Synthesis of terbium and silica modified graphene quantum dots for targeted bioimaging of breast cancer cells using folate receptors

Abstract

The overexpression of folate receptors (FR) on the surface Michigan Cancer Foundation 7 (MCF-7) cancer cells offers a strategic target for enhancing cancer imaging and diagnostic accuracy due to their high affinity toward folic acid (FA). Graphene quantum dots (GQDs) were synthesized via thermal pyrolysis and chelated with terbium (Tb) ions. These Tb-GQDs were then coated with a silica layer and functionalized with FA, resulting in a fluorescent nanoprobe specifically designed for targeted bioimaging of FR-positive cells. The synthesized Tb-GQDs-SiO₂-APTES-NH₂-FA nanoprobe demonstrated intense fluorescence emission at 425 nm upon excitation at 310 nm. Characterization of the nanoprobe revealed a high quantum yield (QY) of 29%, along with excellent photostability and favourable optical properties, establishing its efficacy as a tool for specifically targeting FR on MCF-7 cancer cells. Qualitative analysis via fluorescence microscopy confirmed the successful and specific uptake of Tb-GQDs-SiO₂-APTES-NH₂-FA by MCF-7 cells, which was evaluated across varying incubation times and concentrations. Cytotoxicity assays further confirmed the biocompatibility of the nanoprobe at concentrations up to 1000 µg/mL, with cell viability remaining above 90%. These findings collectively suggest that Tb-GQDs-SiO₂-APTES-NH₂-FA holds significant potential for future in vivo cancer cell imaging applications. In conclusion, the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoprobe represents a promising and biocompatible platform for the targeted bioimaging of cancer cells, with strong implications for improving diagnostic precision in breast cancer.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33188-z.

Introduction

Breast cancer is one of the leading causes of mortality worldwide, with a substantial impact on families, societies, and economies. According to the World Health Organization (WHO), breast cancer cases are expected to increase significantly by 2030¹. In 2020 alone, more than 2.3 million new cases were diagnosed, leading to around 685,000 deaths. By 2040, these figures are estimated to rise to over 3 million new cases and 1 million deaths yearly, largely as a global population growth and aging². Given the rising incidence of breast cancer and concerns among oncologists, it is vital to ensure early and accurate tumor detection and identification of tumors to enhance patient outcomes. Various breast cancer screening methods, including X-ray mammography³ and low-dose computed tomography (CT)⁴, have been implemented for early detection. While these techniques are relatively fast and cost-effective, they suffer from limited resolution and pose risks due to ionizing radiation exposure⁵. Other diagnostic approaches such as magnetic resonance imaging (MRI), ultrasound, and immunohistochemistry also face significant challenges, including restricted resolution, the need for complex sample preparation, non-targeted diagnostics, and limited capability for monitoring therapeutic responses. Additionally, these methods often fail to provide detailed cellular-scale information and may introduce potential side effects^6,7.

Fluorescence detection methods offer several significant advantages over traditional diagnostic techniques, including straightforward preparation, reliable outcomes, real-time detection, and high sensitivity⁸. Unlike many other approaches, fluorescence detection is non-destructive, preserving the integrity of target molecules and requiring minimal sample preparation^6,9–11. For the detection of disease in quantities, they are therefore highly helpful⁹. Folic acid (FA), a vital vitamin for one-carbon metabolism and nucleotide synthesis, plays a crucial role in cellular function. In humans, folate receptors (FRs (FRα, FRβ, and FRγ)) can bind with high specificity, enhancing cellular uptake in cancer cells than in normal cells¹². Although FRs are typically expressed at low levels in normal cells, they are significantly upregulated in numerous cancers, including breast cancer. This upregulation occurs to meet the heightened folate demand of rapidly dividing cancer cells, especially under conditions of low folate availability¹³. The interaction between FA molecules and FRs allows for the precise identification of breast cancer cells, such as Michigan Cancer Foundation 7 (MCF-7), making FA a valuable tool in cancer diagnostics and targeted therapy^12,13.

Nanotechnology offers a promising solution to these challenges, with significant advantages in biomedicine¹⁴. Nanoparticles, with their modifiable surfaces, high surface area, and reduced toxicity, can be precisely engineered to target specific analytes^15,16. Compared to conventional fluorescent dyes and proteins, quantum dots (QDs) have many advantages: they have brightness levels that are orders of magnitude higher, photostability that is thousands of times better than conventional options, and broad excitation spectra for luminescence along with narrow and symmetrical emission spectra with significant Stokes shifts^17–20. QDs are characterized by wide absorption spectra, allowing them to efficiently transfer energy to lanthanide ions and thereby enhance their characteristically weak luminescence. Owing to their high quantum yields (QYs) and high molar absorption coefficients, QDs can be excited without the need for strong photon beams, which reduces the possibility of photodamage to biological samples during imaging²¹. Furthermore, as their spectral characteristics are tunable based on their core size and composition, QDs exhibit an exceptionally broad fluorescence range, spanning from the near-ultraviolet (NUV) to the near-infrared (NIR) regions. In particular, NIR-emitting QDs hold significant potential for both in vitro and in vivo deep tissue bioimaging applications²⁰. The combination of QDs and lanthanide ions synergistically generates a strong luminescence effect via energy transfer, making this hybrid system a highly useful tools for bioimaging and sensing²².

