WO2016090509A1 - Aqueous synthesis of nanoparticles of cadmium sulphide - Google Patents

Aqueous synthesis of nanoparticles of cadmium sulphide Download PDF

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Publication number
WO2016090509A1
WO2016090509A1 PCT/CL2014/000075 CL2014000075W WO2016090509A1 WO 2016090509 A1 WO2016090509 A1 WO 2016090509A1 CL 2014000075 W CL2014000075 W CL 2014000075W WO 2016090509 A1 WO2016090509 A1 WO 2016090509A1
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phosphate
nps
cds
qds
concentration
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PCT/CL2014/000075
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Spanish (es)
French (fr)
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José Manuel PEREZ-DONOSO
Felipe VENEGAS FAUNDEZ
Juan Pablo MONRÁS CHARLES
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Universidad Andrés Bello
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G11/00—Compounds of cadmium
    • C01G11/02—Sulfides
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/54—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing zinc or cadmium

Definitions

  • the present invention relates to a method of production in aqueous medium of semiconductor and fluorescent nanoparticles (hereinafter NPs) (called quantum dots, hereinafter QDs) of cadmium sulphide (CdS), using phosphorylated molecules in the synthesis process.
  • NPs semiconductor and fluorescent nanoparticles
  • QDs quantum dots
  • CdS cadmium sulphide
  • the proposed method allows to produce semiconductor and fluorescent NPs, of the type, Qds of CdS, by means of a methodology that includes adding an inorganic salt containing Cd.
  • the proposed method comprises mixing a thiol as a source of sulfur and as a wrapping agent, adding cadmium chloride, and including a compound containing inorganic phosphate (P ⁇ ).
  • P ⁇ inorganic phosphate
  • the objective of the addition of a phosphate group donor compound within the reaction is to favor the formation of CdSa QDs at low temperatures (between 15 and 80 ° C) and in a shorter time, since phosphate is the base of the synthesis since it is the initiating element of the nucleation of CdS crystals.
  • phosphate is the base of the synthesis since it is the initiating element of the nucleation of CdS crystals.
  • it is also possible to develop the synthesis using phosphorylated molecules found in biological organisms such as, but not limited to, glucose-6-phosphate, glycerol-2-phosphate, fructose-1, 6-bisphosphate, adenosine monophosphate, among others.
  • the NPs of CdS produced by the method of the present invention have advantageous characteristics: they are semiconductors,
  • the described method corresponds to a simple and efficient methodology, since first a lower concentration is used and therefore a smaller quantity of certain reagents, such as the CdCI 2 salt.
  • the presence of phosphate within the synthesis procedure favors the formation reaction of the CdS QDs allowing the synthesis to occur in a shorter period of time and temperatures lower than other methods described.
  • the latter makes the method simpler, avoiding intrinsic complications related to work at high temperatures such as the possible decomposition of some reagents, and more importantly, avoiding possible accidents of the user, also dispensing with inert atmospheres such as Ar or N 2 .
  • the fact that the method can be carried out at low temperatures, thanks to the presence of phosphate allows to dispense with machinery and equipment of incubation at high temperatures.
  • the QDs of CdS produced by this method have thiol and phosphate groups on their cover, which gives them greater solubility, and therefore, gives them benefits in terms of in vivo use if they wanted to incorporate the QDs into cells or organisms. Additionally, by being able to incorporate phosphorylated intermediates into the synthetic process, the NPs can eventually be directed to a particular cell receptor to mark cells, and even, allow visualizing the amount of these receptors by fluorescence microscopy (exciting the QDs).
  • CdS QDs produced by the disclosed method Another possible application of the CdS QDs produced by the disclosed method is its use in the localization of enzymes capable of recognizing phosphorylated substrates, since the QDs could bind to this type of enzyme, and be monitored live / n inside a cell . Also the QDs produced by this method could be used in solar cells sensitized by QDs (QDSSC).
  • WO 2012090161 A1 refers to a method for the synthesis of CdTe-GSH QDs in an aqueous medium.
  • the method comprises the steps: a) preparing a cadmium precursor solution in a citrate buffer solution, b) adding GSH to the preexisting mixture by stirring, c) adding a tellurium oxyanion (potassium or sodium telluride to the previous mixture) ) as a tellurium donor, d) wait for the mixture to react, e) stop the reaction by incubating at low temperature.
  • the cadmium precursor corresponds to a soluble cadmium salt (CdCI 2 , CdSO4 or Cd (CH3CO2) 2) and the buffer solution can correspond to a buffer solution of citrate, phosphate, Tris-HCI and microorganism culture media such as LB ( Tryptone, NaCI and yeast extract) or M9 (Na 2 HPO 4 , KH 2 PO4, NaCly NH 4 CI).
  • the reaction temperature is 37- 130 ° C
  • This document points to the synthesis of CdTe QDs with a GSH cover as a weathering agent, and does not refer to the synthesis of CdS.
  • US 8491818 B2 presents among the scope of the invention the aqueous preparation of CdS QDs.
  • the methodology proposed in this document basically consists in reacting an aqueous solution of Cd (NO3) 2 with MSA (mercaptosuccinic acid), under stirring, and adding NH 4 OH to adjust to a pH close to 7-9. Subsequently, a certain amount of aqueous Na 2 S solution is added under stirring for 3-5 min. The entire reaction is carried out in an oxygen free environment. After this the suspension is ultrasonic and filtered, to be incubated at 0 ° C and then stored at 4 ° C.
  • the scope of the invention includes the synthesis of CdS QDs, the use of inorganic phosphate or molecules presenting phosphate groups is not established in the methodology. The only thing similar with this method could arise of the idea of using thiols as cover agents, however, in the present invention the thiol compound in addition to being used as a cover agent, is also used as a source of the sulfide necessary to form the CsS NPs.
  • CN103215042A a method of synthesis of CdSe QDs in aqueous phase is disclosed, which generally comprises mixing CdC, sodium selenite and MSA (mercaptosuccinic acid), adding a buffer solution, stirring, adjusting pH to 5, then adding NaBH 4 , heat at a temperature of 60 ° C, and allow to react for one hour.
  • the resulting QDs correspond to QDs of type MSA-CdSe.
  • the methodology includes the use of a stabilizing or covering agent derived from a compound containing thiol groups in its structure
  • a stabilizing or covering agent derived from a compound containing thiol groups in its structure
  • WO 2013019090 A1 discloses a composition that includes hydrophilic NPs that have adhered to their surface monosaccharides-phosphate or a derivative thereof, a colloidal solution of the composition, and a contrast agent of RMI (magnetic resonance imaging) that includes the colodial solution.
  • hydrophilic NPs are disclosed that have a surface modified by the addition of monosaccharide phosphate molecules or derivatives thereof on the surface of inorganic particles.
  • the NPs correspond to inorganic particles that can be of the type metal, chalcogenic metal, metal oxide, magnetic materials, magnetic alloys, semiconductor materials and hybrid structures, without reference to NPs formed by cadmium.
  • the NPs generated from the method described in WO 2013019090 have phosphate groups on their cover, the synthesis methodology is different from the one proposed here. invention.
  • the preparation of the NPs is carried out separately from the binding of sugar-phosphate molecules, without the addition of a phosphate group donor molecule being an important component in the synthesis reaction.
  • the addition of phosphate as part of the synthesis procedure favors the reaction efficiency, and allows it to be carried out at room temperature and in the presence of oxygen, the addition of phosphate having a totally different purpose. .
  • the production protocol of the QDsCdS-GSH described includes mixing a solution of CdCI 2 with water and adjusting the pH to 10.2, saturating with N 2 bubbles for about 20 min and then adding GSH (Cd ratio : GSH 1: 1) to the CdCI 2 solution, and keep stirring and constant heat low in an N 2 atmosphere for 10 min.
  • the methodology described in the cited scientific articles differs from the method proposed here since a phosphate group donor molecule is not included in the synthesis reaction.
  • the addition of this type of reagent considers a proposal with characteristics not previously described, since it allows the production of QDs at low temperatures (including room temperature) and the possibility of performing the synthesis in the presence of oxygen. Therefore the The method proposed here is based on the use of phosphate for the formation of the nanostructure.
  • the method is presented as a general protocol for CdZnS structures by mixing cadmium and zinc acetate as precursors.
  • the invention described in this document aims at the formation of QDs by means of a chemical synthesis to which phosphorus-containing compounds are added but which do not consider the release or formation of phosphate groups as part of the NP envelope, not least as a reaction enhancing agent.
  • characteristics of the proposed method such as, performing the synthesis at room temperature, in the presence of oxygen and in less time to produce CdSle QDs, gives the methodology characteristics not described above.
  • the NPs produced by this method have unique characteristics in terms of their solubility and bioavailability, which allows their use in the in vivo monitoring of cell markers, having a potential use in the area of medicine and research.
  • Figure 1 Effect of phosphate, cadmium and temperature in the in vitro synthesis of Qds of CdS.
  • the solutions contained 10 mM P ⁇ (inorganic phosphate), 54.5 ⁇ C CdCI 2 and 5 mM GSH and were incubated at 37 ° C for 1 to 4 days.
  • the solutions contained 10 mM Pi, CDCI February 30, 150 and 300 ⁇ and 5 mM MSA.
  • the incubation time ranges from 1 to 6 days for 37 ° C, and 0.5; 1.0; fifteen; 2.0; 2.5; 3 and 4 h for 80 ° C.
  • the samples contain 1 mL of final volume and were exposed to a UV transilluminator (at 365 nm).
  • Figure 2 Characterization of NPs produced in vitro.
  • a and B Absorbance and fluorescence spectra (350 nm excitation) of blue NPs (solid line) and orange NPs (dashed line).
  • Figure 3 FTIR spectrum of NPs synthesized in vitro.
  • FIG. 4 Synthesis of cadmium sulfide QDs using phosphorylated organic molecules.
  • the synthesis of QDs was evaluated using different phosphorylated molecules as a phosphate source: adenosine triphosphate (ATP), adenosyphosphate (ADP), adenosine monophosphate (AMP), Fructose 1, 6 bisphosphate (F1, 6 BP), glucose-2-phosphate (G2P) , glucose-6-phosphate (G6P).
  • ATP adenosine triphosphate
  • ADP adenosyphosphate
  • AMP adenosine monophosphate
  • F1, 6 BP F1, 6 bisphosphate
  • G2P glucose-2-phosphate
  • G6P glucose-6-phosphate
  • the present invention relates to a method of synthesis of fluorescent semiconductor NPs (QDs) of CdS.
  • the proposed method consists in the production of CdS QDs by a process in which phosphorylated molecules are used as a phosphate source, which is carried out in an aqueous medium, in the presence of oxygen, at room temperature, and more particularly, at temperatures between 15 and 80 ° C.
  • the proposed method comprises the steps of: a) Mix: i. A thiol as a source of sulfur and as a wrapping compound; ii. A cadmium salt; iii. A compound that contains or generates a phosphate source; b) Add a buffer solution to the mixture obtained in step (a), at a pH between 7-10, in the presence or absence of oxygen and at a temperature between 15-80 ° C; c) Incubate the solution obtained in (b) at a temperature between 15-80 ° C, for an incubation period of 1-9 days, until fluorescence is obtained. d) Evaluate the fluorescence of CdS NPs, exposing them to a UV transilluminator (at 365 nm).
  • the regulation of the concentrations and proportions of the reagents allows to control the type of fluorescence (color) and the size of the NPs of CdS.
  • the reaction mixture is carried out in an aqueous environment at a pH between 7-10 thanks to the addition of a buffer solution that allows maintaining this pH, in the presence or absence of oxygen and at low temperatures.
  • a buffer solution that allows maintaining this pH, in the presence or absence of oxygen and at low temperatures.
  • different buffer solutions commonly used in Molecular Biology and Biochemistry, can be used at a concentration of 1 to 100 mM, which have a cell pH of 7.4, which include:
  • PBS phosphate buffered saline: KH2PO4 / K2HPO4.
  • Tris-HCI Tris: (HOCH 2 ) 3 CNH 2 ).
  • the preferred buffer corresponds to the PBS buffer at a concentration of between 1 to 100 mM, preferably 10 mM, as it showed the best results (data not shown).
