Characterization and Application of the Fluorescent APTA-nano Biosensors Based on CdTe-TGA QDs for Determination of 25-hydroxyvitamin D3

doi:10.21203/rs.3.rs-4703158/v1

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Characterization and Application of the Fluorescent APTA-nano Biosensors Based on CdTe-TGA QDs for Determination of 25-hydroxyvitamin D₃

https://doi.org/10.21203/rs.3.rs-4703158/v1

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Medical studies have shown that vitamin D deficiency is strongly associated with several metabolic disorders, including diabetes, cardiovascular diseases, and cancer. So, it is crucial to regularly check its concentration in the blood serum. Traditional methods for detecting 25-hydroxyvitamin D₃ [25(OH)D₃] as a marker of vitamin D status are expensive, time-consuming, and require a skilled workforce and specialized laboratory. This study developed a simple and cost-effective fluorescence system for 25-hydroxyvitamin D₃determination. The fluorescent APTA-nano biosensors were fabricated using cadmium telluride quantum dots modified with thioglycolic acid (CdTe-TGA QDs) and functionalized with thiol-25(OH)D₃-aptamer through ligand exchange. The thiol-25(OH)D₃-aptamer interacted directly with CdTe-TGA QDs, increasing fluorescence intensity. However, it decreased when the target molecules of 25-hydroxyvitamin D₃ were introduced. The structural and morphological characteristics of APTA-nano biosensors were confirmed by various analytical methods such as UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FESEM), energy dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), and dynamic light scattering (DLS). According to the typical Stern-Volmer equation, the relationship between fluorescent quenching and target concentration was linear with a detection limit of 1.35 × 10^-8M, a quantification limit of 4.50 × 10^-8M, and a relative standard deviation of 1.75%. The optimized APTA-nano biosensor demonstrated high specificity towards the target and stability over 28 days. Furthermore, it detected 25-hydroxyvitamin D₃ in human serum with a recovery rate of up to 99.77%. The results indicate that the fluorescent APTA-nano biosensors could be valuable in developing robust sensing technology for low-concentrated analytes.

APTA-nano biosensor

CdTe-TGA quantum dots

25-hydroxyvitamin D3

Fluorescence quenching

There are two types of fat-soluble vitamin D - vitamin D₂ and vitamin D₃. Vitamin D is commonly known as the sunshine vitamin because it is synthesized by the body during exposure to sunlight as the most efficient source of vitamin D₃. It can also be obtained from oily fish, eggs, fortified food products, and supplements (both vitamin D₂ and vitamin D₃) (Schwalfenberg, 2011). It plays a vital role in various physiological activities, the immune system, bone metabolism, and the regulation of calcium and phosphate homeostasis (Mitchell et al., 2019). The hydroxylase enzyme in the liver converts vitamin D₃ to a half-activated state, 25-hydroxyvitamin D₃ [25(OH)D₃], whose receptors can be found in almost every tissue. When the human body tissue needs vitamin D functions, it goes into the tissue and converts to a fully activated form [1,25(OH)₂D₃] (Norman, 2012). So 25-hydroxyvitamin D₃, a great circulating form of vitamin D in blood serum, reflects vitamin D status. Due to environmental and socio-economic factors, people are unable to derive sufficient quantity of vitamin D. According to the most recent studies, up to 50% of adults and children worldwide suffer from vitamin D deficiency (≤ 29 ng mL^-1), which is closely related to different kinds of metabolic diseases such as type 2 diabetes, cancer, cardiovascular disease, rheumatoid arthritis, depression, and a reduction in life expectancy with indirect symptoms. Meanwhile, excess vitamin D in the body, known as hypervitaminosis D (> 150 ng mL^-1), results in anorexia, fatigue, muscle pain, irregular heartbeat, and hypercalcemia (Dudenkov et al., 2015). Hence, periodic monitoring of 25-hydroxyvitamin D₃ concentration is critical to maintain an optimum level of vitamin D and early diagnosis of vitamin D deficiency or toxicity. The conventional techniques used for the assessment of vitamin D statuses, such as radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), and high-performance liquid chromatography (HPLC), are expensive, time-consuming, and require skilled lab technicians and elaborate machinery. They may suffer from false and large deviations in quantitative results (Fraser & Milan, 2013; Lazzarino et al., 2017; Wallace et al., 2010). Previously reported electrochemical biosensors and surface Plasmon resonance (SPR) have drawbacks, including high cost, rapid denaturation or instability at room temperature, and a shorter shelf life (Carlucci et al., 2013). Therefore, there is a great need to develop an appropriate vitamin D detection system to overcome these limitations.