Lanthanide ions, particularly europium and Tb, exhibit distinctive photoluminescent properties^23–25. Tb, a rare element with atomic number 65, exhibits remarkable optical and magnetic characteristics that make it invaluable for various technological applications such as fluorescent labelling, drug delivery, bioimaging, and cancer therapy. Tb³⁺ ions emit light through a special process known as f-f transition luminescence; however, when sensitized via an antennae effect, they can also exhibit upconversion luminescence, where high-energy photons are released upon the absorption of low-energy photons. This capability improves imaging quality and helps to avoid the issue of photobleaching associated with traditional fluorophores. Furthermore, Tb-based nanoparticles (TbNPs) demonstrate pronounced emission peaks within the visible spectra, thereby reducing spectral interference from biological autofluorescence and improving the signal-to-background ratio in imaging applications. This pronounced emission facilitates more accurate detection and imaging of target molecules or structures within biological specimens. Tb ions possess a relatively extended luminescence lifetime, typically spanning the millisecond range. This prolonged lifetime is beneficial in time-resolved imaging methodologies, such as time-gated luminescence imaging, as it aids in diminishing short-lived background noise and enhancing image contrast and resolution. Moreover, Tb-based complexes and nanoparticles exhibit exceptional photostability, meaning they can endure extended exposure to excitation light without considerable degradation or photobleaching. This characteristic guarantees consistent and reliable imaging outcomes across prolonged experimental sessions, rendering TbNPs particularly well-suited for long-term biological investigations. TbNPs can be tailored to be biocompatible and stabile within physiological conditions, thereby ensuring minimal cytotoxic effects while preserving imaging efficacy in biological systems. Additionally, TbNPs can be functionalized or combined with other imaging agents or modalities, including QDs or organic dyes, to facilitate multiplexed imaging of multiple targets concurrently²⁶. A key challenge, however, is that lanthanide ions have inherent weak absorption bands that make direct excitation difficult. Despite this, they have gained attention due to their high internal quantum efficiencies and robust luminescence. Consequently, they have found valuable applications in various photoluminescent technologies²⁷. To overcome the excitation challenges, these ions are often formed chelates with ligands that act as “antennas”, containing specific functional groups, such as carboxylic acids, to exhibit enhanced luminescence. In this antenna process, the ligand absorbs energy and transfers it to the lanthanide ion, which then emits light. The excitation wavelength is therefore attributed to the ligand, while the emission wavelength is characteristic of the lanthanide ion²⁸. The incorporation of Ln³⁺ ions into host matrices aids in the development of fluorescent probes, leveraging their 4f–4f or 5d–4f transition bands. Tb³⁺ ions, in particular, act as effective activators when excited within a wavelength range of 300 to 380 nm, facilitating multi-emission detection for biomedical applications. Their emission spectrum is dominated by the ⁵D₄–⁷F_J (green) and ⁵D₃–⁷F_J (blue) transitions²⁹. Silane coupling agents, which are biocompatible and often non-toxic material, are extensively utilized in the encapsulation of various fluorescent components and the fabrication of multifunctional fluorescence nanoparticles^21,30. For example, when applied to A375 cells, the luminous characteristics of citrate-capped Tb-functionalized carbonated apatite nanomaterials allowed for the detection of their cytoplasmic uptake after approximately 12 h of treatment via fluorescence confocal microscopy and flow cytometry⁹.

Herein, the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles were produced as a highly fluorescent probe for bioimaging of MCF-7 cells, demonstrating excellent biocompatibility and high fluorescence intensity (Fig. 1). Although various methods and materials have been used to study imaging of various cells, most of them have limitations such as leaching of materials in real environment, use of toxic heavy metal in the material composition and high relative toxicity, low quantum yield (QY), etc.^31–33. Meanwhile, Tb-GQDs-SiO₂-APTES-NH₂-FA uses Tb-GQDs as the base material, which has excellent biocompatibility and low toxicity compared to quantum dots containing heavy metals. By doping them with Tb³⁺ ions, we have achieved better fluorescence properties and overcome the toxicity limitation. Also, Tb-GQDs-SiO₂-APTES-NH₂-FA is a multifunctional system in which the silica shell (SiO₂) enhances stability and biocompatibility, while the amine groups on the surface (-NH₂) allow for easy attachment to FA as well as the possibility of attaching therapeutic agents for future therapeutic applications. The synthesis started with the preparation of Tb-GQDs nanoparticles, which were subsequently coated with a silane layer. Finally, FA molecules were conjugated to the Tb-GQDs-SiO₂-APTES-NH₂ under the N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) procedure, resulting in the creation of fluorescent Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles.