  • the reaction mixture comprises: i. A thiol as a source of sulfur and as a wrapping compound of the QDs, in concentration ranges between 0.25-15 mM, such as:
  • a cadmium salt at a concentration of 30-300 ⁇ such as:
  • a phosphorylated compound or molecule as a phosphate source at concentrations between 10 ⁇ and 100 mM, such as:
  • G2P Glycerol-2-phosphate
  • ADP Adenosine diphosphate
  • the reaction mixture comprises mixing a PBS buffer solution at a concentration of 10 mM with a solution of CdCI 2 at a concentration of 54.5 ⁇ , a solution of MSA at a concentration of 5 mM and one glucose-6-phosphate solution at a concentration of 10 mM.
  • the mixture is incubated at 37 ° C for 1-9 days to assess its fluorescence when exposed to a UV transilluminator (at 365 nm).
  • the reaction mixture is filtered on Amicon filters with a pore size of 3 kDa ( 0.3-0.5 nm) and centrifuged in a range of 3000 and 10000 G (preferably at 7000 G) until a solution concentrated in NPs is obtained, preferably for 30 minutes, or more depending on the initial reaction volume.
  • the concentrated solutions are washed with miliQ distilled water by the same centrifugation procedure in the Amicon tubes.
  • thiol refers without being limited to GSH, MSA, CYS, among others.
  • the thiol compound corresponds to the source of sulfur for the formation of CdS QDs, and is also part of the shell that surrounds NPs.
  • the method works using other thiols such as: dithiothreitol, thioglycolic acid, among others, at different operating temperatures and with a variable production yield.
  • phosphorylated molecules When it refers to phosphorylated molecules, it corresponds to molecules that contain at least one phosphate group in their structure, and may be, without limitation, phosphorylated molecules such as; glucose-6- phosphate, glycerol-2-phosphate, fructose-1, 6-bisphosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, among others.
  • the proposed methodology makes it possible to synthesize QDs of CdS more efficiently, in the presence of oxygen and in low temperature conditions.
  • low temperatures they correspond to temperatures between 15 and 80 ° C. More particularly, it refers to lower temperatures at 65 ° C.
  • the temperature for carrying out the methodology is 37 ° C.
  • the quantum dots of CdS generated from the presented method have particular characteristics with respect to other nanoparticles.
  • the QDs produced are composed of cadmium, sulfur, organic molecules corresponding to the thiol sheath but also to phosphate molecules on its surface.
  • X-ray diffraction confirmed that they corresponded to cadmium sulphide crystals.
  • the CdS QDs synthesized from the method of the invention have important advantageous characteristics and potential utilities. Due to their cover composed of thiol-like molecules and phosphate groups, these QDs are biocompatible. The latter allows the inclusion of CdS QDs in cells, and can be monitored according to the affinity in interaction of phosphorylated groups and phosphate groups with certain receptors and cellular enzymes.
  • the synthesized QDs can be used in the detection and monitoring of receptors, proteins, enzymes and cellular structures in cell culture in vitro, also in the marking and monitoring of receptors, proteins, enzymes and cellular structures in a way in vivo and as imaging probes. Examples:
  • Example 1 Procedure for preparing cadmium sulphide QDs in vitro and evaluating the concentrations of Pi and CdCI ⁇ and incubation temperatures in synthesis efficiency:
  • This example describes the methodology for the preparation of CdS QDs and the evaluation of different concentrations of the inorganic phosphate and CdCI 2 components in the reaction mixture. Additionally, the most efficient incubation temperatures for the method were determined.
  • the proposed Quantum dots synthesis procedure of CdS has the following steps: a) Mix: i. A thiol as a source of sulfur and as a wrapping compound; ii. A cadmium salt; iii. A compound that contains or generates a phosphate source; b) Add a buffer solution to the mixture obtained in step (a), at a pH between 7-10, in the presence of oxygen and at a temperature between 15-80 ° C; c) Incubate the solution obtained in (b) at a temperature between 15-80 ° C, during an incubation period of 1-9 days, if a temperature of 80 ° C is used the reaction occurs in hours, until obtaining fluorescence.
  • the main components in the proposed synthesis methodology are inorganic phosphate and cadmium salt.
  • inorganic phosphate and cadmium salt were evaluated as cadmium salt.
  • inorganic phosphate (Pi) was used in the form of K 2 HP0 4 , mercaptosuccinic acid as a source of thiol since it is the fastest thiol in the appearance of fluorescence and less precipitation (data not shown), and the concentration was varied of P ⁇ like that of Cd and the synthesis temperature.
  • the incubation period ranges from 1 to 6 days for a temperature of 37 ° C, and 0.5; 1.0; fifteen; 2.0; 2.5; 3 and 4 h for a temperature of 80 ° C.
  • the samples contain 1 ml_ of final volume and were exposed to a UV transilluminator (at 365 nm).
  • the P ⁇ is capable of generating solutions that change fluorescence color over time (Fig. 1A), and that the concentration of P ⁇ regulates the fluorescence color obtained at a fixed time (in mM concentrations) (Fig. B).
  • the amount of Cd added also regulates the fluorescence color obtained (in ⁇ concentrations) (Fig. 1 C), fluorescence being observed after one day when low temperatures are used, 37 ° C and even up to 15 ° C ( data not shown), or at incubation hours, at temperatures of 80 ° C (Fig. 1 C).
  • Example 2 Characterization of cadmium sulphide QDs produced using compounds of the thiol type as envelope and inorganic phosphate in its synthesis:
  • the spectroscopic properties of the synthesized NPs were determined using a spectrophotometer / fluorimeter with plate reader, Synergy H1 (Biotek).
  • the absorbance profile of solutions Concentrates of QDs synthesized in vitro were measured between 300 and 700 nm, and for recording the fluorescence emission they were excited at 350 nm with a 100% gain and the fluorescence collected between 400 and 700 nm.
  • the DLS measurements in the NPs were carried out in 4-step disposable plastic cuvettes using a Zetasizer Nano ZS dispersion kit (Malvern Instruments Limited, UK).
  • Electron microscopy confirmed the presence of agglomerated particles smaller than 20 nm (Fig. 2E), therefore a dilution of these allowed to observe individual NPs with sizes ranging from 5 to 12 nm (Fig. 2F), for an orange solution of QDs.
  • FTIR Fourier transform infrared spectroscopy
  • the QDs of CdS produced were also characterized according to their IR spectrum. For this, samples of QDs were taken, which were lyophilized to dryness to remove water from the solution.
  • the NPs powder was mixed with KBr, making tablets that were measured in a solid FTIR equipment, Spectrum Two (Perkin Elmer), between 4000 and 400 cm “1. Additionally, the relevant controls were performed, measuring the IR spectrum of the separate reagents (thiols, phosphate, CdCI 2 , phosphorylated molecules, among others), using the same equipment and with the same preparation.
  • Example 3 Synthesis of quantum dots of CdS / n in vitro by adding phosphate group donor molecules:
  • the methodology for the synthesis of cadmium sulphide QDs from the addition of phosphate group donor molecules in the reaction mixtures is presented. Therefore, the P ⁇ , of the above-mentioned synthesis, was replaced by phosphorylated molecules found in biological organisms; glucose-6-phosphate (G6P), glycerol-2-phosphate (G2P), fructose-1, 6-bisphosphate (F1.6BP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), between others.
  • G6P glucose-6-phosphate
  • G2P glycerol-2-phosphate
  • F1.6BP adenosine monophosphate
  • AMP adenosine diphosphate
  • ATP adenosine triphosphate
  • a reaction mixture was prepared between MSA (5 mM), CdCl 2 (54.5 ⁇ ) and the different phosphate group donor molecules (10 mM). The mixtures were incubated for 12 days, the appearance of fluorescence being evaluated day by day when exposed to a UV transilluminator (at 365 nm).
  • the NPs obtained presented an absorbance spectrum as fluorescence corresponding to that expected for CdS QDs; shoulder absorbance between 350 and 400 nm and a fluorescence emission of at 500 nm corresponding to green NPs (Fig. 4B).
  • the green fluorescent solution featured the CH stretch bands and CH 2 (between 3000 and 1800 cm “1 ) and CS stretch (1390 cm “ 1 ) characteristic of MSA, and as in QDs synthesized with P ⁇ , the band corresponding to the sulfhydryl group is not observed, suggesting that thiol It is covalently linked to the QDs through the sulfhydryl group in the sample, plus between 1090-1100 cm "1 the phosphate group signal, and the asymmetric and symmetrical stretches of the POC are found both in the control (G6P only) and in the NPs between 970-980 and ⁇ 816 cm “1 respectively (Fig.

Abstract

The invention relates to a method for the synthesis of fluorescent, semiconductive nanoparticles (NPs) of cadmium sulphide (CdS), comprising: a) mixing i. a thiol as a source of sulphur and as a covering compound; ii. a cadmium salt; iii. a compound containing or generating a source of phosphate; b) adding a buffer solution to the mixture obtained in step (a), at a pH of between 7 and 10, in the presence or absence of oxygen and at a temperature of between 15 and 80°C; c) incubating the solution produced in b) at a temperature of between 15 and 80°C, for an incubation period of between 1 and 9 days, until fluorescence is obtained; and d) evaluating the fluorescence of the NPs of CdS, exposing them to a UV transilluminator (at 365 nm).

Description

SÍNTESIS ACUOSA DE NANOPARTÍCULAS DE SULFURO DE  WATERY SYNTHESIS OF SULFIDE NANOPARTICLES OF
CADMIO.  CADMIUM.
La presente invención se refiere a un método de producción en medio acuoso de nanopartículas (en adelante.NPs) semiconductoras y fluorescentes (denominadas quantum dots, en adelante QDs) de sulfuro de cadmio (CdS), utilizando moléculas fosforiladas en el proceso de síntesis. Dicho método permitiría generar NPsútiles en el monitoreo y mareaje in vivo de moléculas fosforiladas al interior de una célula, permitiendo localizar receptores, enzimas y moléculas que presenten en su estructura o que metabolicen moléculas fosforiladas o grupos fosfato. The present invention relates to a method of production in aqueous medium of semiconductor and fluorescent nanoparticles (hereinafter NPs) (called quantum dots, hereinafter QDs) of cadmium sulphide (CdS), using phosphorylated molecules in the synthesis process. This method would allow the generation of NPs useful in the monitoring and in vivo mapping of phosphorylated molecules inside a cell, allowing to locate receptors, enzymes and molecules that present in their structure or that metabolize phosphorylated molecules or phosphate groups.
El método propuesto permite producir NPs semiconductoras y fluorescentes, del tipo, QDs de CdS, mediante una metodología que incluye adicionar una sal inorgánica que contenga Cd.la adición de moléculas fosforiladas como dador degrupos fosfatos, y la adición de tioles. En particular, elmétodo propuesto comprende mezclar un tiol como fuente de azufre y como agente de envoltura, adicionar cloruro de cadmio, e incluir un compuesto que contenga fosfato inorgánico (P¡). La regulación de las concentraciones y proporciones de los reactivos permite controlar el tipo de fluorescencia (color) y el tamaño de las NPs de CdS. El objetivo de la adición de un compuesto dador de grupos fosfato dentro de la reacción, es favorecer la formación de QDs de CdSa bajas temperaturas (entre 15 y 80 °C) y en un menor tiempo, pues el fosfato es la base de la síntesis ya que es el elemento iniciador de la nucleación de los cristales de CdS. En este sentido, también es posible desarrollar la síntesis utilizando moléculas fosforiladas encontradas en organismos biológicos como por ejemplo, pero sin limitarse, glucosa-6-fosfato, glicerol-2-fosfato, fructosa-1 ,6-bisfosfato, adenosínamonofosfato, entre otros. The proposed method allows to produce semiconductor and fluorescent NPs, of the type, Qds of CdS, by means of a methodology that includes adding an inorganic salt containing Cd. The addition of phosphorylated molecules such as phosphate group donors, and the addition of thiols. In particular, the proposed method comprises mixing a thiol as a source of sulfur and as a wrapping agent, adding cadmium chloride, and including a compound containing inorganic phosphate (P¡). The regulation of the concentrations and proportions of the reagents allows to control the type of fluorescence (color) and the size of the NPs of CdS. The objective of the addition of a phosphate group donor compound within the reaction is to favor the formation of CdSa QDs at low temperatures (between 15 and 80 ° C) and in a shorter time, since phosphate is the base of the synthesis since it is the initiating element of the nucleation of CdS crystals. In this sense, it is also possible to develop the synthesis using phosphorylated molecules found in biological organisms such as, but not limited to, glucose-6-phosphate, glycerol-2-phosphate, fructose-1, 6-bisphosphate, adenosine monophosphate, among others.