Nano biosensors offer an accurate, reliable, portable, convenient, cost-effective, time-saving, and user-friendly method for realizing the quantitative detection of 25-hydroxyvitamin D₃ directly from biological fluids (Jo et al., 2021). These nano biosensors are chemical sensors that convert biological responses into detectable signals with high sensitivity, selectivity, and excellent linear concentration range (Elmizadeh et al., 2023). A wide variety of nanomaterials, such as mesoporous nano-silica, carbon nanotubes, and quantum dots (QDs), owing to high surface-to-volume ratio, are functionalized with different biomolecules like enzymes, antibodies, and nucleic acids to fabricate such biosensors (Lin et al., 2008). Fluorescent quantum dots are semiconductor nanocrystals applied in various nano biosensors to detect low concentrations of different analytes, like pharmaceutical molecules. They exhibit superior optical, chemical, mechanical, and electron transport performances, broad absorption spectra, narrow emission peaks, and size-tunable properties unmatched by other fluorescent nanomaterials (Bardajee et al., 2022). For designing QDs-based nano biosensors, various strategies, including ligand exchange, amphiphilic compound, sulfhydryl group, and dendrimer coupling reactions, are employed to modify QDs surfaces. The aim of QDs surface modification is the improvement of the optical properties for achieving the desired selectivity, biodegradability, biocompatibility, and nontoxicity to be used in biochemistry and biomedical fields (Elmizadeh et al., 2018). Classical cadmium-telluride quantum dots (CdTe QDs) are known for their strong fluorescence and ease of synthesis compared to other QDs. They are often coated with glutathione or thioglycolic acid (TGA) and used in various applications such as cell imaging, determining biological macromolecules, developing sensors, and drug delivery (Wang et al., 2023). CdTe QDs have recently been functionalized with aptamers to create modern nano biosensors, which are easily synthesized, highly selective, low-cost, stable to temperature and pH changes, and characterized through analytical techniques like UV-visible absorption spectroscopy and fluorescence spectroscopy (Bertrand, 2023). Aptamers have many advantages over traditional antibodies. They are single-stranded nucleic acid sequences that are highly sensitive and stable. They are also cost-effective as they can be chemically synthesized in vitro. Aptamers can bind to a wide range of target ligands, from small molecules and peptides to proteins and cells, making them highly versatile (Weng et al., 2018). The SELEX method (systematic evolution of ligands via an exponential enrichment) is used to isolate high affinity and specificity DNAs or RNAs from a library with random sequences. Sometimes, the original aptamer with long oligonucleotide sequences can bind analogs of its targets through primer regions or additional fragments, which can compromise its selectivity. However, truncating the non-binding region of the original aptamer may result in an aptamer with better performance in terms of affinity, selectivity, and cost (Liu et al., 2014). Aptamers generate signals when interacting with the analyte of interest, enabling accurate, rapid, and simple analytical methods. This makes them widely used as recognition elements, such as in the case of 25-hydroxyvitamin D₃ (Kaushik et al., 2018). Based on a literature review, Lee et al. developed an aptamer-based graphene oxide (GO-SELEX) nano biosensor that demonstrated specific affinity to 25-hydroxyvitamin D₃ with a limit of detection of 1 µM (Lee & Gu, 2017). On the other hand, Qiao et al. designed a PicoGreen-based fluorescence strategy to develop truncated affinity-improved aptamers for the quantitative detection of 25-hydroxyvitamin D₃ with a detection limit of 0.04 mg/mL. It had an improvement over the original long aptamer that could not reach this fluorescent detection (Qiao et al., 2020).

Herein, APTA-nano biosensors based on CdTe-TGA QDs were prepared to develop a simple and low-cost fluorescence technique for detecting 25-hydroxyvitamin D₃. To the best of our knowledge, this is the first report on the fluorescent APTA-nano biosensors based on CdTe QDs for the rapid and ultrasensitive detection of vitamin D₃. The fluorescent intensity of the biosensors increased significantly after the interaction of aptamer with CdTe-TGA QDs. Then, it was linearly quenched by adding the specific concentration of the target molecule (25-hydroxyvitamin D₃). This work aimed to improve the Limit of Detection (LOD) and Limit of Quantitation (LOQ) of 25-hydroxyvitamin D₃ by optimizing the variable parameters, such as fluorophore and analyte concentrations. The results of the typical Stern–Volmer equation showed that the optimized APTA-nano biosensor was highly effective in detecting 25-hydroxyvitamin D₃ due to its high sensitivity, selectivity, stability, reproducibility, low detection limit, and broad linear range. It also provided reliable results when used with human serum samples. The photo-induced electron transfer (PET) mechanism was investigated in detail for turning on and quenching the fluorophore. Additionally, the synthesized APTA-nano biosensors were characterized by UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FESEM), energy dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), and dynamic light scattering (DLS).

2.1. Materials

All the chemical reagents, including tellurium powder (Te, 127.6 g/mol), sodium borohydride (NaBH₄, 37.8 g/mol), cadmium chloride hemipentahydrate (CdCl₂.2 ½ H₂O, 228.3 g/mol), thioglycolic acid (C₂H₄O₂S, 92.1 g/mol), sodium hydroxide (NaOH, 39.9 g/mol), and 25-hydroxyvitamin D₃ monohydrate (C₂₇H₄₆O₃, 418.6 g/mol) were purchased from Sigma-Aldrich International GmbH (Germany), while analytical grade and chemically purified. DNA oligonucleotide, purified by high-performance liquid chromatography, was obtained from Metabion International AG (Germany). The sequence information of thiol-25(OH)D₃-aptamer was as follows:

5′-Thiol-C6-AGCAGCACAGAGGTCATGGGGGGTGTGACTTTGGTGTGCCTATGCGTGCTACGGAA-3′.

A human serum sample was collected from the local pathology lab (Tehran, Iran) and stored at 4°C in a refrigerator. The ultrapure RNase-free water used throughout the experiments was produced by the Milli-Q water purification system (Millipore, Molsheim, France) in the lab.

2.2. Synthesis

2.2.1. Synthesis of CdTe-TGA QDs

CdTe-TGA QDs were synthesized in an aqueous phase, which is essential for their use in biological applications. The synthesis process was based on the previously reported works of Bardajee et al. (Bardajee et al., 2022; Elmizadeh et al., 2018; Elmizadeh et al., 2019), and involved dispersing a mixture of Te powder (0.025 g) and NaBH₄ (0.025 g) in ultrapure water (2.5 mL) at room temperature. The mixture was preserved under a pure argon atmosphere with magnetic stirring for 120 min to achieve a colorless solution of sodium hydrogen telluride. Next, a mixture of thioglycolic acid (TGA, 1.4 mmol) and CdCl₂ (0.11 g) in ultrapure water (80 mL) was prepared while maintaining a pure argon gas environment and magnetic stirring. The pH level was adjusted to 8–9 using NaOH aqueous solution (1 M), after which the first mixture containing the Te precursor was added. The resulting mixture was then refluxed at 100°C for 1 h. Finally, the orange colloidal solution of CdTe-TGA QDs was purified using Ultra-4 Centrifugal filters (5 kDa MWCO) at 10,000 rpm for 15 min and stored at 4°C in a refrigerator.