Fig. 1.

(a) Synthesis of Tb-GQDs-SiO₂-APTES-NH₂-FA, (b) schematic representation of cellular uptake, and (c) fluorescence microscopy of the uptake of Tb-GQDs-SiO₂-APTES-NH₂-FA to the MCF-7 cells.

Experimental details

Reagents and materials

Tb (III) salt (TbCl₃.6H₂O, 99.9%) was bought from Thermo Scientific Chemicals (USA). The citric acid (CA, C₆H₈O₇, 98.0%), tetraethyl orthosilicate (TEOS, SiC₈H₂₀O₄, 98.1%), aminopropyltriethoxysilane (APTES, C₉H₂₃NO₃Si, 98.2%), NHS (C₄H₅NO₃, 99.0%), EDC (C₈H₁₇N₃, 99.3%), sodium hydroxide (NaOH, 99.0%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 97.0%), sodium dihydrogen phosphate (NaH₂PO₄, 99.0%), sodium chloride (NaCl, 99.0%), disodium hydrogen phosphate (Na₂HPO₄, 99.2%), and potassium chloride (KCl, 98.0%) were obtained from Merck (Germany). In addition, FA was sourced from Mehr Darou Company (98.0%, Iran). Furthermore, trypsin-EDTA (25%) and Roswell Park Memorial Institute 1640 growth medium (RPMI) were purchased from Gibco BRL Life Technologies (USA). Penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Biowest Company (France). Dimethyl sulfoxide (DMSO, C₂H₆OS, 99.0%) was obtained from DAEJUNG Company (98.0%, South Korea). MCF-7 cancer cell was provided by the National Cell Bank of Iran (NCBI) (Tehran, Iran). All standards were produced using pure deionized (DI) water.

Apparatus

Tb-GQDs-SiO₂-APTES-NH₂ and their FA-conjugated counterpart were characterized using advanced instruments. Fourier Transform Infrared (FTIR) spectra of the materials were acquired employing a Bruker FTIR spectrometer model Tensor 27 (Massachusetts, USA) in the wavenumber range of 4000 to 400 cm^{- 1}. The fluorescence properties and absorbance measurements of the produced nanomaterials were subjected to a JASCO FP 750 fluorimeter (Tokyo, Japan) and Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer, respectively. To visualize the fluorescence bioimages, an Olympus Corporation microscope (BX64 model, Tokyo, Japan) was utilized. The surface charge and size of the produced nanomaterials were measured by the Malvern particle size analyzer (Malvern, UK). Atomic force microscopy (AFM) of the materials was recorded using Nanosurf mobile S (AFM; Nanosurf mobile S instrument, Switzerland). Moreover, field emission scanning electron microscopy (FESEM) of the particles was recorded at 15 kV using Tescan, MIRA3 FEG-SEM model (Beno, Czech Republic) to investigate the morphology of Tb-GQDs-SiO₂-APTES-NH₂ and their energy dispersive X-ray (EDX) analysis.

Synthesis Tb-GQDs-SiO₂-APTES-NH₂-FA

Preparation of Tb-GQDs-SiO₂-APTES-NH₂

In brief, 2.0 g of CA was mixed with 5.0 mL of TbCl₃ (0.1 M) were mixed in a beaker and heated to 180 °C using a mantle for approximately 8 h until it transformed into an orange liquid. The solution was then left to cool to room temperature. The resulting product (Tb-GQDs) were dissolved in ultrapure water and neutralized to reach pH 7 by gradually adding NaOH (1 M) drop by drop, with continuous vigorous stirring. The mixture was dialyzed using a dialysis bag (3 kDa molecular weight cut-off), with the water being replaced every 6 h until a precipitate formed. Finally, the Tb-GQDs were redispersed in 5 mL of DI water and kept at 4 °C^28,34,35.

To coat the Tb-GQDs with a silica shell, a modified Stöber method using APTES was applied³⁶, which introduced amino groups onto the surface. Specifically, 44.5 mg/mL of the GQDs solution was mixed in ethanol. Subsequently, 60 µL of TEOS and APTES were added to the mixture. The mixture was agitated at 40 °C for 2 h and was subsequently allowed to cool to room temperature. The resulting Tb-GQDs-SiO₂-APTES-NH₂ were purified by washing several times with water and ethanol^37,38.

