Folic acid (FA) is a known water-soluble B-vitamin comprising three sub-components: A pterin ring, p-aminobenzoyl residue, and L-glutamic acid (Figure 1
]. It is linked with different diseases; FA prevents birth defects of the brain and spinal cord, especially spina bifida, and decreases serum homocysteine levels, reducing the risk of heart diseases [2
]. FA is an essential vitamin that is nowadays prescribed to pregnant women and is indicated in the prevention of certain types of anemia [4
]. FA is also used in bioimaging as an element of orientation toward cancer cells, as folate receptors are highly over-expressed on the surface of many tumors [6
]. FA plays an important role in the proper development of physiological processes in the body [8
The human body is not able to synthesize FA, and its presence in foods such as spinach, cereals, citrus, rice, and beans may not be enough to meet the daily requirement for this vitamin [3
]. The daily consumption of foods fortified with FA, such as citrus beverages, can provide the amount required for human health. Therefore, having a simple and fast method for determining FA in commercial beverages is of great interest.
Different methods have been reported in the literature for the quantification of FA, including spectrophotometric methods [9
], electrochemical techniques [11
], high-performance liquid chromatography (HPLC) [13
] and ultrahigh HPLC, and ion chromatography [16
]. In general, these methods are based on time-consuming reactions that involve laborious and slow procedures. In particular, the chromatographic methods require expensive equipment and the use of large volumes of organic solvents. Therefore, the development of a simple, accurate, and low-cost method with high sensitivity and selectivity for the determination of FA is of great importance for monitoring the quality of food products. Fluorescence methods have gained considerable attention in recent years, due to their selectivity, sensitivity, easy sample preparation, and rapid analysis. As a kind of new fluorophore, quantum dots (QDs) are a favorable alternative as they have advantages over conventional organic fluorescent dyes. They have unusual optical and electronic properties as a consequence of the quantum confinement effect. QDs have been proven to be effective in different applications as biological biomarkers, sensors, drug delivery systems, and solar cells [17
In recent years, methods for the determination of FA based on the use of nanomaterials have been reported. Graphene oxide/Ag nanoparticle hybrids with surface-enhanced Raman spectroscopy (SERS) detection [22
], a bovine serum albumin (BSA)-modified gold nanocluster with fluorescence detection [23
], and a ZrO2
nanoparticle-modified carbon paste electrode with voltammetry detection [24
] have been used in the determination of FA. Other works show the determination of FA using QDs capped with thiolated ligands as thioglycolic (TGA) [25
] and mercaptopropionic (MPA) acids [26
]. However, in these nanoparticles, at pH 8, the carboxylate groups are dissociated, giving a negative charge to the surface of the QDs. Under these conditions, FA is also negatively charged, and the interaction between the QDs and FA is not favored, due to electrostatic repulsions. Therefore, QDs coated with amino groups, which, at pH 8, are positively charged, could improve the analytical characteristics of the sensor.
Cysteamine (CA) is a bifunctional ligand, with thiol groups that bind strongly to metal ions on the surface of QDs and hydrophilic amino groups that render the QDs biocompatible and dispersible in water [27
]. In addition, the amino groups of CA are protonated at physiological pH and can interact with negatively charged molecules, favoring the formation of a stable assembly by electrostatic interactions.
There are some works based on amino-coated QDs as nanosensors for the determination of different molecules [27
]. However, to our knowledge, there are no reports based on CdTe/CA QDs for the determination of FA. Therefore, the objective of the present work is the development of a sensor based on QDs coated with amino groups and its application in the determination of FA in commercial beverages. The proposed design should improve the analytical characteristics of the sensor.
In this work, we present a system based on water-soluble CdTe QDs coated with CA for the determination of FA. CdTe/CA was prepared and characterized by high-resolution transmission electron microscopy, FT-IR spectroscopy, the zeta potential, and UV-visible and fluorescence spectroscopy. The analytical response of CdTe QDs capped with CA toward FA was studied and compared to the results obtained using other thiol-capped QDs. As CdTe/CA QDs showed the largest quenching efficiency, this system was further studied for FA determination. The principle of this nanosensor is illustrated in Scheme 1
. The probe is based on the fluorescence quenching of CdTe/CA QDs by FA at pH 8. A probable mechanism of fluorescence quenching based on the coordinated behavior of electrostatic interactions between positively charged CdTe/CA QDs (ethylamine groups, pKa = 10.52) and negatively charged FA (carboxylic groups, pKaα
= 3.46 and pKaγ
= 4.83) and the electronic transfer of QDs to FA is proposed. The nanosensor is rapid, easy to obtain, and shows a low detection limit, high selectivity, and low cost. The method showed excellent results in detecting FA contents in beverages (recoveries from 98.3 to 103.9%). The system provides a new approach for the construction of nanosensors with high application potential in different areas.