2.2.2. Synthesis of APTA-nano biosensors

A ligand exchange process was used to create the APTA-nano biosensor, wherein the thiol-labeled aptamer was replaced by a surface-bounded TGA molecule on QDs. The process involved adding a solution of CdTe-TGA QDs dropwise to RNase-free water and stirring it magnetically for 30 min at room temperature. Then, thiol-25(OH)D₃-aptamer was added and the sealed system was incubated for 24 h under slow magnetic stirring. The resulting APTA-nano biosensor was then stored at 4°C in a refrigerator. To investigate the effect of ionic strength on the efficacy of the APTA-nano biosensor, NaCl solution was diluted to different concentrations and used instead of RNase-free water, following the above synthesis route. Table 1 provides details of the materials used to prepare APTA-nano biosensors based on CdTe-TGA QDs.

Table 1

Details of materials used to prepare APTA-nano biosensors based on CdTe-TGA QDs.
APTA-nano biosensors	CdTe-TGA QDs	Thiol-25(OH)D₃- aptamer	RNase-free water	NaCl (990 µL)
Sample A	10 µL	10 µL	1000 µL	---
Sample B	10 µL	20 µL	990 µL	---
Sample C	10 µL	30 µL	980 µL	---
Sample D	10 µL	20 µL	---	100 × 10^− 9 M
Sample E	10 µL	20 µL	---	80 × 10^− 9 M
Sample F	10 µL	20 µL	---	60 × 10^− 9 M
Sample G	10 µL	20 µL	---	40 × 10^− 9 M
Sample H	10 µL	20 µL	---	20 × 10^− 9 M

→ Table 1←

2.3. Screening of APTA-nano biosensors for 25-hydroxyvitamin D₃

A simple fluorescent method was used to screen APTA-nano biosensors for 25-hydroxyvitamin D₃. The sensitivity of this method was determined by recording the fluorescent spectra of APTA-nano biosensors at an excitation wavelength of 325 nm and an emission wavelength range of 420–720 nm. To see the dose-dependent responses to the target by APTA-nano biosensors, a stock solution of 25-hydroxyvitamin D₃ was diluted to different concentrations (0.6 × 10^− 6 M, 0.6 × 10^− 8 M, and 0.6 × 10^− 10 M). To conduct the experiments, 170 µL of the APTA-nano biosensor was stirred slowly and incubated for 24 h at room temperature. Then, 0.5 µL of 25-hydroxyvitamin D₃ was added gradually to it at 5-min intervals and the mixture was shaken gently for 5 min after each addition. The binding affinity of the APTA-nano biosensor to the target molecules was determined by measuring the fluorescence intensity. Each experiment was repeated five times to obtain average values for analysis.

2.4. Validation of the fluorescent method for detection of 25-hydroxyvitamin D₃

Different parameters such as linearity, sensitivity, repeatability, reproducibility, and stability validated the fluorescent method for sensing 25-hydroxyvitamin D₃. By plotting fluorescence intensity against 25-hydroxyvitamin D₃ concentration, the linearity of the response was revealed and evaluated by the correlation coefficient value (R²), the standard deviation (σ), the slope of the regression line (S), and the sample mean (x̄). The sensitivity of the fluorescent method was estimated in terms of the Limit of Detection (LOD) and Limit of Quantitation (LOQ) calculated by the following formulae:

LOD = 3σ / S (1)

LOQ = 10σ / S (2)

Accuracy and repeatability of the optimized APTA-nano biosensor were determined by the relative standard deviation (RSD%) for five successive replicates as follows:

RSD% = 100σ / x̄ (3)

The reproducibility of the optimized APTA-nano biosensor was studied through the fabrication of five sensors independently. They were then employed to measure samples containing the same concentration of 25-hydroxyvitamin D₃. The stability of the optimized biosensor was tested by sensing the same concentration of 25-hydroxyvitamin D₃ every 7 days for 28 days using the stored APTA-nano biosensor.

2.5. Optimization of the optical properties of APTA-nano biosensors

This study focused on testing the efficiency of APTA-nano biosensors in detecting 25-hydroxyvitamin D₃ using a fluorescent method. The experiment involved optimizing the optical properties of the biosensors by analyzing various independent variables, such as the concentration of aptamer and 25-hydroxyvitamin D₃, adding the sequence of CdTe-TGA QDs, NaCl salt, incubation time, temperature, and pH level.

2.6. Selectivity of the APTA-nano biosensor by interference studies

An essential parameter for the well-performing of the APTA-nano biosensor is its selectivity for the specific analyte of 25-hydroxyvitamin D₃. For this purpose, the optical responses of the APTA-nano biosensor to some common interferences, including inorganic compounds like Na⁺, Cl⁻, Mg²⁺, Fe³⁺, NO₃⁻, and organic compounds such as glucose and fructose, were studied. The concentrations of the interfering components were 100 times higher than the analyte.