Synthesis of Tb-GQDs-SiO₂-APTES-NH₂-FA

Initially, FA moieties were activated using an EDC/NHS protocols. Briefly, 1.1 mg of EDC, 1.6 mg of NHS, and 2.5 mg of FA were dissolved in PBS (50 mM, pH 7.4) and stirred for 0.5 h at room temperature. During this process, the carboxylic group of FA was activated by EDC, forming an O-acylisourea intermediate, which readily reacts with the primary amines to form an amide bond. However, this intermediate is unstable and can undergo hydrolysis in an aqueous solution, thereby regenerating the carboxyl groups. To prevent this, NHS was added, resulting in the formation of a more stable NHS ester that allows for effective conjugation to the primary amine functional groups at neutral pH. The prepared FA-NHS solution was then added to the Tb-GQDs-SiO₂-APTES-NH₂ and stirred for 24 h. The amine groups on APTES reacted with the activated FA molecules, forming the final product, Tb-GQDs-SiO₂-APTES-NH₂-FA^19,39.

Biological studies

General cell culture procedure and cell viability assay

Cells were cultured in RPMI medium supplemented with 10% FBS and 1% PS at 37 °C in a 5% CO₂ incubator. The cells were then rinsed with PBS to eliminate excess RPMI, followed by trypsinization and incubating for 4 min at 37 °C in a 5% CO₂ atmosphere. The trypsinized cells were transported to a tube and washed for 2 min to remove any remaining trypsin. After centrifugation, the cells were suspended in fresh RPMI medium for the next tests. All tests were done when the cells’ confluency reached 70–80%. Additionally, the cells were passaged at least three to four times before further analysis.

Cellular uptake study

Different concentrations and exposure times were applied to explore the cellular internalization of Tb-GQDs-SiO₂-APTES-NH₂-FA by MCF-7 cells. Initially, about 5 × 10⁵ of the MCF-7 cancer cells were seeded in a six-well culture wells. Then, the cells were incubated with the produced materials to facilitate cellular uptake. Finally, fluorescence microscopy was applied to study cellular uptake and provide a broad evaluation of the internalization process in different times and concentrations.

Cell viability analysis

The cytotoxicity of the Tb-GQDs-SiO₂-APTES-NH₂-FA on the MCF-7 cancer cells was assessed using the MTT assay. Initially, 7.0 × 10³ cells per well were seeded into the wells of a 96-well plate and incubated for 24 h (at 37 °C in a 5% CO₂) to allow for cell adhesion and proliferation. Subsequently, the cells were exposed to different concentrations of the synthesized nanomaterials, ranging from 1.0 to 1000 µg/mL, with each concentration tested in triplicate to ensure statistical reliability. The plates were then kept again at 37 °C in a 5% CO₂ atmosphere for another 24 h to replicate physiological conditions. Following the incubation, to determine cell viability, about 10 µL of MTT reagent (6 mg/mL) was added to each well and the plates were incubated for 4 h to allow the reduction of MTT reagent to formazan by metabolically active cells. Afterward, the culture medium was removed and replaced with DMSO (200 µL) to dissolve the produced formazan crystals. The plates were incubated for an additional 30 min to ensure complete dissolution. Finally, the absorbance of the wells was measured at 570 nm using a microplate reader. The resulting absorbance values provided a quantitative assessment of cell viability, where the intensity of the color was directly proportional to the number of viable cells.

Statistical analysis

In this study, statistical analysis was utilized to assess the collected data, presenting results as mean ± standard error. A T-test was used to determine statistical significance between the two groups, with P-values below 0.05 deemed statistically significant. The analyses were performed using GraphPad Prism version 9.5.1.

Results and discussions

Characterization of the synthesized materials

The FESEM analysis shows that the synthesized Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles exhibit a quasi-spherical morphology with excellent water dispersibility (Fig. 2(a)). In similar studies, the size of the GQDs was reported to be approximately 4 nm ^39,41. The size of the Tb-GQDs-SiO₂-APTES-NH₂-FA was approximately 17 nm. These FESEM images provide clear evidence of the nanocomposite’s morphology, confirming the attachment of the SiO₂ layer and the conjugation of FA. FESEM analysis is less ideal for colloidal nanomaterials due to potential structural alterations during sample preparation, such as aggregation or distortion.

Fig. 2.

(a) FESEM and (b) AFM image of Tb-GQD-SiO₂-APTES-NH₂-FA.

Furthermore, AFM imaging provided a detailed view of the structural characteristics and morphology of the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles, highlighting its quasi-spherical shape (Fig. 2(b)). The AFM analysis revealed that the height of the nanoparticles was approximately 99 nm. This data complements previous findings and reinforces the understanding of the structural integrity of the Tb-GQDs-SiO₂-APTES-NH₂-FA nanocomposite. While both AFM and FE-SEM provide structural information, they differ in their strengths. AFM excels at obtaining precise vertical measurements, whereas FE-SEM offers high-resolution imaging in the lateral plane. Discrepancies in size measurements primarily arise from the nature of these techniques. FE-SEM involves imaging dried samples under a high vacuum, a process that often leads to particle aggregation or shrinkage due to dehydration, potentially resulting in an underestimation of the true size. This phenomenon of aggregation was further supported by the surface roughness observed in AFM images and the high polydispersity index (PDI) of 0.7 obtained from DLS analysis. Conversely, AFM measures nanoparticles in a more natural state, potentially including adsorbed layers, which yields resulting in larger and more reliable size estimates. FE-SEM measurements may also be subject to uncertainties caused by electron beam interference, the approximate nature of size measurement from images, and noise in analysis methods like pattern matching^41–43.

