buffer.

DETAILED DESCRIPTION OF THE INVENTION

(6) The synthesis of nanoparticles (NPs) or quantum dots (QDs) of CdTe-GSH in aqueous phase is conducted following this protocol:

(7) An aqueous CdCl.sub.2 solution is prepared (it can be any other salt of Cd.sup.+2 salt, such as cadmium sulphate, acetate or per-chlorate) up to a final concentration of 4 mM, in 50 ml 15 mM citrate buffer (it can be also Tris-HCl, phosphate, borax citrate Luria Bertani bacterial culture media or M9, among others), at pH9 (the pH may vary in the range of pH 9-13), at ambient temperature. M9 is a culture medium of microorganisms with the following composition:

(8) For 1 L of M9 medium, salts 100 mL 10× autoclave (Na.sub.2HPO.sub.4 anhydrous 60 g, KH.sub.2PO.sub.4 30 g, NaCl 5 g, NH4Cl 10 g in 1 L of nanopure water pH 7.2), nanopure sterile H.sub.2O 894 mL, MgSO.sub.4*4 mL 7H2O, CaCl.sub.2*0.1 mL 2H2O 1 M, glucose 20% 10 mL and hydrochloride thiamine 3 mL 0.1 M is required. MgSO.sub.4*7H2O, CaCl.sub.2*2H.sub.2O, glucose and the hydrochloride thiamine are all previously sterilized with 0.2 μm filters under a laminar flow cabinet.

(9) GSH up to a final concentration of 10 mM is added, under strong stirring or agitation (avoiding the forming of a white precipitate of Cd°).

(10) After 5-10 minutes ( ) a tellurium oxyanion is added as K.sub.2TeO.sub.3 (or Na.sub.2TeO.sub.3) at a final concentration of 1 mM. The component ratio in the final synthesis mixture CdCl.sub.2:GSH:K.sub.2TeO.sub.3 is 4:10:1, (however, the synthesis also allows other ratios as 1:2:1, and 6:10:1). At this stage, the solution turns to a light green color, which is indicative of the generation of the first nanoparticle “seeds”, that will be able to start the nucleation of the QDs, and increasing their size as the treatment time increases.

(11) To start the nucleation process (synthesis) of the QDs, the mixture should be heated up to 90° C. (T°); (the protocol is valid in the temperature range of 37-130° C.). The synthesis kinetic (velocity) of the QDs is proportional to the T° of the test: to a higher temperature (T°) corresponds a higher production kinetic of big sized QDs.

(12) From this point onward, the color of the solution changes with time, and its spectroscopic properties (absorption and fluorescence) vary as a consequence of QDs formation At different times samples can be collected to obtain QDs having the desired color and/or size. If the synthesis is conducted at 90° C., after 4 hours, the solution has presented different colors, and it stabilizes in a red color, which indicates the presence of the CdTe-GSH NPs of bigger size. The sizes of the QDs are comprised in the range of 2.5-3 nm diameter at the first time (green color) up to approximately 5-6.5 nm diameter for the red color suspension.

(13) If the synthesis is conducted at lower temperatures, for example 60° C., after 2 h the solution is a green fluorescence, and after approximately 20 h it turns to a reddish color (during this time, the solution has changed to different colors, different levels and intensities of green, yellow and red; as it occurs in the system at 50° C.).

(14) To stop the reaction it is necessary to reduce the temperature of the solution to 4° C. (keep the tubes in a glass case or in ice during at least 30 min). To maintain the properties of the synthesized QDs they should be stored at low temperature and in the dark. However, NPs which have been synthesized with this method, remain fluorescent for at least 6 months, when exposed to room temperature and/or to day light. To store and determine the mass of the synthesized QDs, it is possible to precipitate nanoparticles from the synthesis solution (or suspension) by treating it with 2 volumes of isopropanol and centrifuge during 20 min at 13,000× g. In this way, a highly fluorescent precipitate is obtained, which corresponds to CdTe-GSH QDs.

(15) Characteristics of the QDs Generated According to this Protocol

(16) With this process it is possible to obtain CdTe-GSH nanoparticles with characteristic absorption and fluorescence spectra (see FIGS. 1 and 2, respectively). The synthesized QDs have an absorption that varies in the range of 450-560 nm, with emission in the range of 500 and 650 nm (in other synthesis protocols ranges between 520 and 620 nm have been reported). The absorption maximum has a width of approximately 40-50 nm, whereas the emission maximum is comprised between approximately 50-70 nm.

(17) The quantum efficiency of the produced QDs, according to this protocol is approximately 25-30%, depending of the nanoparticle size, being similar with that described in other synthesis methods.

(18) The composition of the nanoparticles was estimated by means of an EDAX analysis (Energy Dispersive X-Rays Analysis). It was determined that they contain approximately 35% C, 12% 0, 15.5% N, 5% S, 23.6% Cd, and 72% Te. The C, O, N, and S contents are those expected for the QDs which comprise a tripeptide as GSH. The Cd:Te ratio: was 3.3:1, which agrees with that described for CdTe-GSH nanoparticles synthesized by other methods. Together with the above cited analysis, an Atomic Force Microscopy (AFM) analysis and Dynamic Light Scattering (DLS) determined that the size of the QDs vary between 3-6 nm. The green, yellow, and red QDs displayed diameters of approximately 3, 4.2, and 5 nm, respectively.

(19) A direct application of these QDs, is their joining to a protein, particularly to antibodies, and their use in a one-step detection of specific antigens.

(20) The CdTe QDs were synthesized at 60° C. during 6 h, by using 15 mM of citrate buffer, at pH 9.0, 1 mM K.sub.2TeO.sub.3, 4 mM CdCl.sub.2 and 10 mM GSH, according to the previously described instructions.

(21) The CdTe-GSH QDs were joined to the protein with the help of 2 imino thiolane. The protein was an antibody (Ab) recognizing the food-contaminating agent Salmonella enterica serovar Typhimurium. This complex Ab+NPs was used for detecting the pathogenic agent. FIG. 4 shows that Ab+NPs complex, fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions stained with Comassie blue (4A) and revealed in an UV transluminator to determine protein fluorescence s (4 B). A Dot blot was made, using the Ab+NPS complex, and associated fluorescence was observed with the complex, specifically in those dishes where protein extracts of the microorganisms to be detected were added (FIG. 4B). This novel method, based on our QDs is highly specific and allows detecting antigens in only one step, since it does not require the use of secondary antibodies, nor developing solutions.