2.7. Spiking human serum with 25-hydroxyvitamin D₃

In this research, the standard addition method was used for the accurate detection of 25-hydroxyvitamin D₃ in human serum by the APTA-nano biosensor. The serum sample was collected from the local pathology lab (Tehran, Iran) and stored at 4°C in a refrigerator. For preparing the pre-analyzed samples, human serum (3 mL) was titrated with nitric acid (20 µL, 0.01 M) as a serum protein sedimentation agent. The mixture was then vortexed for 1 min and centrifuged at 10,000 rpm for 5 min. The clear protein-free supernatant was diluted 20 times with distilled water, neutralized with NaOH (0.01 M), and stored in the refrigerator. The diluted serum samples were mixed with the APTA-nano biosensor, spiked with different known concentrations of 25-hydroxyvitamin D₃, and studied using the fluorescent method. The experiment data was then statistically analyzed using the following formula to validate the proposed method and determine the recovery percentage (Wang et al., 2018):

Recovery (%) = (Recovered concentration / Injected concentration) × 100 (4)

2.8. Characterization

Fluorescence studies were performed on a SHIMADZU RF-6000 fluorescence spectrophotometer using a quartz cell with a slit width of 3 nm, an excitation wavelength of 325 nm, and an emission wavelength range of 420–720 nm. UV-vis absorption spectra were taken at room temperature from a GBC CINTRA 20 double-beam spectrophotometer using quartz cuvettes. The baseline-corrected spectra were scanned in a wavelength range of 200–600 nm. Fourier-transform infrared (FT-IR) spectra were recorded on a SHIMADZU IRPrestige-21 IR spectrophotometer within the 4000 − 500 cm^-1 range. Samples were transferred to a watch glass, dried in an oven, mixed with an appropriate amount of KBr, and ground evenly with a mortar to obtain a fine powder. Field emission scanning electron microscopy (FESEM) and energy dispersive x-ray spectroscopy (EDX) were checked by a MIRA III VEGA-TESCAN-XMU instrument at the acceleration voltage of 20 kV to examine the surface morphology and the elemental composition of samples, respectively. Transmission electron microscopy (TEM) images were obtained using a PHILIPS CM 120 apparatus to analyze the particle size distribution of samples. To prepare each specimen, a small drop of an aqueous sample solution was placed onto a carbon-coated copper TEM grid and then each grid was dried in air. Dynamic light scattering (DLS) particle size and Zeta potential analyses were carried out at a 90° scattering angle using an SZ-100 HORIBA Scientific instrument.

3.1. Synthesis and characterization of CdTe-TGA QDs nano biosensor

3.1.1. Nano biosensor fabrication

CdTe-TGA QDs were synthesized according to a one-step hydrothermal protocol under argon at 100°C for 1h. The QDs were capped with thioglycolic acid to endow them with high colloidal stability in water. Finally, the CdTe-TGA QDs nano biosensor was obtained after functionalization with thiol-25(OH)D₃-aptamer through ligand exchange. The nano biosensor sensitivity for 25-hydroxyvitamin D₃ was assessed by a simple fluorescent quenching. The preparation procedures of the APTA-nano biosensor and the fluorescent sensing mechanism are schematically illustrated in Fig. 1.

→Figure 1←

3.1.2. UV-visible spectroscopy

The UV-vis spectra of CdTe-TGA QDs before and after conjugation with thiol-25(OH)D₃-aptamer are revealed in Fig. 2a. The unique spectral characteristics of QDs have gained a lot of interest among researchers as their emission can cover a broader light spectrum, from visible to infrared. The maximum absorbance of the samples is observed within the wavelength range of 300–600 nm, as reported in the literature. The UV-vis spectrum of the APTA-nano biosensor reveals a slight red shift in the band-edge absorption at 320 nm and 475 nm due to the successful replacement of TGA ligands from the surface of CdTe QDs with thiol-25(OH)D₃-aptamer (Elmizadeh et al., 2023; Sharma et al., 2024).

→Figure 2←

3.1.3. FT-IR Chemical analysis

The chemical structures and functional groups of CdTe-TGA QDs before and after conjugation with thiol-25(OH)D₃-aptamer, using FT-IR spectroscopy, are presented in Fig. 2b. The changes in molecular structures, determined by the bonds between elements, are characterized by infrared transmittance intensity at certain wavenumbers. The apparent elongation of the APTA-nano biosensor spectrum and the slight shift of stretching vibrations in some bands ascertain the interaction between CdTe-TGA QDs and thiol-25(OH)D₃-aptamer (Wang et al., 2023). As shown in both spectra, the absorption bands around 642 cm^− 1 and 850 cm^− 1 are assigned to the bond formation between Cd and Te, confirming the formation of CdTe QDs and the C–H bending vibration, respectively. The peak at 1142 cm^− 1 corresponds with the C–O stretching motion. The peaks at 1377 cm^− 1 and 1587 cm^− 1 arise from the presence of C = O stretching mode (Elmizadeh & Bardajee, 2023). The distinct absorption bands at around 2585 cm^− 1 and 2665 cm^− 1 are related to the S–H vibrations of sulfur in TGA molecules on CdTe surfaces and thiol-25(OH)D₃-aptamer (Ayodhya & Veerabhadram, 2018). The peaks at 2900 cm^− 1 to 3000 cm^− 1 correspond to the CH₂ asymmetric stretching vibrations, which were sharpened in the spectrum of the APTA-nano biosensor, indicating the occurrence of ligand exchange (Elmizadeh et al., 2023). A broad absorption band around 3450 cm^− 1 corresponds with the stretching vibrations of the hydroxyl groups in CdTe-TGA QDs, which were overlapped with the N-H vibrations of the bases in aptamer, indicating the amide formation between CdTe QDs and thiol-25(OH)D₃-aptamer. So, the peak at 3700 cm^− 1 is related to the N-H and O-H stretching vibrations in the APTA-nano biosensor (Elmizadeh et al., 2020; Hedayati et al., 2016). Moreover, the peaks around 1650 cm^− 1 to 1907 cm^− 1 and 900 cm^− 1 to 1090 cm^− 1 in the spectrum of the APTA-nano biosensor are associated with the symmetric stretching vibrations of the N-H and the PO₂⁻ groups on the sugar-phosphate backbone of aptamer, respectively (Qiao et al., 2020).