Further structural validation is provided by EDX analysis, which confirms the composition of the Tb-GQDs-SiO₂-APTES-NH₂-FA nanocomposite, verifying the successful coating, functionalization, and conjugation of the synthetic nanomaterials (Fig. S1 and Table S1). The FTIR spectra of the Tb-GQDs-SiO₂-APTES-NH₂ and Tb-GQDs-SiO₂-APTES-NH₂-FA are depicted in Fig. 3. The broad absorption peak at approximately 3416 cm^{− 1} can be related to the presence of the hydroxyl groups on the surface of the Tb-GQDs⁴⁴. Peaks observed at around 1616 cm^{− 1} are due to the C = C stretching vibrations and 1392 cm^{− 1} corresponds to the symmetric carboxylate stretch (-COO⁻). Furthermore, 3416 cm^{− 1}, 3547 cm^{− 1}, and 3672 cm^{− 1} are also associated with Si-O-Si, indicating the availability of a silica monolayer on the Tb-GQDs surface. Additionally, the peak at 472 cm^{− 1} corresponds to the Si–O stretching vibration^40,45. Peaks below 800 cm^{− 1} are usually attributed to the metal-O vibrations like Tb-O. In addition, the stretching vibration of –CH at 2926 cm^{− 1} confirms the successful attachment of APTES and TEOS on the Tb-GQDs surface⁴⁰.

Fig. 3.

FT-IR spectra of (a) Tb-GQDs-SiO₂-APTES-NH₂ (b) Tb-GQDs-SiO₂-APTES-NH₂-FA.

Following functionalization with FA, new peaks emerged or shifted, particularly within the 1300–1700 cm^− 1 region. Specifically, peaks appeared at 1387 cm^− 1 and 1563 cm^− 1 emerged following EDC/NHS coupling, which are correspond to C–N and N–H stretching vibrations, respectively. Additionally, new peaks in the 3400–3800 cm⁻¹ region were attributed to N–H vibrations. A peak around 1638 cm^− 1 corresponds to the amide (–NH–CO–) stretching vibration that further suggests the development of amide groups in Tb-GQDs-SiO₂-APTES-NH₂. This detailed FTIR analysis confirms the successful functionalization and modification of Tb-GQDs with both silica and FA⁴⁶.

Dynamic Light Scattering (DLS) analysis revealed that the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles (200 µg/mL) had a mean size of nearly 282 nm with a PDI of 0.7 (Fig. S2(a)), indicating a polydisperse nanocomposite. The discrepancy between the DLS analysis and FE-SEM/AFM measurements can be attributed to the formation of a hydration layer on the synthetic nanoparticles or their potential agglomeration. Such inconsistencies between DLS and morphological analysis, such as FE-SEM or AFM, have also been reported in previous studies^43,47. Additionally, the final zeta potential of the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles were measured to be -14.4 mV (Fig. S2(b)). This negative value was attributed to the attachment of FA molecules (which have a zeta potential of -31.6 mV) to the amino ends on the surface of the Tb-GQDs, as discussed in Sect. 2.3.1¹⁹. Furthermore, the presence of APTES on the Tb-GQDs contributed a positive charge due to groups, which also indicated a shift in zeta potential⁴⁰. These findings confirmed the successful attachment of FA to the amino groups on the surface of the Tb-GQDs, validating the functionalization process of the nanocomposite.

UV and fluorescence spectra analysis of Tb-GQDs-SiO₂-APTES-NH₂-FA

The photophysical features of the Tb-GQDs-SiO₂-APTES-NH₂ nanocomposites were evaluated before and after the conjugation of FA molecules. The UV-vis absorption spectrum of Tb-GQDs-SiO₂-APTES-NH₂-FA is depicted in Fig. S3. The Tb-GQDs-SiO₂-APTES-NH₂ nanoparticles exhibited absorption bands around 220 nm and 290 nm (Fig. S3(a)), which could be attributed to the excitation energy of Tb ions on the surface and the π-π* electronic transitions of C = C bonds in the sp² hybridized carbon atoms, respectively. The presence of C = C bonds is further supported by the FTIR spectrum, which shows a characteristic peak at approximately 1616 cm^{− 119,48}. A blue shift in the absorption peak from 290 nm to 265 nm was observed by UV-Vis spectroscopy, confirming the successful conjugation of FA to the primary amine groups on the surface of Tb-GQDs-SiO₂-APTES-NH₂.