Las NPs de CdS producidas mediante el método de la presente invención, presentan ventajosas características: son semiconductoras, The NPs of CdS produced by the method of the present invention have advantageous characteristics: they are semiconductors,
i fluorescentes y biocompatibles. El método descrito corresponde a una metodología sencilla y eficiente, pues en primer lugar se utiliza una menor concentración y por tanto una menor cantidad de ciertos reactivos, como por ejemplo la sal CdCI2. En segundo lugar, la presencia de fosfato dentro del procedimiento de síntesis favorece la reacción de formación de los QDs de CdS permitiendo que la síntesis ocurra en un menor período de tiempo y temperaturas menores a otros métodos descritos. Esto último, provoca que el método sea más sencillo, evitando complicaciones intrínsecas relacionadas con el trabajo a altas temperaturas tales como la posible descomposición de algunos reactivos, y más importante aún, evitando posibles accidentes del usuario, además prescindiendo de atmosferas inertes como Ar o N2. El hecho de que el método pueda ser llevado a cabo a bajas temperaturas, gracias a la presencia de fosfato, permite prescindir de maquinaria y equipos de incubación a altas temperaturas. i fluorescent and biocompatible. The described method corresponds to a simple and efficient methodology, since first a lower concentration is used and therefore a smaller quantity of certain reagents, such as the CdCI 2 salt. Secondly, the presence of phosphate within the synthesis procedure favors the formation reaction of the CdS QDs allowing the synthesis to occur in a shorter period of time and temperatures lower than other methods described. The latter, makes the method simpler, avoiding intrinsic complications related to work at high temperatures such as the possible decomposition of some reagents, and more importantly, avoiding possible accidents of the user, also dispensing with inert atmospheres such as Ar or N 2 . The fact that the method can be carried out at low temperatures, thanks to the presence of phosphate, allows to dispense with machinery and equipment of incubation at high temperatures.
Otra importante ventaja del método de la presente invención son las potenciales aplicaciones de los QDs generados. Los QDs de CdS producidos mediante este método presentan en su cubierta grupos tiol y fosfato, lo que les confiere mayor solubilidad, y por tanto, les otorga beneficios en cuanto a su utilización in vivo si es que se quisieran incorporar los QDs al interior de células u organismos. Adicionalmente, al poder incorporar dentro del proceso sintético intermediarios fosforilados, pueden eventualmente las NPs ser direccionadas a un receptor celular en particular para marcar células, e incluso, permitir visualizar la cantidad de estos receptores mediante microscopía de fluorescencia (excitando los QDs). Otra posible aplicación de los QDs de CdS producidos mediante el método divulgado, es su uso en la localización de enzimas capaces de reconocer sustratos fosforilados, pues los QDspodrían unirse a este tipo de enzima, y ser monitoreadas de forma/n vivo dentro de una célula. También los QDs producidos por este método podrían ser utilizados en celdas solares sensibilizadas por QDs (QDSSC). Another important advantage of the method of the present invention is the potential applications of the generated QDs. The QDs of CdS produced by this method have thiol and phosphate groups on their cover, which gives them greater solubility, and therefore, gives them benefits in terms of in vivo use if they wanted to incorporate the QDs into cells or organisms. Additionally, by being able to incorporate phosphorylated intermediates into the synthetic process, the NPs can eventually be directed to a particular cell receptor to mark cells, and even, allow visualizing the amount of these receptors by fluorescence microscopy (exciting the QDs). Another possible application of the CdS QDs produced by the disclosed method is its use in the localization of enzymes capable of recognizing phosphorylated substrates, since the QDs could bind to this type of enzyme, and be monitored live / n inside a cell . Also the QDs produced by this method could be used in solar cells sensitized by QDs (QDSSC).
Antecedentes del Estado del Arte: Background of the State of the Art:
Se han descrito con anterioridad métodos para la síntesis de QDs de Cdque involucren dentro de la metodología la adición de un compuesto parte de la envoltura del QDspara estabilizar la síntesis y aumentar su biocompatibilidad. Sin embargo, no existen antecedentes previos deun método realizado a temperatura ambiente para generar QDs de CdS que utilicen dentro de la síntesis moléculas de fosfato para favorecer la eficacia de reacción en estas condiciones de temperatura. Adicionalmente, no existe un método que permita producir QDs de CdS que incorporen en su cubierta grupos tiol y fosfato para aumentar su biocompatibilidad e interacción con células, lo que favorece sus potenciales aplicaciones ya sea en cultivo celular {in vitro), en mareaje in vivo, entre muchas otras aplicaciones. Methods for the synthesis of Cd QDs that involve within the methodology the addition of a compound part of the QD envelope to stabilize the synthesis and increase its biocompatibility have been previously described. However, there is no previous history of a method carried out at room temperature to generate CdS QDs that use phosphate molecules within the synthesis to favor the reaction efficiency in these temperature conditions. Additionally, there is no method to produce CdS QDs that incorporate thiol and phosphate groups on their deck to increase their biocompatibility and interaction with cells, which favors their potential applications either in cell culture {in vitro), in in vivo tide , among many other applications.
A continuación, se presentan algunas invenciones e investigaciones relacionadas con métodos de síntesis de QDs de Cd: Below are some inventions and research related to methods of synthesis of Cd QDs:
El documento WO 2012090161 A1 se refiere a un método para la síntesis de QDs de CdTe-GSH en un medio acuoso. El método comprende los pasos: a) preparar una solución precursora de cadmio en una solución tampón de citrato, b) adicionar GSH a la mezcla preexistente aplicando agitación, c) adicionar a la mezcla anterior un oxianión de teluro (teluro de potasio o de sodio) como dador de teluro, d) esperar que la mezcla reaccione, e) detener la reacción incubándola a baja temperatura. El precursor de cadmio corresponde a una sal soluble de cadmio (CdCI2, CdSO4 o Cd(CH3CO2)2) y la solución tampón puede corresponder a una solución tampón de citrato, fosfato, Tris-HCI y medios de cultivo de microorganismos como LB (triptona, NaCI y extracto de levadura) o M9 (Na2HPO4, KH2PO4, NaCly NH4CI). La temperatura de reacción es de 37- 130°C. Este documento apunta a la síntesis de QDs de CdTe con una cubierta de GSH como agente capeador, y no hace referencia a la síntesis de CdS. Cabe destacar, que la metodología propuesta en la presente solicitud presenta nuevas características respecto a lo reportado en WO 2012090161 como lo son: utilizar concentraciones de una sal de cadmio en el orden micromolar lo que resulta en un método más económico y menos tóxico, el tiempo de obtención de los QDs a bajas temperaturas es más rápido obteniéndose NPs en días, siendo que el tiempo de síntesis del arte previo es del rango de semanas. Más aun, la principal diferencia es que en la metodología de la presente invención se utiliza fosfato para la síntesis, no así en el caso del documento. Esto es una diferencia fundamental relacionada con el tipo de NP formada y el mecanismo de síntesis química involucrada, además de las características finales de biocompatibilidad y toxicidad que presentara el QDs final. Esta característica no es posible desprenderla del arte del estado previo, pues no es evidente adicionar moléculas fosforiladas en el proceso de síntesis para permitir quela reacción se realice a bajas temperaturas de forma eficiente. WO 2012090161 A1 refers to a method for the synthesis of CdTe-GSH QDs in an aqueous medium. The method comprises the steps: a) preparing a cadmium precursor solution in a citrate buffer solution, b) adding GSH to the preexisting mixture by stirring, c) adding a tellurium oxyanion (potassium or sodium telluride to the previous mixture) ) as a tellurium donor, d) wait for the mixture to react, e) stop the reaction by incubating at low temperature. The cadmium precursor corresponds to a soluble cadmium salt (CdCI 2 , CdSO4 or Cd (CH3CO2) 2) and the buffer solution can correspond to a buffer solution of citrate, phosphate, Tris-HCI and microorganism culture media such as LB ( Tryptone, NaCI and yeast extract) or M9 (Na 2 HPO 4 , KH 2 PO4, NaCly NH 4 CI). The reaction temperature is 37- 130 ° C This document points to the synthesis of CdTe QDs with a GSH cover as a weathering agent, and does not refer to the synthesis of CdS. It should be noted that the methodology proposed in this application presents new characteristics with respect to what is reported in WO 2012090161 such as: use concentrations of a cadmium salt in the micromolar order resulting in a more economical and less toxic method, time to obtain the QDs at low temperatures it is faster obtaining NPs in days, since the synthesis time of the prior art is in the range of weeks. Moreover, the main difference is that phosphate is used in the methodology of the present invention for synthesis, but not in the case of the document. This is a fundamental difference related to the type of NP formed and the chemical synthesis mechanism involved, in addition to the final biocompatibility and toxicity characteristics that the final QDs will present. This feature is not possible to detach from the prior art, as it is not obvious to add phosphorylated molecules in the synthesis process to allow the reaction to be carried out at low temperatures efficiently.
El documento US 8491818 B2 presenta entre los alcances de la invención la preparación acuosa de QDs de CdS. La metodología propuesta en este documento consiste básicamente en hacer reaccionar una solución acuosa de Cd(NO3)2 Con MSA (ácido mercaptosuccínico), en agitación, y adicionar NH4OH para ajusfar a un pH cercano a 7-9. Posteriormente, se adiciona una cantidad determinada de solución acuosa de Na2S en agitación durante 3-5 min. Toda la reacción se realiza en un ambiente libre de oxígeno. Tras esto la suspensión es ultrasonicada y filtrada, para ser incubada a 0°C y luego almacenada a 4°C. Si bien, el alcance de la invención incluye la síntesis de QDs de CdS, en la metodología no se establece el uso de fosfato inorgánico o moléculas que presenten grupos fosfato. Lo único similar con este método podría surgir de la idea de utilizar tioles como agentes de cubierta, sin embargo, en la presente invención el compuesto de tiol además de utilizarse como agentede cubierta, también se utiliza como fuente del sulfuro necesario para formar las NPs de CdS. US 8491818 B2 presents among the scope of the invention the aqueous preparation of CdS QDs. The methodology proposed in this document basically consists in reacting an aqueous solution of Cd (NO3) 2 with MSA (mercaptosuccinic acid), under stirring, and adding NH 4 OH to adjust to a pH close to 7-9. Subsequently, a certain amount of aqueous Na 2 S solution is added under stirring for 3-5 min. The entire reaction is carried out in an oxygen free environment. After this the suspension is ultrasonic and filtered, to be incubated at 0 ° C and then stored at 4 ° C. Although, the scope of the invention includes the synthesis of CdS QDs, the use of inorganic phosphate or molecules presenting phosphate groups is not established in the methodology. The only thing similar with this method could arise of the idea of using thiols as cover agents, however, in the present invention the thiol compound in addition to being used as a cover agent, is also used as a source of the sulfide necessary to form the CsS NPs.
En CN103215042Ase divulga un método de síntesis de QDs de CdSe en fase acuosa, el que de forma general comprende mezclar CdC , selenito de sodio y MSA (ácido mercaptosuccínico), adicionar una solución tampón, agitar, ajustar pH a 5, luego adicionar NaBH4, calentar a una temperatura de 60°C, y dejar reaccionar por una hora. Los QDs resultantes corresponden a QDs del tipo MSA-CdSe. A pesar de que en esta invención la metodología incluye el uso de un agente estabilizador o de cubierta derivado de un compuesto que contenga grupos tiol en su estructura, no se hace referencia a la adición de un compuesto que aporte grupos fosfato en la reacción para obtener QDs con una capa externa de tiol y fosfato que le otorga nuevas propiedades. Adicionalmente, en este documento se sintetizan NPs de CdSe y no CdS. In CN103215042A a method of synthesis of CdSe QDs in aqueous phase is disclosed, which generally comprises mixing CdC, sodium selenite and MSA (mercaptosuccinic acid), adding a buffer solution, stirring, adjusting pH to 5, then adding NaBH 4 , heat at a temperature of 60 ° C, and allow to react for one hour. The resulting QDs correspond to QDs of type MSA-CdSe. Although in this invention the methodology includes the use of a stabilizing or covering agent derived from a compound containing thiol groups in its structure, reference is not made to the addition of a compound that provides phosphate groups in the reaction to obtain QDs with an external layer of thiol and phosphate that gives it new properties. Additionally, this document synthesizes NPs of CdSe and not CdS.