3.1.4. EDX spectroscopy

The elemental composition of the APTA-nano biosensor, evaluated through the EDX detector, is shown in Fig. 2c. The elemental analysis reveals the peaks for seven different elements, namely C, N, O, P, S, Cd, and Te with the proper energy level. The presence of the P element is associated with the sugar-phosphate backbone, which forms the structural framework of the aptamer (Ayodhya & Veerabhadram, 2018). The inset of the elemental graphs shows the composition and weight percentage of the elements.

3.1.5. Morphological studies (FESEM & TEM)

The surface morphologies of CdTe-TGA QDs and the APTA-nano biosensor, examined through the FESEM technique, are presented in Fig. 3(a, b). The FESEM image of CdTe-TGA QDs in Fig. 3a reveals spherical nanoparticles in a mass structure. On the other hand, the FESEM image of the APTA-nano biosensor in Fig. 3b displays a stacked lamellar structure with numerous spherical nanoparticles. Although there are differences between CdTe-TGA QDs before and after their bio-conjugation with thiol-25(OH)D₃-aptamer, their morphologies do not change significantly in TEM images. As seen, both CdTe-TGA QDs (Fig. 3c) and the APTA-nano biosensor (Fig. 3d) have a globosity morphology and uniform distribution.

→Figure 3←

3.1.6. Dynamic light scattering (DLS)

The particle size and Zeta potential of CdTe-TGA QDs before and after conjugation with thiol-25(OH)D₃-aptamer, characterized by dynamic light scattering (DLS), are shown in Fig. 4(a-d). The DLS analyzer is a user-friendly system used for characterizing particle size distribution and particle Zeta potential, which relates to the magnitude of the electrical charge on the particle surface dispersed in water. The particle sizes of CdTe-TGA QDs and the APTA-nano biosensor are 2.7 nm and 2.8 nm, respectively (Fig. 4a, b). The slight increase is due to the larger size of the aptamer molecule compared to TGA. Additionally, the Zeta potential of CdTe-TGA QDs and the APTA-nano biosensor is − 19.6 mV and − 33.4 mV, respectively, which indicates the proper attachment of thiol-25(OH)D₃-aptamer to the surface of CdTe QDs (Fig. 4c, d). The negative charge increase for the APTA-nano biosensor is mainly due to the negatively charged oligonucleotide chains on the surface of CdTe QDs (Elmizadeh et al., 2023; Paimard et al., 2015).

→Figure 4←

3.2. Analytical applications

It is worth noting that CdTe-TGA QDs alone cannot be considered for biological target sensing such as 25-hydroxyvitamin D₃ and require surface modification with aptamers, leading to the improvement of their optical properties (Muñoz et al., 2021). The design and fabrication of APTA-nano biosensors in detecting 25-hydroxyvitamin D₃ were studied using a fluorescent assay. Optimization of various independent variables improved the efficiency of APTA-nano biosensors. These variables included the concentration of thiol-25(OH)D₃-aptamer and 25-hydroxyvitamin D₃, the addition of the sequence of CdTe-TGA QDs, NaCl salt, incubation time, temperature, and pH level. This study examined the effects of these variables on the fluorescence intensity in detail.

3.2.1. Concentration of thiol-25(OH)D₃-aptamer

The ratio of CdTe-TGA QDs to thiol-25(OH)D₃-aptamer had a significant impact on the sensitivity and selectivity of APTA-nano biosensors. As described in Table 1, three-volume ratios of QDs to aptamer (10:10, 10:20, and 10:30 (µL)) were used to fabricate APTA-nano biosensors A, B, and C to study the effect of thiol-25(OH)D₃-aptamer concentration in the absence of target (25-hydroxyvitamin D₃). The interaction between thiol-25(OH)D₃-aptamer and CdTe-TGA QDs through the ligand exchange process resulted in a remarkable increase in fluorescence intensity (Fig. 5a-c). The turning-on phenomenon could be due to the forming of strong interactions between the sulfur electron donor atoms from aptamer and Cd²⁺ ions from QDs. Additionally, the weak bonding of heavy sulfur atoms improved the spin-orbit coupling and intersystem crossing transitions between two electron spin multiplicities (Elmizadeh et al., 2023). Before the ligand exchange, the emission peak of CdTe-TGA QDs was centered at 525 nm, while following that, it shifted to the higher wavelengths. The redshift in the emission spectra is consistent with the results reported in the literature (Lin et al., 2008). The optimized APTA-nano biosensor for further detection of 25-hydroxyvitamin D₃ was selected based on the high fluorescence intensity recorded in 24 h. The APTA-nano biosensor B revealed a high, stable, sharp, and symmetrical peak, indicating the suppression of the non-radiative localized electron-hole pairs and increment of the electron transfer process. It suggested that 20 µL of the electron-donating thiol-25(OH)D₃-aptamer was sufficient for binding to 10 µL of CdTe-TGA QDs through the ligand exchange, reforming the electronic conjugation on quantum dots, and enhancing fluorescence emission. From this time on, all experiments were conducted following a 24-hour interaction period between the aptamer and quantum dots for utmost accuracy and precision.