Furthermore, fluorescence spectra were recorded in PBS buffer (pH 7.4). The characteristic fluorescence emission wavelength of Tb-GQDs-SiO₂-APTES-NH₂ was notably changed after FA conjugation, with the peak shifting from 400 nm to 410 nm (Fig. S3(b)) under 310 nm excitation. This shift is corresponded with an n-π* electronic transition. This transition leads to a high QY fluorescence emission due to the direct bandgap transition inherent to Tb-GQDs⁴⁹. Additionally, the fluorescence intensity at 600 nm increased significantly. These changes further validated the successful chemical modification of Tb-GQDs-SiO₂-APTES-NH₂ with FA molecules, highlighting the impact of FA conjugation on the optical properties of the nanocomposites.

Photoluminescence of Tb-GQDs-SiO₂-APTES-NH₂-FA

The emission behavior of the Tb-GQDs-SiO₂-APTES-NH₂ and Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles was studied over a wide excitation wavelength range, as illustrated in Fig. S4. The luminescence intensity of Tb-GQDs-SiO₂-APTES-NH₂ increased as the excitation wavelength was raised from 270 nm to 330 nm, after which it decreased as the wavelength increased to 370 nm. A similar trend was observed for the Tb-GQDs-SiO₂-APTES-NH₂-FA sample, with its maximum luminescence intensity was recorded at 310 nm. Beyond this wavelength, the intensity declined steadily, reaching its minimum at 320 nm (Fig. S4). Therefore, an excitation wavelength of 310 nm was chosen as the optimal for further investigations.

The observed variations in luminescence intensity across all three samples are consist with Kasha’s rule. This rule states that fluorescence typically occurs from the lowest vibrational level of the first excited singlet state, irrespective of the excitation wavelength. Additionally, the maximum photoluminescence peaks shifted across the different samples, suggesting that the chemical modifications introduced to the Tb-GQDs are the primary cause. These spectral shifts could result from changes in nanoparticle size or variations in the surface emission traps, which further highlights the significant impact of surface modifications on the optical properties of the nanoparticles⁵⁰.

The influence of pH on the absorbance and fluorescence of the Tb-GQDs-SiO₂-APTES-NH₂-FA

The absorbance behavior of Tb-GQDs-SiO₂-APTES-NH₂-FA is significantly affected by changes in pH, as illustrated in Fig. S5(a). The study explored a pH range from 3 to 11, revealing a gradual increase in absorbance from pH 3 to 8, with minimal difference observed between pH 4 and 6.4. In highly acidic conditions, the n-π* absorption of Tb-GQDs-SiO₂-APTES-NH₂-FA was decreased. However, after pH 6.4, the absorbance increased significantly until reaching pH 8, beyond which it decreased. Notably, no blue or red shift was observed in the absorption maximum, indicating that pH variation did not alter the spectral position of the absorbance peak.

Similarly, the fluorescence emission of Tb-GQDs-SiO₂-APTES-NH₂-FA was influenced by pH changes. As shown in Fig. S5(b), the fluorescence intensity increased from pH 3 to 7.4, which can be attributed to the protonation of free amine functional groups under acidic conditions. After a slight decrease, the intensity decreased slightly before increasing again to pH 11. The maximum fluorescence emission was recorded at pH 7.4 and 11, highlighting the suitability of Tb-GQDs-SiO₂-APTES-NH₂-FA for applications in both physiological and alkaline environments. Furthermore, Fig. S5(c) demonstrates that the emission peak wavelength remained stable across the pH range, indicating that pH variation did not affect the emission peak position. This stability represents a key advantage for the use of Tb-GQDs-SiO₂-APTES-NH₂-FA in bioimaging applications, particularly in cancer cell imaging, where consistent fluorescence behavior is critical.

The influence of the amount of Tb-GQDs-SiO₂-APTES-NH₂-FA on the absorbance and fluorescence

The influence of concentration on the absorbance and fluorescence of the synthetic Tb-GQDs-SiO₂-APTES-NH₂-FA nanocomposite was analyzed. As shown in Fig. S6(a), a linear increase in absorbance was observed over the concentration range of 100 µg/mL to 1000 µg/mL, consistent with the Beer-Lambert law, which establishes a direct correlation between sample absorption and concentration. Furthermore, Fig. S6(b) and Fig. S6(c) illustrate the relationship between the fluorescence peak wavelength, its intensity, and the concentration of the Tb-GQDs-SiO₂-APTES-NH₂-FA nanocomposite. The analysis of these data revealed a slight red shift in the fluorescence peak, moving from 408 nm at 100 µg/mL to 412 nm at 1000 µg/mL. This red shift suggests that at lower concentrations, the nanoparticles exhibited minimal interactions, while at higher concentrations, self-absorption of the emitted fluorescence likely occurred due to increased proximity of the molecules. The highest intensity was recorded at 800 µg/mL, indicating an optimal concentration for fluorescence emission. Beyond this point, the intensity decreases slightly, likely due to self-quenching effects as the concentration increases further. These results highlight the importance of optimizing concentration in applications where fluorescence properties are critical.