En el documento WO 2013019090 A1 se divulga una composición que incluye NPs hidrofílicas que tienen adherido a su superficie monosacáridos-fosfato o un derivado de estos, una solución coloidal de la composición, y un agente de contraste de RMI (resonancia magnética de imagen) que incluye la solución colodial. Específicamente, se divulgan NPs hidrofílicas que presentan una superficie modificada por adición de moléculas de monosacáridos-fosfato o derivados de estos en la superficie de partículas inorgánicas. Las NPs corresponden a partículas inorgánicas que pueden ser del tipo metal, metal calcogénico, óxido metálico, materiales magnéticos, aleaciones magnéticas, materiales semiconductores y estructuras híbridas, sin hacer referencia a NPs conformadas por cadmio. Si bien, las NPs generadas a partir del método descrito en WO 2013019090 presentan grupos fosfato en su cubierta, la metodología de síntesis es diferente a la propuesta en la presente invención. Así, en WO 2013019090 A1 la preparación de las NPsse realiza de forma separada de la unión de moléculas azúcar-fosfato, sin ser la adición de una molécula dadora de grupos fosfato un componente importante en la reacción de síntesis. En el método aquí propuesto, la adición de fosfato como parte del procedimiento de síntesis, favorece la eficacia de reacción, y permite que ésta pueda ser llevada a cabo a temperatura ambiente y en presencia de oxígeno, teniendo la adición de fosfato un propósito totalmente diferente. WO 2013019090 A1 discloses a composition that includes hydrophilic NPs that have adhered to their surface monosaccharides-phosphate or a derivative thereof, a colloidal solution of the composition, and a contrast agent of RMI (magnetic resonance imaging) that includes the colodial solution. Specifically, hydrophilic NPs are disclosed that have a surface modified by the addition of monosaccharide phosphate molecules or derivatives thereof on the surface of inorganic particles. The NPs correspond to inorganic particles that can be of the type metal, chalcogenic metal, metal oxide, magnetic materials, magnetic alloys, semiconductor materials and hybrid structures, without reference to NPs formed by cadmium. Although the NPs generated from the method described in WO 2013019090 have phosphate groups on their cover, the synthesis methodology is different from the one proposed here. invention. Thus, in WO 2013019090 A1 the preparation of the NPs is carried out separately from the binding of sugar-phosphate molecules, without the addition of a phosphate group donor molecule being an important component in the synthesis reaction. In the method proposed here, the addition of phosphate as part of the synthesis procedure, favors the reaction efficiency, and allows it to be carried out at room temperature and in the presence of oxygen, the addition of phosphate having a totally different purpose. .
Existen diversos artículos de divulgación científica que se refieren a la síntesis de QDs de Cd que comprende un agente de cubierta o molécula del tipo tiol como envoltura. Así, en el documento científico "Synthesis of GlutathioneCoatedQDs" (referencia) se presentan los tipos de QDs más utilizados, sus metodologías de preparación, y principalmente, la estrategia metodológica de adición de un agente de cubierta como GSH a los QDs.De forma específica, en el punto 4.1.3 se reseña lo establecido en el estado del arte acerca de NPs de CdS-GSH, describiéndose la preparación de NPs de CdS a partir de precursores como CdC y su reacción con diferentes agentes como CH3CSNH2 a temperatura ambiente y la adición final de una solución de GSH (Liangef al. 2010). En este artículo de revisión se indica que en el estudio de Thangaduraí y colaboradores se prepararon NPs de CdS con un compuesto de envoltura del tipo tiol. En este trabajo se probaron las moléculas 1 ,4-ditiotreitol (DTT), 2-mercaptoetanol, CYS, metionina y GSH (Thangaduraí et al. 2008). El agente de cubierta se adicionó en forma de solución acuosa a una solución acuosa de Cd(NO3)2. El trabajo concluye que el mejor compuesto para conformar la envoltura de la NPses GSH. Sin embargo, no se hace referencia a la adición de fosfato inorgánico o moléculas que comprendan grupos fosfato para mejorar la eficiencia de síntesis. En el documento científico "Surface modification of CdS QDs using thiols— structural and photophysical studies" (Thangadurai P.ef al, 2008a) se presenta un estudio de las características estructurales, térmicas y de emisión de diferentes tipos de tioles orgánicos para QDs de CdS. Se evaluaron 5 tioles orgánicos como cubierta de QDs de CdS: (i) 1 ,4- ditiotreitol (DTT), (¡i) 2-mercaptoetanol (ME), (iii) CYS, (iv) metionina, and (v) GSH. Al comparar QDs sin cubierta de tipo tiol y QDs con tioles en la superficie, la presencia de moléculas tipo cubierta en la estructura de los QDs le confieren un mayor tamaño y por ende una mayor superficie de contacto a los QDs, y mejora sus propiedades fluorescentes. Nuevamente, este tipo de documento no se refiere a la adición de moléculas fosforiladas o fosfato inorgánico como parte de la mezcla de reacción con el objetivo de aumentar la eficiencia de la síntesis. There are several articles of scientific disclosure that refer to the synthesis of Cd QDs comprising a thiol-like covering agent or molecule as a shell. Thus, in the scientific document "Synthesis of GlutathioneCoatedQDs" (reference) the most commonly used types of QDs, their preparation methodologies, and mainly, the methodological strategy of adding a cover agent such as GSH to QDs are presented. , in section 4.1.3 the provisions of the state of the art about CdS-GSH NPs are described, describing the preparation of CdS NPs from precursors such as CdC and their reaction with different agents such as CH 3 CSNH 2 at temperature environment and the final addition of a GSH solution (Liangef al. 2010). This review article indicates that in the study by Thangaduraí et al., NPs of CdS were prepared with a thiol-type wrapping compound. In this work the molecules 1, 4-dithiothreitol (DTT), 2-mercaptoethanol, CYS, methionine and GSH were tested (Thangaduraí et al. 2008). The coating agent was added as an aqueous solution to an aqueous solution of Cd (NO 3 ) 2 . The work concludes that the best compound to form the envelope of the GSH NPses. However, no reference is made to the addition of inorganic phosphate or molecules comprising phosphate groups to improve synthesis efficiency. In the scientific paper "Surface modification of CdS QDs using thiols— structural and photophysical studies" (Thangadurai P.ef al, 2008a) a study of the structural, thermal and emission characteristics of different types of organic thiols for CdS QDs is presented . Five organic thiols were evaluated as a cover of CdS QDs: (i) 1, 4-dithiothreitol (DTT), (i) 2-mercaptoethanol (ME), (iii) CYS, (iv) methionine, and (v) GSH . When comparing uncoated QDs of the thiol type and QDs with thiols on the surface, the presence of covered type molecules in the structure of the QDs gives it a larger size and therefore a greater contact surface to the QDs, and improves its fluorescent properties . Again, this type of document does not refer to the addition of phosphorylated molecules or inorganic phosphate as part of the reaction mixture in order to increase the efficiency of the synthesis.
Adicionalmente, en el artículo de divulgación científica "Synthesis of glutathione-capped CdS QDs and preliminary studies on protein detection and cell fluorescence image" (Jiang C. et al, 2007) se presenta una metodología de preparación de QDs de CdS-GSH. En términos generales, el protocolo de producción de los QDsCdS-GSH descrito incluye mezclar una solución de CdCI2 con agua y ajustar el pH a 10,2, saturar con burbujas de N2 por cerca de 20 min y luego adicionar GSH (relación Cd:GSH de 1 :1 ) a la solución de CdCI2, y mantener agitación y calor constante bajo en una atmósfera de N2 durante 10 min. Additionally, in the scientific dissemination article "Synthesis of glutathione-capped CdS QDs and preliminary studies on protein detection and cell fluorescence image" (Jiang C. et al, 2007) a methodology for the preparation of CdS-GSH QDs is presented. In general terms, the production protocol of the QDsCdS-GSH described includes mixing a solution of CdCI 2 with water and adjusting the pH to 10.2, saturating with N 2 bubbles for about 20 min and then adding GSH (Cd ratio : GSH 1: 1) to the CdCI 2 solution, and keep stirring and constant heat low in an N 2 atmosphere for 10 min.
La metodología descrita en los artículos científicos citados difiere del método aquí propuesto puesto que dentro de la reacción de síntesis no se incluye como reactivo una molécula dadora de grupos fosfato. La adición de este tipo de reactivo considera una propuesta con características anteriormente no descritas, pues permite la producción de QDs a bajas temperaturas (incluyendo temperatura ambiente) y la posibilidad de realizar la síntesis en presencia de oxígeno. Por tanto el método aquí propuesto se basa en el uso del fosfato para la formación de la nanoestructura. The methodology described in the cited scientific articles differs from the method proposed here since a phosphate group donor molecule is not included in the synthesis reaction. The addition of this type of reagent considers a proposal with characteristics not previously described, since it allows the production of QDs at low temperatures (including room temperature) and the possibility of performing the synthesis in the presence of oxygen. Therefore the The method proposed here is based on the use of phosphate for the formation of the nanostructure.
Otros documentos como el artículo de divulgación científica "Low temperature synthesis of ZnS and CdZnS shells on CdSe quantum dots" (Zhu H. et al, 2010) describen protocolos en los que se adicionan compuestos inorgánicos que presentan átomos de fósforo en su estructura. En este documento se presenta un método para sintetizar QDs de ZnS, CdZnS yCdSe a temperaturas de entre 65°C y 180°C. Se destaca el uso de pequeñas cantidades de trioctilfosfina y óxido de trioctilfosfina en las soluciones precursoras, y en el cual se utiliza tiourea como fuente de azufre (grupos sulfuro). El método se presenta como un protocolo general para estructuras CdZnS mediante la mezcla de cadmio y acetato de zinc como precursores. La invención descrita en este documento apunta a la formación de QDs mediante una síntesis química a la que se le adicionan compuestos que contienen fósforo pero que no consideran la liberación o formación de grupos fosfato como parte de la envoltura de la NP, ni menos como un agente potenciador de la reacción. Other documents such as the popular science article "Low temperature synthesis of ZnS and CdZnS shells on CdSe quantum dots" (Zhu H. et al, 2010) describe protocols in which inorganic compounds that have phosphorus atoms in their structure are added. This document presents a method to synthesize QDs of ZnS, CdZnS and CdSe at temperatures between 65 ° C and 180 ° C. The use of small amounts of trioctylphosphine and trioctylphosphine oxide in precursor solutions is highlighted, and in which thiourea is used as a source of sulfur (sulfide groups). The method is presented as a general protocol for CdZnS structures by mixing cadmium and zinc acetate as precursors. The invention described in this document aims at the formation of QDs by means of a chemical synthesis to which phosphorus-containing compounds are added but which do not consider the release or formation of phosphate groups as part of the NP envelope, not least as a reaction enhancing agent.
En conclusión, características del método propuesto, tales como, realizar la síntesis a temperatura ambiente, en presencia de oxígeno y en menor tiempo para producir QDs de CdSle confiere a la metodología características no descritas anteriormente. De forma particular, las NPs producidas mediante este método presentan características únicas en cuanto a su solubilidad y biodisponibilidad, lo que permite su uso en el monitoreo in vivo de marcadores celulares, teniendo un potencial uso en el área de la medicina y de la investigación. In conclusion, characteristics of the proposed method, such as, performing the synthesis at room temperature, in the presence of oxygen and in less time to produce CdSle QDs, gives the methodology characteristics not described above. In particular, the NPs produced by this method have unique characteristics in terms of their solubility and bioavailability, which allows their use in the in vivo monitoring of cell markers, having a potential use in the area of medicine and research.