→Figure 5←

3.2.2. Concentration of 25-hydroxyvitamin D₃

The three APTA-nano biosensors, A, B, and C, were used to detect different concentrations of 25-hydroxyvitamin D₃ solution (0.6 × 10^− 6 M, 0.6 × 10^− 8 M, and 0.6 × 10^–10 M) at room temperature and normal pH of 7.4. Each biosensor was tested five times for each concentration of 25-hydroxyvitamin D₃ solution to ensure the consistency of results. As shown in Fig. 6a-i, the fluorescence intensities of APTA-nano biosensors were remarkably decreased in the presence of 25-hydroxyvitamin D₃. It was due to the specific affinity of the aptamer to the target analyte, which led to the creation of a bio-conjugate of 25(OH)D₃-aptamer-QDs and the subsequent turning-off of its fluorescence intensity (Salehian et al., 2019). Furthermore, the small redshift in the emission spectra was due to the electronic coupling between the aggregated 25-hydroxyvitamin D₃ molecules on the surface of quantum dots. The dose dependency of each APTA-nano biosensor to its target was studied using fluorescence quenching. Fluorescence quenching is an important phenomenon, achieved through different mechanisms, including dynamic quenching, static quenching, or a combination of both. Dynamic quenching is due to collisional encounters between the fluorophore and quencher, while static quenching results from a non-fluorescent complex formed between the two in a ground state. Both static and dynamic quenching require some molecular contact between the fluorophore and the quencher and can be distinguished by their varying dependence on temperature and concentration (Callan et al., 2008). As can be seen, the quenching efficiency of the APTA-nano biosensors A and C is lower than that of kind B. The reason can be attributed to a decrease or increase in the number of oligonucleotide chains on the surface of CdTe QDs, resulting in the ineffective interaction between 25(OH)D₃-aptamer and molecules of 25-hydroxyvitamin D₃ (Bardajee et al., 2021).

→Figure 6←

3.2.2.1. Calibration curves and fluorescence quenching mechanism

When 25(OH)D₃-aptamer interacted with 25-hydroxyvitamin D₃, it caused a structural change in the biosensor, forming a non-conductive complex based on the molecular orbital theory (Qiao et al., 2020). It led to a fluorescence quenching described by a fast and fully reversible photo-induced electron transfer mechanism (PET). According to the PET mechanism, when a molecule of fluorophore (APTA-nano biosensor) is excited by a photon, the electron is excited from the valence band to the conduction band. Subsequently, it returns to the valence band, resulting in the production of the fluorescence signal. The presence of a quencher (25-hydroxyvitamin D₃) disrupts this electron transfer process. The excited electrons move from the conduction band of the complex, containing suitable electron donor groups of hydroxyl, phosphate, and amines, to the ground state of 25-hydroxyvitamin D₃ acceptor with no fluorescence, leading to the fluorescence quenching (Wadhwa et al., 2020). Fluorescence quenching is a technique used to determine the concentration of a quencher of interest with the help of the Stern-Volmer equation:

F ₀ / F = K_SV [Q] + 1 (5)

Where F₀ and F are fluorescence emission signals at band maxima in the absence and presence of the quencher, respectively. K_SV is the Stern-Volmer quenching constant and Q is the quencher concentration. When the F₀ / F is plotted against the various concentrations of the quencher, a linear curve is obtained. The linear relationship suggests that only one type of quencher is affecting the fluorophore, meaning that fluorescence quenching can be used to determine the quencher. In this case, the slope of the line is proportional to K_SV, provided the y-axis intercept is at level 1 (Elmizadeh et al., 2020). Figure 7a-c shows the Stern-Volmer plots of each APTA-nano biosensor in different concentrations of 25-hydroxyvitamin D₃. According to the plots, it is clear that the APTA-nano biosensor B with the Stern-Volmer equation of F₀ / F = 0.557Q + 0.890 (R² = 0.987) has a high affinity to 25-hydroxyvitamin D₃ (0.6 × 10^− 8 M) compared with others (Fig. 7b). Therefore, this biosensor is known as the optimized APTA-nano biosensor. Table 2 provides details of the Stern-Volmer plots of each APTA-nano biosensor in detecting 25-Hydroxyvitamin D₃.

Table 2

Details of the Stern-Volmer plots of each APTA-nano biosensor in detecting 25-hydroxyvitamin D₃.
25-hydroxyvitamin D₃	APTA-nano biosensor	Stern-Volmer equation	K_SV	R²
0.6 × 10^− 6 M	Sample A	F0/F = 1.566Q + 0.758	1.566	0.979
0.6 × 10^− 6 M	Sample B	F0/F = 1.162Q + 0.808	1.162	0.978
0.6 × 10^− 6 M	Sample C	F0/F = 0.748Q + 0.667	0.748	0.932
0.6 × 10^− 8 M	Sample A	F0/F = 0.630Q + 0.875	0.630	0.982
0.6 × 10^− 8 M	Sample B	F0/F = 0.557Q + 0.890	0.557	0.987
0.6 × 10^− 8 M	Sample C	F0/F = 0.652Q + 0.733	0.652	0.952
0.6 × 10^− 10 M	Sample A	F0/F = 0.833Q + 0.758	0.833	0.964
0.6 × 10^− 10 M	Sample B	F0/F = 0.669Q + 0.726	0.669	0.957
0.6 × 10^− 10 M	Sample C	F0/F = 0.620Q + 0.737	0.620	0.949

→Figure 7←

→ Table 2←

Figure 7d illustrates the Stern-Volmer plot of the optimized APTA-nano biosensor. It was used to detect different concentrations of 25-hydroxyvitamin D₃, ranging from 0.01–0.10 µM. The high K_SV value indicates that the measurement was conducted with greater sensitivity. The equation is F₀ / F = 26.255Q + 0.998 (R² = 0.984), with a limit of detection (LOD) of 1.35 × 10^− 8 M using the 3σ rule, and a limit of quantification (LOQ) of 4.50 × 10^− 8 M using the 10σ rule. Several parameters, such as repeatability, reproducibility, and stability, were studied to validate the proposed fluorescence method for detecting 25-hydroxyvitamin D₃. Five consecutive replicates of the APTA-nano biosensor were employed to detect a 25-hydroxyvitamin D₃ sample. The relative standard deviation (RSD) was calculated at 1.75%, indicating high precision and repeatability. Five APTA-nano biosensors were applied to detect the same concentration of 25-hydroxyvitamin D₃. The results were almost identical, with an RSD value of 1.15%, indicating satisfactory reproducibility. The stability of the APTA-nano biosensor was assessed by monitoring fluorescence quenching under the same concentration of 25-hydroxyvitamin D₃ at a regular interval of 7 days for over 28 days using the stored APTA-nano biosensor. The response did not decay and retained 98% of the first recorded response. Table 3 provides details of the Stern-Volmer plot of the optimized APTA-nano biosensor in detecting 25-hydroxyvitamin D₃, including K_SV, R², LOD, LOQ, and RSD. Table 4 presents the findings of studies that employed fluorescent sensors for detecting 25-hydroxyvitamin D₃. The proposed APTA-nano biosensor based on CdTe-TGA QDs, fabricated using simple and low-cost procedures, has a low detection limit compared to other previously reported sensing systems.