QY of Tb-GQDs-SiO₂-APTES-NH₂-FA

The QY of Tb-GQDs-SiO₂-APTES-NH₂-FA was estimated using rhodamine B (RhB) as the reference molecule with a QY of approximately 31% in water (Fig. S7). To determine the relative QY of Tb-GQDs-SiO₂-APTES-NH₂-FA, a series of concentrations were prepared for both Tb-GQDs-SiO₂-APTES-NH₂-FA (10, 50, 80, and 130 µg/mL) and RhB (0.2, 1, 3, and 5 µg/mL), adjusting the absorbance to 0.1. The QY of Tb-GQDs-SiO₂-APTES-NH₂-FA was subsequently calculated using the following equation:

1

Where “S” and “R” refer to Tb-GQDs-SiO₂-APTES-NH₂-FA and RhB, respectively. The symbols QY_S and QY_R represent the QY of Tb-GQDs-SiO₂-APTES-NH₂-FA and RhB. Furthermore, F, A, and η refer to the fluorescence peak area, absorbance, and the refractive index of the solvent (where η is 1 for water), respectively. By applying the given formula, the QY of the Tb-GQDs-SiO₂-APTES-NH₂-FA nanocomposite was calculated to be approximately 29.1%. This value is significantly higher than those previously reported for similar nanomaterials in the literature^12,35,50.

Stability of the Tb-GQDs-SiO₂-APTES-NH₂-FA

The photostability of the Tb-GQDs-SiO₂-APTES-NH₂-FA composite, a crucial property for its application in fluorescence-based detection in both in vitro and in vivo settings, was evaluated. The stability of the fluorescent emission was assessed under continuous UV irradiation (350 nm) over various time intervals (1, 5, 10, 20, 30, 45, 60, 80, and 120 min). The results showed that the fluorescence emission of the composite was sensitive to UV light. A significant decrease in the fluorescence emission was observed after just 1 min of UV exposure, which is attributed to photobleaching. However, following this initial decline, the fluorescence intensity stabilized and remained relatively constant with no significant changes (p-value > 0.05) for exposure times of up to 45 min, as depicted in Fig. S8(a).

Additionally, the influence of incubation time on the fluorescence intensity was examined. As shown in Fig. S8(b), the fluorescence emission intensity of Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles‒which was enhanced in the presence of carboxyl (–COOH) and hydroxyl (–OH) functional groups‒ remained stable over time without significant changes. This observation indicates high photostability, which is advantageous for bioimaging applications¹⁰. The impact of temperature on fluorescence intensity was tested over a range of temperatures (0–50 ℃). Fig. S8(c) shows that higher temperatures reduced the fluorescence intensity for Tb-GQDs-SiO₂-APTES-NH₂-FA nanocomposite. This decline is likely due to increased intermolecular interactions leading to non-radiative relaxation processes. Finally, the fluorescence spectrum of the Tb-GQDs-SiO2-APTES-NH2-FA was recorded after 365 days. Fig. S8(d) shows that its intensity has not decreased significantly, which indicates the stability of the compound.

Cell targeting

Toxicity test

To evaluate the biocompatibility of Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles for potential in vivo bioimaging applications, a cell viability assay was conducted. The viability of MCF-7 cancer cells in the presence of Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles was assessed using the MTT assay protocol at various concentrations from 10 to 1000 µg/mL. The results demonstrated that cell viability remained around 90% after a 24-hour incubation, even at the highest concentration of 1000 µg/mL, indicating minimal interference with cell growth and no significant increase in cell death (Fig. 4). Statistical analysis of cell viability was performed using a T-test, with a p-value > 0.05, confirming that there was no significant difference in cell viability across the tested concentrations. The MTT assay results suggest that the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles possessed excellent biocompatibility, likely due to the silica layer and FA conjugation⁵¹. Consequently, Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticle is a promising candidate for biological testing and in vivo applications, offering potential benefits for bioimaging in cancer diagnostics and therapy.

Fig. 4.

Cell viability of Tb-GQDs-SiO₂-APTES-NH₂-FA on MCF-7 cells in various concentrations of 0 (Blank), 10, 25, 50, 100, 200, 400, 600, 800, and 1000 µg/mL for 24 h.