Descripción de las figuras: Description of the figures:
Figura 1 : Efecto de fosfato, cadmio y la temperatura en la síntesis in vitro de QDs de CdS. A) Las soluciones contenían P¡ (fosfato inorgánico) 10 mM, CdCI2 54,5 μΜ y GSH 5 mM y fueron incubadas a 37 °C de 1 a 4 días. Figure 1: Effect of phosphate, cadmium and temperature in the in vitro synthesis of Qds of CdS. A) The solutions contained 10 mM P¡ (inorganic phosphate), 54.5 μC CdCI 2 and 5 mM GSH and were incubated at 37 ° C for 1 to 4 days.
B) Las soluciones contenían P¡: 0; 7,5; 10; 20 y 75 mM; CdCI2 150 μΜ más MSA 5 mM y fueron incubadas a 37 °C por 6 días. B) The solutions contained P¡: 0; 7.5; 10; 20 and 75 mM; CdCI 2 150 μΜ plus 5 mM MSA and were incubated at 37 ° C for 6 days.
C) Las soluciones contenían P¡ 10 mM, CdCI230, 150 y 300 μΜ y MSA 5 mM. El tiempo de incubación va desde 1 a 6 días para 37 °C, y de 0,5; 1 ,0; 1 ,5; 2,0; 2,5; 3 y 4 h para 80°C. C) The solutions contained 10 mM Pi, CDCI February 30, 150 and 300 μΜ and 5 mM MSA. The incubation time ranges from 1 to 6 days for 37 ° C, and 0.5; 1.0; fifteen; 2.0; 2.5; 3 and 4 h for 80 ° C.
En A), B) y C) las muestras contienen 1 mL de volumen final y fueron expuestas a un transiluminador UV (a 365 nm). In A), B) and C) the samples contain 1 mL of final volume and were exposed to a UV transilluminator (at 365 nm).
Figura 2: Caracterización de las NPs producidas in vitro. Figure 2: Characterization of NPs produced in vitro.
A y B) Espectros de absorbancia y fluorescencia (excitación a 350 nm) de NPs azules (línea continua) y NPs naranja (línea discontinua). A and B) Absorbance and fluorescence spectra (350 nm excitation) of blue NPs (solid line) and orange NPs (dashed line).
C y D) Determinación del tamaño de NPs azules y naranjas mediante DLS. C and D) Determination of the size of blue and orange NPs by DLS.
E y F) TEM de NPs naranjas. E and F) TEM of orange NPs.
E) Visión general de la solución de NPs donde se observa aglomerados nanométricos y E) Overview of the NP solution where nanometric agglomerates are observed and
F) NPs individuales a una mayor magnificación de la imagen. Las reglas son de 100 y 20 nm, respectivamente. F) Individual NPs at a higher magnification of the image. The rules are 100 and 20 nm, respectively.
Figura 3: Espectro FTIR de NPs sintetizadas in vitro. Figure 3: FTIR spectrum of NPs synthesized in vitro.
De arriba abajo se registra el espectro FTIR de P¡, MSA y NPs (naranjas) respectivamente. Las señales características de cada compuesto han sido registradas en las gráficas en magnitudes en cm"1. The FTIR spectrum of P¡, MSA and NPs (oranges) respectively is recorded from top to bottom. The characteristic signals of each compound have been recorded in the graphs in magnitudes in cm "1 .
Figura 4: Síntesis de QDs de sulfuro de cadmio utilizando moléculas orgánicas fosforiladas. Se evaluó la síntesis de QDs utilizando diferentes moléculas fosforiladas como fuente de fosfato: adenosintrifosfato (ATP), adenosindifosfato (ADP), adenosinmonofosfato (AMP), Fructosa 1 ,6 bifosfato (F1 ,6 BP), glucosa-2-fosfato (G2P), glucosa-6-fosfato (G6P). Figure 4: Synthesis of cadmium sulfide QDs using phosphorylated organic molecules. The synthesis of QDs was evaluated using different phosphorylated molecules as a phosphate source: adenosine triphosphate (ATP), adenosyphosphate (ADP), adenosine monophosphate (AMP), Fructose 1, 6 bisphosphate (F1, 6 BP), glucose-2-phosphate (G2P) , glucose-6-phosphate (G6P).
En A) se observan los resultados de la síntesis de QDs con las diferentes fuentes de fosfato entre 1 y 12 días de incubación. In A) the results of the synthesis of QDs with the different phosphate sources are observed between 1 and 12 days of incubation.
En B) se presentan los espectros de absorbancia (arriba) y fluorescencia (abajo) de NPs verdes purificadas de una reacción entre MSA (5 mM), CdCI2 (54,5 μΜ) y G6P (10 mM). In B) the absorbance (above) and fluorescence (below) spectra of purified green NPs from a reaction between MSA (5 mM), CdCI 2 (54.5 μΜ) and G6P (10 mM) are presented.
En C) se presenta el espectro FTIR de G6P (control) y las NPs verdes sintetizadas con G6P. In C) the FTIR spectrum of G6P (control) and green NPs synthesized with G6P are presented.
Descripción de la invención: Description of the invention:
La presente invención se refiere a un método de síntesis de NPs semiconductoras fluorescentes (QDs) de CdS. The present invention relates to a method of synthesis of fluorescent semiconductor NPs (QDs) of CdS.
El método propuesto consiste en la producción de QDs de CdS mediante un procedimiento en el que se utilizan moléculas fosforiladas como fuente de fosfato, el cual se lleva a cabo en un medio acuoso, en presencia de oxígeno, a temperatura ambiente, y más particularmente, a temperaturas entre 15 y 80 °C. The proposed method consists in the production of CdS QDs by a process in which phosphorylated molecules are used as a phosphate source, which is carried out in an aqueous medium, in the presence of oxygen, at room temperature, and more particularly, at temperatures between 15 and 80 ° C.
El método propuesto comprende los pasos de: a) Mezclar: i. Un tiol como fuente de azufre y como compuesto de envoltura; ii. Una sal de cadmio; iii. Un compuesto que contenga o genere una fuente de fosfato; b) Agregar una solución tampón a la mezcla obtenida en la etapa (a), a un pH entre 7-10, en presencia o ausencia de oxígeno y a una temperatura entre 15 - 80°C; c) Incubar la solución obtenida en (b) a una temperatura entre 15 - 80°C, durante un período de tiempo de incubación de 1 - 9 días, hasta obtener fluorescencia. d) Evaluar la fluorescencia de las NPs de CdS, exponiéndolas a un transiluminador UV (a 365 nm). The proposed method comprises the steps of: a) Mix: i. A thiol as a source of sulfur and as a wrapping compound; ii. A cadmium salt; iii. A compound that contains or generates a phosphate source; b) Add a buffer solution to the mixture obtained in step (a), at a pH between 7-10, in the presence or absence of oxygen and at a temperature between 15-80 ° C; c) Incubate the solution obtained in (b) at a temperature between 15-80 ° C, for an incubation period of 1-9 days, until fluorescence is obtained. d) Evaluate the fluorescence of CdS NPs, exposing them to a UV transilluminator (at 365 nm).
La regulación de las concentraciones y proporciones de los reactivos permite controlar el tipo de fluorescencia (color) y el tamaño de las NPs de CdS. The regulation of the concentrations and proportions of the reagents allows to control the type of fluorescence (color) and the size of the NPs of CdS.
La mezcla de reacción es llevada a cabo en un ambiente acuoso a un pH entre 7-10 gracias a la adición de una solución tampón que permita mantener este pH, en presencia o ausencia de oxígeno y a bajas temperaturas. En el método se pueden utilizar diferentes soluciones tampón, utilizadas comúnmente en Biología Molecular y Bioquímica, a una concentración de 1 a 100 mM, los que poseen un pH celular de 7,4, dentro de los cuales se pueden citar: The reaction mixture is carried out in an aqueous environment at a pH between 7-10 thanks to the addition of a buffer solution that allows maintaining this pH, in the presence or absence of oxygen and at low temperatures. In the method, different buffer solutions, commonly used in Molecular Biology and Biochemistry, can be used at a concentration of 1 to 100 mM, which have a cell pH of 7.4, which include:
> PBS (tampón fosfato salino: KH2PO4/K2HPO4). > PBS (phosphate buffered saline: KH2PO4 / K2HPO4).
> Ácido 3-(N-morfolino) propanosulfónico (MOPS,C7Hi5N04S). > 3- (N-morpholino) propanosulfonic acid (MOPS, C 7 Hi 5 N0 4 S).
> Ácido (4-(2-hidroxietil)-1-piperazinetanosulfónico (HEPES,
Figure imgf000012_0001
> (4- (2-Hydroxyethyl) -1-piperazinethanesulfonic acid (HEPES,
Figure imgf000012_0001
> Tris-HCI (Tris: (HOCH2)3CNH2). > Tris-HCI (Tris: (HOCH 2 ) 3 CNH 2 ).
> Citrato de sodio (Na3C3H50(COO)3). El tampón preferido corresponde al tampón PBS a una concentración de entre 1 a 100 mM, preferentemente 10 mM, pues es el que mostró mejores resultados (datos no mostrados). > Sodium citrate (Na3C 3 H 5 0 (COO) 3). The preferred buffer corresponds to the PBS buffer at a concentration of between 1 to 100 mM, preferably 10 mM, as it showed the best results (data not shown).
La mezcla de reacción comprende: i. Un tiol como fuente de azufre y como compuesto de envoltura de los QDs, en rangos de concentración entre 0,25-15 mM, tales como: The reaction mixture comprises: i. A thiol as a source of sulfur and as a wrapping compound of the QDs, in concentration ranges between 0.25-15 mM, such as:
> Glutatión (GSH: C10H17N3O6S). > Glutathione (GSH: C 10 H 17 N3O 6 S).
> Cisteína (CYS: C3H7N02S). > Cysteine (CYS: C 3 H 7 N0 2 S).
> Ácido mercaptosuccinico (MSA: C4H604S). > Mercaptosuccinic acid (MSA: C 4 H 6 0 4 S).
ii. Una sal de cadmio a una concentración de 30-300 μΜ tales como:  ii. A cadmium salt at a concentration of 30-300 μΜ such as:
> CdCI2.> CdCI 2 .
Cd(N03)2 x 4H20.Cd (N0 3 ) 2 x 4H 2 0.
Cd(CH3COO)2 x 2H20. Cd (CH 3 COO) 2 x 2H 2 0.
iii. Y un compuesto o molécula fosforilada como fuente de fosfato a concentraciones entre 10 μΜ y 100 mM, tales como:  iii. And a phosphorylated compound or molecule as a phosphate source at concentrations between 10 μΜ and 100 mM, such as:
> Fosfato inorgánico en forma de K2HP0 . > Inorganic phosphate in the form of K 2 HP0.
> Glucosa-6-fosfato (G6P).  > Glucose-6-phosphate (G6P).
> Glicerol-2-fosfato (G2P).  > Glycerol-2-phosphate (G2P).
> Fructosa-1 ,6-bisfosfato (F1.6BP).  > Fructose-1, 6-bisphosphate (F1.6BP).
> Adenosina monofosfato (AMP).  > Adenosine monophosphate (AMP).
> Adenosina difosfato (ADP).  > Adenosine diphosphate (ADP).
> Adenosina trifosfato (ATP).  > Adenosine triphosphate (ATP).
En una forma preferida de la invención, la mezcla de reacción comprende la mezcla de una solución tampón de PBS a una concentración de 10 mM con una solución de CdCI2 a una concentración de 54,5 μΜ, una solución de MSA a una concentración de 5 mM y una solución de glucosa-6-fosfato a una concentración de 10 mM. La mezcla es incubada a 37°C durante 1-9 días para evaluar su fluorescencia al ser expuestas a un transiluminador UV (a 365 nm). In a preferred form of the invention, the reaction mixture comprises mixing a PBS buffer solution at a concentration of 10 mM with a solution of CdCI 2 at a concentration of 54.5 μΜ, a solution of MSA at a concentration of 5 mM and one glucose-6-phosphate solution at a concentration of 10 mM. The mixture is incubated at 37 ° C for 1-9 days to assess its fluorescence when exposed to a UV transilluminator (at 365 nm).