2. Materials and Methods
Cadmium chloride hemi(pentahydrate), potassium tellurite, cysteamine (CA), mercaptopropionic acid (MPA), glutathione (GSH), sodium borohydride (NaBH4), sodium hydroxide, ascorbic acid, citric acid, glutamic acid, tartaric acid, glucose, bovine serum albumin (BSA), and fluorescein (quantum yield (QY), 79%) were purchased from Sigma-Aldrich, Mexico. All chemicals (analytical grade) and solvents (spectroscopic grade) were used without further purification. Deionized water was used in the experiments. Beverage samples were obtained from a local supermarket.
2.2. Characterization of Thiol-Capped Nanoparticles
Fourier-transform infrared (FT-IR) spectra (650–4000 cm−1) were recorded in the transmission mode on a Perkin Elmer FT-IR Spectrum 400 spectrophotometer (Perkin Elmer, Mexico). UV-visible spectra were collected on a Shimadzu spectrophotometer model UV 2700 (Shimadzu, Japan) using a 1 cm path-length quartz cell. Fluorescence spectra were recorded from 300 to 700 nm on a Horiba Nanolog fluorescence spectrophotometer (HORIBA Scientific, Edison, NJ, USA) using a xenon lamp as the excitation source and a 1 cm path-length fluorescence quartz cell. The morphologies and sizes of nanoparticles were characterized by JEM-2200FS (JEOL, Akishima, Japan) transmission electron microscopy (TEM). The microscope was capable of spherical aberration correction in a scanning transmission electron microscope (STEM) mode working at an accelerating voltage of 200 keV. The images were acquired by a high-angle annular dark-field (HAADF) detector. Zeta potential measurements were acquired using a Horiba Scientific SZ-100 nanoparticle analyzer (Horiba Scientific, Japan). A Thermo Scientific pH meter was utilized to measure the pH of the solutions (Thermo Fisher Scientific, Mexico City, Mexico).
2.3. Preparation of GSH, MPA, and CA-Capped CdTe QDs
GSH, MPA, and CA-capped CdTe nanocrystals were synthesized according to the methodology used in previous work from the group [31
]. Experimental details for the preparation and characterization by UV-visible and fluorescence spectroscopy of QDs modified with MPA, GSH, and CA are described in the Supplementary Material
The relative quantum yield (QY) of thiol-capped CdTe QDs was examined by a procedure described in the Supplementary Material
2.4. Stability Study
The photochemistry stability was investigated by exposing the nanomaterial to UV irradiation (365 nm) at a constant temperature with a xenon lamp of 450 W. The dispersed material was prepared in ultrapure water at pH 8 using a phosphate buffer (PBS). The fluorescence intensity was measured using the “kinetic” mode of the Nanolog spectrofluorometer for 60 min.
2.5. Fluorescence Study
The fluorescent response of CdTe/CA QDs toward FA was studied by adding 2 mL of QD solution and 1 mL of PBS buffer solution (pH 8) in a 1 cm optical-path quartz fluorescence cell. The titration was carried out by adding increasing amounts of FA in a concentration range from 0 to 16.4 µM. The fluorescence spectra were obtained in a range of emission wavelengths from 500 to 750 nm, at an excitation wavelength of 440 nm.
2.6. Interference Study
The response of the nanoprobe to other compounds was studied through fluorescence spectra. Competition assays were performed for BSA, GSH, glucose, ascorbic, and citric acids. For competition experiments, the solution of CdTe/CA QDs (3 mL) was placed in a 1 cm optical-path quartz cell and mixed with FA solution in the presence of the possible interference, at a molar ratio of FA:Interference of 1:20. The fluorescence intensity was measured at λem = 597, with excitation at 440 nm.
2.7. Sensitivity Detection
For the construction of the calibration graph, FA stock solution was prepared in phosphate buffer (100 µM, pH 8). QD/CA solution, prepared as shown above, was mixed with different concentrations of FA from 0.16 to 16.4 µM in 5 mL volumetric flasks and diluted to the mark. The calibration curve was the average of three repetitions.
2.8. Detection of FA in Real Samples
The determination of FA was carried out directly in samples of orange beverages without any previous treatment. The QD solution was mixed with 100 µL of the sample in a buffer solution (pH 8). A standard addition method was used for FA determination. The standard addition curve was obtained in a concentration range between 0 and 7 µM and was the result of three repetitions (R2 = 0.9950 sample 1 and R2 = 0.9994 sample 2).