Bioimaging

The cellular uptake of Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles was investigated across various concentrations and incubation times to assess their interaction with MCF-7 cancer cells. This interaction occurs through the specific binding between FRs overexpressed on MCF-7 cells and the FA moieties on the nanoparticle, driven by their high affinity. The fluorescence intensity also increases, resulting in a brighter blue fluorescence emission, which is consistent with previous fluorescence emission studies referenced in subsection 3.3.

Figure 5 illustrates the impact of Tb-GQDs-SiO₂-APTES-NH₂-FA concentration on cellular uptake. In the control sample, no photoluminescence emission was detected, despite the FR overexpression by the cancer cells. Upon incubation with Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles, fluorescence intensity became apparent. An increase in the concentration of Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles provided more binding sites for interaction with the FRs on the cell surfaces, leading to heightened fluorescence. The uptake reached its peak at a concentration of 1000 µg/mL, where the nanoparticles exhibited maximum brightness due to the high-affinity interactions between FR and FA.

Fig. 5.

The qualitative fluorescence microscopy bioimaging of cellular uptake of Tb-GQDs-SiO₂-APTES-NH₂-FA at different concentrations on MCF-7 cells (scale bar 20 μm).

Furthermore, Fig. 6 presents the influence of incubation time on the uptake of Tb-GQDs-SiO₂-APTES-NH₂-FA by MCF-7 cancer cells. To evaluate this, MCF-7 cells cultured in six-well plates were incubated with the nanoparticles at a concentration of 1000 µg/mL over different time intervals (0.5, 1, 2, 4, 16, and 24 h). The results demonstrated a time-dependent increase in fluorescence intensity and nanoparticle uptake, indicating enhanced attachment to the cell surface. Notably, significant uptake and emission were observed within the first 2 h, as MCF-7 cells rapidly bound to the FA residues on the nanoparticles due to FR overexpression. After 2 h, no significant increase in uptake and emission were observed, as the FR-FA binding primarily occurs within a short time frame. Figures 5 and 6 clearly show that the Tb-GQDs-SiO₂-APTES-NH₂-FA nanoparticles predominantly attach to the cell membranes of MCF-7 cancer cells, rather than entering the nucleus. These interactions suggest that the nanoparticles specifically target FRs, which are primarily located on the membrane surface, further confirming their selective binding to FR-overexpressed cancer cells. To assess the selectivity of the method, Tb-GQDs-SiO₂-APTES-NH₂ was incubated with MCF-7 cells at different times. The results indicated that the level of cellular uptake was very low, attributed to the lack of targeting ability of the Tb-GQDs-SiO₂-APTES-NH₂ to the FA on the cell surfaces (Fig. 7).

Fig. 6.

The qualitative fluorescence microscopy bioimaging of cellular uptake of Tb-GQDs-SiO₂-APTES-NH₂-FA (1000 µg/mL) at various times incubation on MCF-7 cells (scale bar 20 μm).

Fig. 7.

Study of selectivity of Tb-GQDs-SiO₂-APTES-NH₂ (1000 µg/mL) towards MCF-7 cells at different times of incubation.

Conclusions

In this study, Tb-GQDs-SiO₂-APTES-NH₂-FA was synthesized through a multi-step process involving the thermal pyrolysis of citric acid to form GQDs, chelation with terbium ions, silica coating via a modified Stöber method, and functionalization with FA molecules using the EDC/NHS coupling method, exhibited favorable physicochemical characteristics, including a high QY, excellent photostability, and pH-stable fluorescence emission. The nanoparticles proved highly biocompatible, maintaining over 90% cell viability in MCF-7 cells even at concentrations up to 1000 µg/mL, indicating minimal cytotoxicity. The resulting nanoparticles served as fluorescent nanoprobes for MCF-7 cancer cell bioimaging, leveraging the high binding affinity between FA and the overexpressed FRs on the cell surface. The fluorescence probe showed selective binding to MCF-7 cells that overexpress FRs, as confirmed by both bright-field and fluorescence imaging, which demonstrated the binding of Tb-GQDs-SiO₂-APTES-NH₂-FA to the tumor cell membranes and resulted in significantly enhanced fluorescence signals in a concentration- and time-dependent manner. These nanoparticles showed high specificity for cellular targeting, excellent QY, and excellent biocompatibility, as validated by MTT assays and bright fluorescence emission. These attributes highlight their potential for in vivo cancer bioimaging applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

H.G. and S.K.D. investigation and writing the first draft, M.G. characterization of materials and writing the first draft, M.E., investigation and writing the first draft, J.S.; conceptualization, method design, reviewing the final version, A.A.M.; resources, reviewing the final version.

Data availability

Data will be available on request by Mr. H.Gh.

Declarations

Competing interests

The authors declare no competing interests.

Ethics statement

This project was approved by the Research Ethics Committee of Tabriz University of Medical Sciences ethics committee with the confirmation code IR.TBZMED.VCR.REC.1402.156. Also, this study complies with all regulations.