Preferentemente luego de la incubación, para eliminar los reactivos que no reaccionaron durante la incubación (excedentes) y también a modo de enriquecer en NPs las mezclas de reacción, se filtra la mezcla de reacción en filtros Amicon con un tamaño de poro de 3 kDa (0,3-0,5 nm) y se centrifuga en un rango de 3000 y 10000 G (preferentemente a 7000 G) hasta obtener una solución concentrada en NPs, preferentemente durante 30 minutos, o más dependiendo del volumen inicial de reacción. Las soluciones concentradas son lavadas con agua destilada miliQ mediante el mismo procedimiento de centrifugación en los tubos Amicon. Preferably after incubation, to remove reagents that did not react during incubation (surpluses) and also to enrich the reaction mixtures in NPs, the reaction mixture is filtered on Amicon filters with a pore size of 3 kDa ( 0.3-0.5 nm) and centrifuged in a range of 3000 and 10000 G (preferably at 7000 G) until a solution concentrated in NPs is obtained, preferably for 30 minutes, or more depending on the initial reaction volume. The concentrated solutions are washed with miliQ distilled water by the same centrifugation procedure in the Amicon tubes.
En la presente invención, tiol se refiere sin limitarse a GSH, MSA, CYS, entre otros. El compuesto tiol corresponde a la fuente de azufre para la formación de los QDs de CdS, y también es parte de la cubierta que envuelve a las NPs. El método funciona utilizando otros tioles tales como: ditiotreitol, ácido tioglicólico, entre otros, a diferentes temperaturas de funcionamiento y con un rendimiento de producción variable. In the present invention, thiol refers without being limited to GSH, MSA, CYS, among others. The thiol compound corresponds to the source of sulfur for the formation of CdS QDs, and is also part of the shell that surrounds NPs. The method works using other thiols such as: dithiothreitol, thioglycolic acid, among others, at different operating temperatures and with a variable production yield.
Cuando se refiere a moléculas fosforiladas corresponde a moléculas que contengan al menos un grupo fosfato en su estructura, pudiendo ser, sin limitarse, moléculas fosforiladas como; glucosa-6- fosfato, glicerol-2-fosfato, fructosa-1 ,6-bisfosfato, adenosina monofosfato, adenosina difosfato, adenosina trifosfato, entre otros. When it refers to phosphorylated molecules, it corresponds to molecules that contain at least one phosphate group in their structure, and may be, without limitation, phosphorylated molecules such as; glucose-6- phosphate, glycerol-2-phosphate, fructose-1, 6-bisphosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, among others.
En la presente invención, la metodología propuesta permite sintetizar QDs de CdS de forma más eficiente, en presencia de oxígeno y en condiciones de bajas temperaturas. Cuando se refiere a bajas temperaturas corresponden a temperaturas de entre 15 y 80°C. Más particularmente, se refiere a temperaturasmenores a 65°C. De forma preferente, la temperatura de realización de la metodología es de 37°C. Los quantum dots de CdS generados a partir del método presentado tienen características particulares respecto a otras nanopartículas. Para dos muestras (azul y naranja), al ser caracterizadas mediante espectroscopia de absorción y excitadas a una longitud de onda de 350 nm presentan un espectro con picos de absorción a 380 y 360 nm; al ser caracterizadas mediante espectroscopia de fluorescencia y ser excitadas a 350 nm presentan un patrón con picos a los 450 y 560 nm; y presentan un tamaño de entre 5-12 nm de acuerdo a análisis de técnicas demicroscopía electrónica de transmisión (TEM) y dispersión dinámica de la luz (DLS). In the present invention, the proposed methodology makes it possible to synthesize QDs of CdS more efficiently, in the presence of oxygen and in low temperature conditions. When it refers to low temperatures they correspond to temperatures between 15 and 80 ° C. More particularly, it refers to lower temperatures at 65 ° C. Preferably, the temperature for carrying out the methodology is 37 ° C. The quantum dots of CdS generated from the presented method have particular characteristics with respect to other nanoparticles. For two samples (blue and orange), when characterized by absorption spectroscopy and excited at a wavelength of 350 nm, they have a spectrum with absorption peaks at 380 and 360 nm; when characterized by fluorescence spectroscopy and excited at 350 nm, they show a peak pattern at 450 and 560 nm; and they have a size of between 5-12 nm according to analysis of transmission electronic demicroscopy (TEM) and dynamic light scattering (DLS) techniques.
Mediante técnicas de microscopía de alta resolución se determinó que los QDs producidos están compuestos por cadmio, azufre, moléculas orgánicas correspondientes a la cubierta de tioles pero además a moléculas de fosfato en su superficie. La difracción de rayos X confirmó que correspondían a cristales de sulfuro de cadmio. Using high resolution microscopy techniques, it was determined that the QDs produced are composed of cadmium, sulfur, organic molecules corresponding to the thiol sheath but also to phosphate molecules on its surface. X-ray diffraction confirmed that they corresponded to cadmium sulphide crystals.
Los QDs de CdS sintetizados a partir delmétodo de la invención, presentan importantes características ventajosas y potenciales utilidades. Debido a su cubierta compuesta por moléculas del tipo tiol y grupos fosfato, estos QDs son biocompatibles. Esto último permite la inclusión de los QDs de CdS en células, pudiendo ser monitoreadas de acuerdo a la afinidad en interacción de los grupos fosforilados y grupos fosfatos con ciertos receptores y enzimas celulares. The CdS QDs synthesized from the method of the invention have important advantageous characteristics and potential utilities. Due to their cover composed of thiol-like molecules and phosphate groups, these QDs are biocompatible. The latter allows the inclusion of CdS QDs in cells, and can be monitored according to the affinity in interaction of phosphorylated groups and phosphate groups with certain receptors and cellular enzymes.
Gracias a las características descritas, es posible realizar el monitoreo de la fluorescencia de los QDs en sistemas biológicos. De esta forma, los QDs sintetizados pueden ser utilizados en la detección y el monitoreo de receptores, proteínas, enzimas y estructuras celulares en cultivo celular de forma in vitro, también en el mareaje y monitoreo de receptores, proteínas, enzimas y estructuras celulares de forma in vivo y como sondas de detección en imagenología. Ejemplos: Thanks to the characteristics described, it is possible to monitor the fluorescence of the QDs in biological systems. In this way, the synthesized QDs can be used in the detection and monitoring of receptors, proteins, enzymes and cellular structures in cell culture in vitro, also in the marking and monitoring of receptors, proteins, enzymes and cellular structures in a way in vivo and as imaging probes. Examples:
Ejemplo 1 : Procedimiento de preparación de QDs de sulfuro de cadmio in vitro y evaluación de las concentraciones de Pi y CdCI^ y temperaturas de incubación en la eficiencia de síntesis: Example 1: Procedure for preparing cadmium sulphide QDs in vitro and evaluating the concentrations of Pi and CdCI ^ and incubation temperatures in synthesis efficiency:
En este ejemplo se describe la metodología de preparación de QDs de CdS y la evaluación de distintas concentraciones de los componentes fosfato inorgánico y CdCI2 en la mezcla de reacción. Adicionalmente, se determinaron las temperaturas de incubación más eficientes para el método. This example describes the methodology for the preparation of CdS QDs and the evaluation of different concentrations of the inorganic phosphate and CdCI 2 components in the reaction mixture. Additionally, the most efficient incubation temperatures for the method were determined.
El procedimiento de síntesis de Quantum dots de CdS propuesto presenta las siguientes etapas: a) Mezclar: i. Un tiol como fuente de azufre y como compuesto de envoltura; ii. Una sal de cadmio; iii. Un compuesto que contenga o genere una fuente de fosfato; b) Agregar una solución tampón a la mezcla obtenida en la etapa (a), a un pH entre 7-10, en presencia de oxígeno y a una temperatura entre 15 - 80°C; c) Incubar la solución obtenida en (b) a una temperatura entre 15 - 80°C, durante un período de tiempo de incubación de 1 - 9 días, si se utiliza una temperatura de 80 °C la reacción ocurre en horas, hasta obtener fluorescencia. d) Evaluar la fluorescencia de las NPs de CdS, exponiéndolas a un transiluminador UV (a 365 nm). Posteriormente, para eliminar los reactivos que no reaccionaron durante la incubación (excedentes) y a modo de enriquecer en NPs las mezclas de reacción, se filtró la mezcla de reacción en filtros Amicon con un tamaño de poro de 3 kDa (0,3-0,5 nm) y se centrifugó a 7000 G hasta obtener una solución concentrada en 200 μΙ_. Las soluciones concentradas fueron lavadas con agua destilada miliQ mediante el mismo procedimiento de centrifugación en los tubos Amicon. The proposed Quantum dots synthesis procedure of CdS has the following steps: a) Mix: i. A thiol as a source of sulfur and as a wrapping compound; ii. A cadmium salt; iii. A compound that contains or generates a phosphate source; b) Add a buffer solution to the mixture obtained in step (a), at a pH between 7-10, in the presence of oxygen and at a temperature between 15-80 ° C; c) Incubate the solution obtained in (b) at a temperature between 15-80 ° C, during an incubation period of 1-9 days, if a temperature of 80 ° C is used the reaction occurs in hours, until obtaining fluorescence. d) Evaluate the fluorescence of CdS NPs, exposing them to a UV transilluminator (at 365 nm). Subsequently, to remove reagents that did not react during incubation (surpluses) and to enrich the reaction mixtures in NPs, the reaction mixture was filtered on Amicon filters with a pore size of 3 kDa (0.3-0, 5 nm) and centrifuged at 7000 G until a solution concentrated in 200 μΙ_ is obtained. The concentrated solutions were washed with miliQ distilled water by the same centrifugation procedure in the Amicon tubes.
Los componentes principales en la metodología de síntesis propuesta son el fosfato inorgánico y la sal de cadmio. Para determinar las mejores condiciones de síntesis de los QDs de CdS se evaluaron distintas concentraciones de fosfato inorgánico y CdC como sal de cadmio. The main components in the proposed synthesis methodology are inorganic phosphate and cadmium salt. To determine the best synthesis conditions of CdS QDs, different concentrations of inorganic phosphate and CdC were evaluated as cadmium salt.
Para esto, se utilizó fosfato inorgánico (Pi) en forma de K2HP04, ácido mercaptosuccinico como fuente de tiol pues es el tiol de mayor rapidez en aparición de fluorescencia y menor precipitación (datos no mostrados), y se varió tanto la concentración de P¡ como la de Cd y la temperatura de síntesis. For this, inorganic phosphate (Pi) was used in the form of K 2 HP0 4 , mercaptosuccinic acid as a source of thiol since it is the fastest thiol in the appearance of fluorescence and less precipitation (data not shown), and the concentration was varied of P¡ like that of Cd and the synthesis temperature.
Entonces, se evaluaron soluciones que contenían: Then, solutions containing:
> P¡ a una concentración de 10 mM, CdCI2 a una concentración de 54,5 μΜ y MSA a una concentración de 5 mM, las que fueron incubadas a una temperatura de 37 °C durante un período de 1 a 4 días. > P¡ at a concentration of 10 mM, CdCI 2 at a concentration of 54.5 μΜ and MSA at a concentration of 5 mM, which were incubated at a temperature of 37 ° C for a period of 1 to 4 days.
> P¡ a distintas concentraciones: 0; 7,5; 10; 20 y 75 mM; CdCI2 a una concentración de 54,5 μΜ y MSA a una concentración de 5 mM, las que fueron incubadas a una temperatura de 37 °C durante un período de 6 días. > P¡ at different concentrations: 0; 7.5; 10; 20 and 75 mM; CdCI 2 at a concentration of 54.5 μΜ and MSA at a concentration of 5 mM, which were incubated at a temperature of 37 ° C for a period of 6 days.
> P¡ a una concentración de 10 mM, CdCI2 a una concentración de 30, 50 y 150 μΜ y MSA a una concentración de 5 mM. El período de incubación va desde 1 a 6 días para una temperatura de 37 °C, y de 0,5; 1 ,0; 1 ,5; 2,0; 2,5; 3 y 4 h para una temperatura de 80 °C. > P¡ at a concentration of 10 mM, CdCI 2 at a concentration of 30, 50 and 150 μΜ and MSA at a concentration of 5 mM. The incubation period ranges from 1 to 6 days for a temperature of 37 ° C, and 0.5; 1.0; fifteen; 2.0; 2.5; 3 and 4 h for a temperature of 80 ° C.