Table 3

Details of the Stern-Volmer plot of the optimized APTA-nano biosensor in detecting 25-hydroxyvitamin D₃.
Stern-Volmer equation	K_SV	R²	LOD	LOQ	RSD
F0/F = 26.255Q + 0.998	26.255	0.984	1.35 × 10^− 8 M	4.50 × 10^− 8 M	1.75%

Table 4

Comparison of the proposed APTA-nano biosensor with other reported fluorescent sensors for detecting 25-hydroxyvitamin D₃.
Fluorescent sensor	LOD	Reference
Truncated VDBA14/PG	0.04 mg/mL	(Qiao et al., 2020)
Graphene QDs-Au	0.28 ng/mL	(Gupta et al., 2021)
Carboxylated Graphene oxide	0.075 µg/mL	(Prante et al., 2019)
VDBA14 aptamer	5.4 nM	(Qin et al., 2022)
APTA-nano biosensor based on CdTe-TGA QDs	1.35 × 10^− 8 M	This work

→ Table 3←

→ Table 4←

3.2.3. Addition of the sequence of CdTe-TGA QDs

This study investigated the effect of adding the sequence of CdTe-TGA QDs on fluorescence quenching. Firstly, 20 µL of thiol-25(OH)D₃-aptamer and 3 µL of 25-hydroxyvitamin D₃ (0.6 × 10^− 8 M) were added to 850 µL of RNase-free water and magnetically stirred at room temperature for 20 min. Then, CdTe-TGA QDs were gradually added every 30 min, starting with 2 µL and fluorescence intensities were measured, as shown in Fig. 8a. A blank solution (without 25-hydroxyvitamin D₃) was also prepared using the same process, as shown in Fig. 8b. The findings revealed that increasing the amount of CdTe-TGA QDs up to 8 µL resulted in fluorescence quenching in the presence of 25-hydroxyvitamin D₃ compared to the blank solution (Fig. 8c). However, higher volumes of CdTe-TGA QDs decreased fluorescence intensities due to the self-quenching or accumulation of CdTe-TGA QDs (Elmizadeh et al., 2023; Elmizadeh et al., 2018).

→Figure 8←

3.2.4. Effect of ionic strength

To investigate the effect of ionic strength on the fluorescence intensity in detecting 25-hydroxyvitamin D₃, different concentrations of NaCl were employed in the fabrication of the optimized APTA-nano biosensor, as outlined in Table 1. As observed in Fig. 9a-f, NaCl strongly inhibited any changes in fluorescence intensities when 25-hydroxyvitamin D₃ (0.6 × 10^− 8 M) was present at 5-minute intervals (0.5–1.5 µL), unlike the RNase-free water used in APTA-nano biosensor B. The results indicated that the impact of ionic strength on fluorescence quenching was the same across different salty solutions. The study showed that Na⁺ and Cl⁻ created an unstable suspension, resulting in negligible changes in fluorescence intensity but a significant decrease in the sensitivity of detecting 25-hydroxyvitamin D₃ (Hashemi & Fatemi, 2019; Renard et al., 1998). The findings further revealed that fluorescence intensity decreased with higher salt concentrations. At higher concentrations of salt, ions could absorb the energy of the excited state, causing a reduction in the fluorescence intensity (Rahman et al., 2019).

→Figure 9←

.3.2.5. Incubation time

It was found that the optimal incubation time for fluorescence sensing was 24 h. It allowed a dynamic equilibrium to be established between CdTe-TGA QDs and thiol-25(OH)D₃-aptamer, enabling complete intermolecular interaction. The fluorescence intensity reached a maximum and remained steady after one day, as shown in Fig. 10a. Additionally, an experiment was conducted to detect 25-hydroxyvitamin D₃ without the 24 h incubation time, as seen in Fig. 10b. The results revealed that the incubation time had a significant impact on increasing fluorescence intensity, as well as on quick fluorescence quenching in the presence of 25-hydroxyvitamin D₃.

3.2.6. Temperature

In the context of sensing, when a quencher molecule interacts with a fluorophore, quenching occurs either through dynamic or static collisions. Dynamic quenching is influenced by temperature, with higher temperatures leading to higher quenching constant values. Conversely, static quenching remains unaffected by temperature changes. Herein, fluorescence quenching of the optimized APTA-nano biosensor at two different temperatures was studied. The APTA-nano biosensor was placed in a mixture of ice and water and an oil bath at 5℃ and 60 ℃, respectively, and then utilized to detect 25-hydroxyvitamin D₃ (0.5–1.5 µL) within 60 min. The findings of the static fluorescence quenching indicated that the impact of temperature on quenching behavior was the same. However, at 5℃, the fluorescence intensity slightly increased compared to 60 ℃ (Fig. 10c), while at 60 ℃, more fluorescence quenching was observed (Fig. 10d). It was because increasing the temperature caused weakly bound complexes to dissociate, resulting in more forceful collisions due to faster molecule movement and reduced fluorescent intensity (Callan et al., 2008).