Las muestras contienen 1 ml_ de volumen final y fueron expuestas a un transiluminador UV (a 365 nm). The samples contain 1 ml_ of final volume and were exposed to a UV transilluminator (at 365 nm).
A partir de esto, se determinó que el P¡ es capaz de generar soluciones que cambian de color de fluorescencia en el tiempo (Fig. 1A), y que la concentración de P¡ regula el color de fluorescencia obtenido a un tiempo fijo (en concentraciones mM) (Fig. B). Por otra parte, la cantidad de Cd agregado también regula el color de fluorescencia obtenido (en concentraciones μΜ) (Fig. 1 C), observándose fluorescencia luego de un día cuando se utiliza bajas temperaturas, 37°C e incluso hasta 15°C (datos no mostrados), o a las horas de incubación, a temperaturas de 80°C (Fig. 1 C). From this, it was determined that the P¡ is capable of generating solutions that change fluorescence color over time (Fig. 1A), and that the concentration of P¡ regulates the fluorescence color obtained at a fixed time (in mM concentrations) (Fig. B). On the other hand, the amount of Cd added also regulates the fluorescence color obtained (in μΜ concentrations) (Fig. 1 C), fluorescence being observed after one day when low temperatures are used, 37 ° C and even up to 15 ° C ( data not shown), or at incubation hours, at temperatures of 80 ° C (Fig. 1 C).
Ejemplo 2: Caracterización de QDs de sulfuro de cadmio producidos utilizando compuestos del tipo tiol como envoltura y fosfato inorgánico en su síntesis: Example 2: Characterization of cadmium sulphide QDs produced using compounds of the thiol type as envelope and inorganic phosphate in its synthesis:
En este ejemplo se presenta la caracterización de las NPs de CdS producidas con el método presentado en el ejemplo 1. In this example the characterization of the NPs of CdS produced with the method presented in example 1 is presented.
Para la caracterización se utilizaron como muestras dos soluciones fluorescentes de distinto color (naranja y verde-azul) las que fueron purificadas para obtener material nanoparticulado (ver metodología). Se determinó el tamaño de los QDs de CdS y se evaluaron sus propiedades espectrofotométricas y de fluorescencia. For the characterization two fluorescent solutions of different color (orange and green-blue) were used as samples, which were purified to obtain nanoparticulate material (see methodology). The size of the CdS QDs was determined and their spectrophotometric and fluorescence properties were evaluated.
Espectroscopia de absorción y fluorescencia, y Dispersión dinámica de la luz (DLS): Absorption and fluorescence spectroscopy, and Dynamic light scattering (DLS):
Las propiedades espectroscópicas de las NPs sintetizadas fueron determinadas usando un espectrofotómetro/fluorímetro con lector de placas, Synergy H1 (Biotek). El perfil de absorbancia de soluciones concentradas de QDs sintetizados in vitro fue medido entre 300 y 700 nm, y para registro de la emisión de fluorescencia fueron excitadas a 350 nm con una ganancia de 100% y la fluorescencia colectada entre 400 y 700 nm. Las mediciones de DLS en las NPs se llevaron a cabo en cubetas plásticas desechables de 4 pasos ópticos utilizando un equipo de dispersión Zetasizer Nano ZS (Malvern Instruments Limited, UK). The spectroscopic properties of the synthesized NPs were determined using a spectrophotometer / fluorimeter with plate reader, Synergy H1 (Biotek). The absorbance profile of solutions Concentrates of QDs synthesized in vitro were measured between 300 and 700 nm, and for recording the fluorescence emission they were excited at 350 nm with a 100% gain and the fluorescence collected between 400 and 700 nm. The DLS measurements in the NPs were carried out in 4-step disposable plastic cuvettes using a Zetasizer Nano ZS dispersion kit (Malvern Instruments Limited, UK).
Se observó que ambas soluciones presentan el típico hombro correspondiente al plasmón de superficie descrito para QDs de CdS solubles en agua, bajo 400, 380 y 360 nm, respectivamente (Fig. 2A). Además, emiten fluorescencia en 450 y 560 nm respectivamente (Fig. 2B). La diferencia en los máximos de fluorescencia está relacionada con el tamaño del material nanoparticulado en las soluciones, encontrándose tamaños promedios de 7,5 y 3,5 nm para la solución naranja y verde-azul respectivamente (Fig. 2D y 2C, respectivamente). It was observed that both solutions have the typical shoulder corresponding to the surface plasmon described for water-soluble CdS QDs, under 400, 380 and 360 nm, respectively (Fig. 2A). In addition, they emit fluorescence at 450 and 560 nm respectively (Fig. 2B). The difference in fluorescence maxima is related to the size of the nanoparticulate material in the solutions, with average sizes of 7.5 and 3.5 nm being found for the orange and green-blue solution respectively (Fig. 2D and 2C, respectively).
Microscopía electrónica de transmisión (TEM): Transmission electron microscopy (TEM):
Las imágenes de microscopía electrónica de transmisión fueron colectadas usando un microscopio a 80 kV marca Phillips Tecnai 12 BioTwin. Para la preparación de las muestras se tomaron entre 1 y 3 pL de soluciones de QDs sintetizados y purificados, los que fueron puestos sobre grillas de cobre (grilla TEM Formvar carbón 300 mesh cobre, tamaño de agujero de grilla 63 pm) y deshidratadas completamente bajo una ampolleta de luz visible convencional. Transmission electron microscopy images were collected using an 80 kV Phillips Tecnai 12 BioTwin brand microscope. For the preparation of the samples, between 1 and 3 pL of synthesized and purified QD solutions were taken, which were placed on copper grids (TEM Formvar grid 300 carbon copper mesh, grid hole size 63 pm) and completely dehydrated a conventional visible light bulb.
Mediante microscopía electrónica (TEM) se corroboró la presencia de partículas aglomeradas de tamaño inferior a 20 nm (Fig. 2E), por consiguiente una dilución de estas permitió observar NPs individuales con tamaños que fluctúan entre 5 a 12 nm (Fig. 2F), para una solución naranja de QDs. Espectroscopia infrarroja con transformada de Fourier (FTIR): Electron microscopy (TEM) confirmed the presence of agglomerated particles smaller than 20 nm (Fig. 2E), therefore a dilution of these allowed to observe individual NPs with sizes ranging from 5 to 12 nm (Fig. 2F), for an orange solution of QDs. Fourier transform infrared spectroscopy (FTIR):
Los QDs de CdS producidos también fueron caracterizados de acuerdo a su espectro IR. Para esto, se tomaron muestras de QDs, las que fueron liofilizadas a sequedad para eliminar el agua de la solución. El polvo de NPs se mezcló con KBr, confeccionando pastillas que se midieron en un equipo de FTIR sólido, Spectrum Two (Perkin Elmer), entre 4000 y 400 cm"1. Adicionalmente, se realizaron los controles pertinentes, midiéndose el espectro IR de los reactivos por separado (tioles, fosfato, CdCI2, moléculas fosforiladas, entre otros), utilizando el mismo equipo y con la misma preparación. The QDs of CdS produced were also characterized according to their IR spectrum. For this, samples of QDs were taken, which were lyophilized to dryness to remove water from the solution. The NPs powder was mixed with KBr, making tablets that were measured in a solid FTIR equipment, Spectrum Two (Perkin Elmer), between 4000 and 400 cm "1. Additionally, the relevant controls were performed, measuring the IR spectrum of the separate reagents (thiols, phosphate, CdCI 2 , phosphorylated molecules, among others), using the same equipment and with the same preparation.
La naturaleza química de las NPs producidas en presencia de P¡ fue evaluada mediante espectroscopia FTIR (Fig. 3). Se observaron señales de P¡ y MSA, sugiriendo que las NPs poseen en su cubierta P¡ y tiol. Para el P¡ se encontró la señal del grupo P04 (1085 cm"1) y la señal que evidencia el estiramiento P-O-P en fosfatos alifáticos (996 cm"1), a su vez para el MSA se observaron las bandas correspondientes a los estiramientos CH y CH2 (entre 3000 y 1800 cm"1), la banda del estiramiento C-S (1390 cm"1) y el cambio desde COOH (MSA puro)→ COO- (NPs) (1700→1563 cm"1). Finalmente, la señal del estiramiento S-H del MSA (2569 y 2566 cm"1) desapareció en la muestra de NPs, sugiriendo una unión covalente del tiol a la NP a través del grupo sulfhidrilo. Además de estas características se observó una robusta banda de estiramiento de O-H a los 3400 cm"1 perteneciente al agua y/o MSA, por lo que probablemente las NPs presenten en su superficie moléculas de agua, característica también previamente descrita para QDs de CdS solubles en agua (Ha y cois., 2011 , Srinivasa y cois., 2011 , Kumar y cois., 2012, Zhang y cois., 2004). The chemical nature of the NPs produced in the presence of P¡ was evaluated by FTIR spectroscopy (Fig. 3). P¡ and MSA signals were observed, suggesting that NPs have P¡ and thiol on their cover. For the P¡, the signal of the P0 4 group (1085 cm "1 ) and the signal evidencing POP stretching in aliphatic phosphates (996 cm " 1 ) were found, in turn for the MSA the bands corresponding to the stretching were observed CH and CH 2 (between 3000 and 1800 cm "1 ), the stretch band CS (1390 cm " 1 ) and the change from COOH (pure MSA) → COO- (NPs) (1700 → 1563 cm "1 ). Finally , the MSA SH stretch signal (2569 and 2566 cm "1 ) disappeared in the sample of NPs, suggesting a covalent binding of thiol to NP through the sulfhydryl group. In addition to these characteristics, a robust OH stretch band at 3400 cm "1 belonging to water and / or MSA was observed, so that NPs probably present water molecules on their surface, a feature also previously described for soluble CdS QDs in water (Ha and cois., 2011, Srinivasa and cois., 2011, Kumar and cois., 2012, Zhang and cois., 2004).
Ejemplo 3: Síntesis de quantum dots de CdS/n vitro mediante la adición de moléculas dadoras de grupos fosfato: En este ejemplo se presenta la metodología de síntesis de QDs de sulfuro de cadmio a partir de la adición de moléculas dadoras de grupos fosfato en las mezclas de reacción. Por tanto, se reemplazó el P¡, de la síntesis anteriormente señalada, por moléculas fosforiladas encontradas en organismos biológicos; glucosa-6-fosfato (G6P), glicerol-2-fosfato (G2P), fructosa-1 ,6-bisfosfato (F1.6BP), adenosina monofosfato (AMP), adenosina difosfato (ADP), adenosina trifosfato (ATP), entre otros. Cada molécula fosforilada quedó a una concentración final de 10 mM. Example 3: Synthesis of quantum dots of CdS / n in vitro by adding phosphate group donor molecules: In this example, the methodology for the synthesis of cadmium sulphide QDs from the addition of phosphate group donor molecules in the reaction mixtures is presented. Therefore, the P¡, of the above-mentioned synthesis, was replaced by phosphorylated molecules found in biological organisms; glucose-6-phosphate (G6P), glycerol-2-phosphate (G2P), fructose-1, 6-bisphosphate (F1.6BP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), between others. Each phosphorylated molecule was at a final concentration of 10 mM.
Se preparó una mezcla de reacción entre MSA (5 mM), CdCl2(54,5 μΜ) y las distintas moléculas dadoras de grupos fosfato (10 mM). Las mezclas se incubaron durante 12 días, evaluándose día a día la aparición de fluorescencia al ser expuestas a un transiluminador UV (a 365 nm). A reaction mixture was prepared between MSA (5 mM), CdCl 2 (54.5 μΜ) and the different phosphate group donor molecules (10 mM). The mixtures were incubated for 12 days, the appearance of fluorescence being evaluated day by day when exposed to a UV transilluminator (at 365 nm).