3.2.7. pH

This study investigated the impact of pH on the fluorescence quenching of the optimized APTA-nano biosensor in the presence of 25-hydroxyvitamin D₃ in varying quantities (0.5–1.5 µL) within 60 min. The results showed that when the pH was 5 (as shown in Fig. 10e), the instability of QDs and the loss of structural conformation significantly reduced the fluorescence emission. Additionally, the acidic conditions protonated the amine groups of the aptamer, resulting in an inefficient interaction with 25-hydroxyvitamin D₃. Conversely, when the pH was 9 (as shown in Fig. 10f), QDs were stable and effective in detecting target molecules. However, the partial ionization of the hydroxyl groups of 25-hydroxyvitamin D₃ in alkaline pH reduced sensitivity compared to pH 7.4 (Fig. 10a) (Sae-Kang & Suphantharika, 2006).

→Figure 10←

3.3. Influence of common interferences on APTA-nano biosensor performance

The selectivity of the optimized APTA-nano biosensor in detecting 25-hydroxyvitamin D₃ validated its practical applications. For this purpose, various inorganic compounds like Na⁺, Cl⁻, Mg²⁺, Fe³⁺, NO₃⁻, and organic compounds such as glucose and fructose, were selected as interferences. The concentrations of these interfering agents (0.6 × 10^− 6 M) were 100 times higher than the analyte of 25-hydroxyvitamin D₃. The graph in Fig. 11 displays the results obtained from different molecules and their corresponding value of fluorescence emission. The findings showed significant fluorescence quenching only in the presence of 25-hydroxyvitamin D₃, whereas interfering agents had no noticeable effect on the quenching performance of the APTA-nano biosensor. Based on these experimental results, the excellent selectivity of APTA-nano biosensor for 25-hydroxyvitamin D₃ was confirmed.

→Figure 11←

3.4. Human serum sample analysis

Herein, the effectiveness of the APTA-nano biosensor in detecting 25-hydroxyvitamin D₃ within human serum samples was studied using the spike method. After deproteinization, dilution, neutralization, and mixing with the APTA-nano biosensor, the diluted serum samples were spiked with five different concentrations of 25-hydroxyvitamin D₃ to record their fluorescence quenching. The recoveries of 25-hydroxyvitamin D₃ in human serum were calculated from Eq. 4 and tabulated in Table 5. The calculated recoveries and RSD values were 96.00-99.77% and 1.06–2.45% (n = 5), respectively. These excellent recoveries confirm that the APTA-nano biosensor is efficient and highly specific in sensing the analyte of interest in human serum samples.

Table 5

Determination of 25-hydroxyvitamin D₃ in human serum samples according to the spike method (n = 5).
Spiked (µM) 25-hydroxyvitamin D₃	Found (µM) 25-hydroxyvitamin D₃	Recovery (%)	RSD (%)
0.0050	0.0048	96.00	2.45
0.0150	0.0149	99.33	1.52
0.0250	0.0247	98.80	1.75
0.0350	0.0348	99.43	1.22
0.0450	0.0449	99.77	1.06

→ Table 5←

This study investigated a sensitive fluorescent APTA-nano biosensor based on CdTe-TGA QDs for detecting 25-hydroxyvitamin D₃. The approach used a fluorescence process, where 25-hydroxyvitamin D₃ could effectively quench the fluorescence intensity of the APTA-nano biosensor by forming a non-fluorescent complex in a ground state (static quenching). The APTA-nano biosensor demonstrated excellent precision, selectivity, and reproducibility for 25-hydroxyvitamin D₃ determination, a linear response in the concentration range of 0.01–0.10 µM, LOD of 1.35 × 10^− 8 M, LOQ of 4.50 × 10^− 8 M, and RSD of 1.75%. The optimized conditions, such as pH, temperature, and incubation time, adding the sequence of CdTe-TGA QDs, and the effect of ionic strength were also studied. The synthesized APTA-nano biosensor revealed excellent specificity against interfering components and stability over 28 days. This simple, low-cost, and non-toxic APTA-nano biosensor detected 25-hydroxyvitamin D₃ within human serum with high selectivity, suitable repeatability and reproducibility, and reasonable response. The APTA-nano biosensor's structural and morphological characteristics were analyzed using various analytical techniques such as UV-visible spectroscopy, FT-IR, FESEM, EDX, TEM, and DLS. The proposed fluorescent nano biosensor has the potential to detect trace amounts of metabolic analytes and drug residues in biological fluids and environmental samples.

Acknowledgment - Payame Noor University and INSF are gratefully acknowledged for their support of this work.

Authors' contributions - We confirm that all authors named in the manuscript have read and approved it. Dr. Ghasem Rezanejade Bardajeeand Dr. Cedric Vancaeyzeele oversaw all experiments and research activities, including experiment design, data interpretation, and manuscript preparation. Farhad Tahriri conducted experiments and data interpretation and prepared figures, tables, and graphical designs.

Funding - Payame Noor University provided financial support.

Availability of data - The data that support the findings of this study are available on request from the corresponding author.

Ethics approval and consent to participate - Ethical approval does not apply to this article.

Consent for publication - All authors approve the manuscript and give their consent for submission and publication.

Competing interests - We confirm that all authors named in the manuscript have read and approved it. In addition, no one meets the criteria for authorship. Throughout the Editorial process (including communication with the Editorial Manager and directly with the office), the Corresponding Author is the sole point of contact. Ghasem Rezanejade Bardajee is responsible for communicating with the other authors about progress, revisions, and final approvals of proofs. The Corresponding Author's email address is confirmed to be current and accurate.

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Characterization and Application of the Fluorescent APTA-nano Biosensors Based on CdTe-TGA QDs for Determination of 25-hydroxyvitamin D3

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1. Introduction

2. Experimental