Se determinó que soluciones de MSA/Cd tratadas con AMP, F1.6BP, G6P o G2P, exhiben un comportamiento de fluorescencia variable en el tiempo (Fig. 4A), sugiriendo que estas moléculas pueden gatillar la síntesis de QDs al igual que el P¡, no así ADP o ATP, que no son capaces de generar fluorescencia en el tiempo en las condiciones mostradas. It was determined that MSA / Cd solutions treated with AMP, F1.6BP, G6P or G2P, exhibit a time-varying fluorescence behavior (Fig. 4A), suggesting that these molecules can trigger the synthesis of QDs as well as P ¡, Not so ADP or ATP, which are not capable of generating fluorescence over time under the conditions shown.
Luego, para comprobar que las soluciones fluorescentes que contienen MSA/Cd/molécula fosforilada, pueden generar QDs de CdS igual que las soluciones tratadas con P¡, una solución (de color verde) tratada con 10 mM de G6P fue concentrada y purificada (ver metodología). Then, to verify that fluorescent solutions containing MSA / Cd / phosphorylated molecule, can generate Qds of CdS as well as solutions treated with P¡, a solution (green) treated with 10 mM G6P was concentrated and purified (see methodology).
Las NPs obtenidas presentaron un espectro de absorbancia como de fluorescencia correspondiente a lo esperado para QDs de CdS; hombro de absorbancia entre 350 y 400 nm y una emisión de fluorescencia de a 500 nm correspondiente a NPs color verde (Fig. 4B). En cuanto a la composición analizada mediante espectroscopia FTIR; la solución fluorescente verde presentó las bandas de estiramiento CH y CH2 (entre 3000 y 1800 cm"1) y estiramiento C-S (1390 cm"1) característicos del MSA, y al igual que en QDs sintetizados con P¡ no se observa la banda correspondiente al grupo sulfhidrilo, lo que sugiere que el tiol se encuentra unido covalentemente al QDs a través del grupo sulfhidrilo en la muestra, además entre 1090-1100 cm"1 la señal del grupo fosfato, y los estiramientos asimétrico y simétrico del P-O-C son encontrados tanto en el control (solo G6P) como en las NPs entre 970- 980 y ~816 cm"1 respectivamente (Fig. 4C). Finalmente, un análisis preliminar de tamaño señaló que la solución contiene material nanoparticulado de aproximadamente 35 nm de diámetro. El cambio de color de fluorescencia en el tiempo, es evidencia de la presencia de QDs. Finalmente, las NPs sintetizadas presentan moléculas de G6P que conforman su capa externa de recubrimiento, aumentando el radio hidrodinámico de las NPs. The NPs obtained presented an absorbance spectrum as fluorescence corresponding to that expected for CdS QDs; shoulder absorbance between 350 and 400 nm and a fluorescence emission of at 500 nm corresponding to green NPs (Fig. 4B). Regarding the composition analyzed by FTIR spectroscopy; the green fluorescent solution featured the CH stretch bands and CH 2 (between 3000 and 1800 cm "1 ) and CS stretch (1390 cm " 1 ) characteristic of MSA, and as in QDs synthesized with P¡, the band corresponding to the sulfhydryl group is not observed, suggesting that thiol It is covalently linked to the QDs through the sulfhydryl group in the sample, plus between 1090-1100 cm "1 the phosphate group signal, and the asymmetric and symmetrical stretches of the POC are found both in the control (G6P only) and in the NPs between 970-980 and ~ 816 cm "1 respectively (Fig. 4C). Finally, a preliminary size analysis indicated that the solution contains nanoparticulate material approximately 35 nm in diameter. The change in fluorescence color over time is evidence of the presence of QDs. Finally, the synthesized NPs have G6P molecules that make up their outer coating layer, increasing the hydrodynamic radius of the NPs.

Claims

REIVINDICACIONES
1) Método para la síntesis de nanopartículas (NPs) semiconductoras, fluorescentes de sulfuro de cadmio (CdS) CARACTERIZADO porque comprende: 1) Method for the synthesis of semiconductor nanoparticles (NPs), cadmium sulphide fluorescent (CdS) CHARACTERIZED because it comprises:
a) Mezclar:  a) Mix:
i. Un tiol como fuente de azufre y como compuesto de envoltura;  i. A thiol as a source of sulfur and as a wrapping compound;
ii. Una sal de cadmio;  ii. A cadmium salt;
iii. Un compuesto que contenga o genere una fuente de fosfato;  iii. A compound that contains or generates a phosphate source;
b) Agregar una solución tampón a la mezcla obtenida en la etapa (a), a un pH entre 7 - 10, en presencia o ausencia de oxígeno y a una temperatura entre 15 - 80°C;  b) Add a buffer solution to the mixture obtained in step (a), at a pH between 7-10, in the presence or absence of oxygen and at a temperature between 15-80 ° C;
c) Incubar la solución obtenida en (b) a una temperatura entre 15 - 80°C, durante un período de tiempo de incubación de 1 - 9 días, hasta obtener fluorescencia;  c) Incubate the solution obtained in (b) at a temperature between 15-80 ° C, for an incubation period of 1-9 days, until fluorescence is obtained;
d) Evaluar la fluorescencia de las NPs de CdS, exponiéndolas a un transiluminador UV (a 365 nm).  d) Evaluate the fluorescence of CdS NPs, exposing them to a UV transilluminator (at 365 nm).
2) Método de acuerdo a la reivindicación 1 CARACTERIZADO porque el tiol como fuente de azufre y como compuesto de envoltura, corresponde a glutatión (GSH), cisteína (CYS) y ácido mercaptosuccínico (MSA). 2) Method according to claim 1 CHARACTERIZED in that the thiol as a source of sulfur and as a wrapping compound corresponds to glutathione (GSH), cysteine (CYS) and mercaptosuccinic acid (MSA).
3) Método de acuerdo a la reivindicación 2 CARACTERIZADO porque la concentración del compuesto de tiol se encuentra entre 0,25 y 15 mM. 3) Method according to claim 2 CHARACTERIZED because the concentration of the thiol compound is between 0.25 and 15 mM.
4) Método de acuerdo a las reivindicaciones anteriores CARACTERIZADO porque la sal de cadmio puede ser seleccionada entre CdCI2, Cd(NO3)2 x 4H2O y Cd(CH3COO)2 x 2H2O. 4) Method according to the preceding claims CHARACTERIZED because the cadmium salt can be selected from CdCI 2 , Cd (NO 3 ) 2 x 4H 2 O and Cd (CH 3 COO) 2 x 2H 2 O.
5) Método de acuerdo a la reivindicación 4 CARACTERIZADO porque la sal de cadmio corresponde preferentemente a una solución de cloruro de cadmio a una concentración de 30-300 μΜ. 5) Method according to claim 4 CHARACTERIZED in that the cadmium salt preferably corresponds to a solution of cadmium chloride at a concentration of 30-300 μΜ.
6) Método de acuerdo a las reivindicacionesanteriores CARACTERIZADO porque el compuesto dador de fosfato puede corresponder a fosfato inorgánico en forma de K2HPO4, y moléculas fosforiladas tales como glucosa-6-fosfato (G6P), glicerol- 2-fosfato (G2P), fructosa-1 ,6-bisfosfato (F1.6BP), adenosina monofosfato (AMP), adenosina difosfato (ADP) y/o adenosina trifosfato (ATP), entre otras. 6) Method according to the preceding claims CHARACTERIZED because the phosphate donor compound can correspond to inorganic phosphate in the form of K 2 HPO 4 , and phosphorylated molecules such as glucose-6-phosphate (G6P), glycerol-2-phosphate (G2P) , fructose-1, 6-bisphosphate (F1.6BP), adenosine monophosphate (AMP), adenosine diphosphate (ADP) and / or adenosine triphosphate (ATP), among others.
7) Método de acuerdo a la reivindicación 6 CARACTERIZADO porque el compuesto dador de fosfato se encuentra en una concentración entre 10 μΜ - 100 mM. 7) Method according to claim 6 CHARACTERIZED in that the phosphate donor compound is in a concentration between 10 μΜ - 100 mM.
8) Método de acuerdo a la reivindicación 7 CARACTERIZADO porque el compuesto dador de fosfato, preferentemente tiene una concentración de 10 mM. 8) Method according to claim 7 CHARACTERIZED in that the phosphate donor compound preferably has a concentration of 10 mM.
9) Método de acuerdo a las reivindicaciones anteriores CARACTERIZADO porque la solución tampón puede corresponder a tampón fosfato salino: KH2PO4/K2HPO4 (PBS), ácido 3-(N- morfolino) propanosulfónico (MOPS), ácido (4-(2-hidroxietil)-1- piperazinetanosulfónico(HEPES), Tris-HCI o citrato de sodio. 9) Method according to the preceding claims CHARACTERIZED because the buffer solution can correspond to phosphate buffered saline: KH 2 PO 4 / K 2 HPO 4 (PBS), 3- (N-morpholino) propanesulfonic acid (MOPS), acid (4 - (2-hydroxyethyl) -1-piperazinethanesulfonic acid (HEPES), Tris-HCI or sodium citrate.
10) Método de acuerdo a la reivindicación 9 CARACTERIZADO porque la solución tampón se encuentra en una concentración entre 1 y 100 mM. 11 ) Método de acuerdo a la reivindicación 10 CARACTERIZADO porque la solución tampón, preferentemente tiene una concentración de 10 mM. 10) Method according to claim 9 CHARACTERIZED in that the buffer solution is in a concentration between 1 and 100 mM. 11) Method according to claim 10 CHARACTERIZED in that the buffer solution preferably has a concentration of 10 mM.
12) Método de acuerdo a las reivindicaciones anteriores CARACTERIZADO porque la temperatura de incubación preferida es de 37°C. 12) Method according to the preceding claims CHARACTERIZED because the preferred incubation temperature is 37 ° C.
13) Método de acuerdo a la reivindicación 1 CARACTERIZADO porque antes de la etapa (d), se filtra la solución obtenida en la etapa (c) de incubación, para eliminar los reactivos que no reaccionaron durante la etapa (c). 13) Method according to claim 1 CHARACTERIZED because prior to step (d), the solution obtained in step (c) of incubation is filtered, to eliminate reagents that did not react during step (c).
14) Método de acuerdo a la reivindicación 13 CARACTERIZADO porque luego de la etapa de filtración, las NPs de CdS también se concentran y se lavan. 14) Method according to claim 13 CHARACTERIZED because after the filtration step, the CdS NPs are also concentrated and washed.
15) Método de acuerdo a la reivindicación 14 CARACTERIZADO porque la etapa de concentración se lleva a cabo a través de una centrifugación a 7000 G, durante 30 minutos. 15) Method according to claim 14 CHARACTERIZED in that the concentration step is carried out through a centrifugation at 7000 G, for 30 minutes.
16) Nanopartículas semiconductoras, fluorescentes de CdS, obtenidas mediante el método de las reivindicaciones anteriores CARACTERIZADAS porque al ser excitadas a 350 nm presentan un espectro de absorción con picos de absorción a 380 y 360 nm (patrón espectroscopia de absorción), al ser excitadas a 350 nm mediante fotoluminiscencia presentan picos a 550 nm y 560 nm (espectroscopia fluorescencia), y de acuerdo microscopía electrónica de transmisión tienen un tamaño de entre 5-12 nm. 16) CdS fluorescent semiconductor nanoparticles, obtained by the method of the preceding claims CHARACTERIZED because when excited at 350 nm they have an absorption spectrum with absorption peaks at 380 and 360 nm (absorption spectroscopy pattern), when excited at 350 nm by photoluminescence show peaks at 550 nm and 560 nm (fluorescence spectroscopy), and according to transmission electron microscopy they are between 5-12 nm in size.
17) Uso de NPs semiconductoras fluorescentes de CdSde acuerdo a la reivindicación 16 CARACTERIZADO porque sirven para la detección y monitoreo de receptores, proteínas, enzimas y estructuras celulares en cultivo celular de forma in vitro; en el mareaje y monitoreo de receptores, proteínas, enzimas estructuras celulares de forma in vivo; sondas de detección imagenología y dispositivos fotovoltaicos. 17) Use of CdS fluorescent semiconductor NPs according to claim 16 CHARACTERIZED because they serve for the detection and monitoring of receptors, proteins, enzymes and cellular structures in cell culture in vitro form; at marking and monitoring of receptors, proteins, enzymes cellular structures in vivo; imaging probes and photovoltaic devices.
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