Introduction

The burgeoning growth of cancer research demands the creation of innovative analytical methodologies for accurately assessing chemotherapy medications [1]. It is imperative to prioritize the development of personalized treatment strategies that take into account individual patient responses and the unique characteristics of their disease [2]. This tailored approach aims to enhance treatment effectiveness while minimizing adverse effects. The successful execution of personalized medicine hinges on numerous factors, notably real-time patient health supervision through continuous monitoring of disease-related biomarkers and the amount of anti-cancer drugs in the bloodstream [3]. Administering micro-doses of drugs aims to attain optimal effects while minimizing adverse reactions. However, accurately detecting these drugs poses significant challenges, particularly when dealing with minimal concentrations in the bloodstream resulting from micro-dosing. This necessitates the utilization of highly sensitive detection techniques to ensure precise monitoring of drug levels. Gemcitabine, an anticancer medication, finds extensive application in treating various tumors including breast, ovarian, and pancreatic cancers [4]. Its mechanism involves DNA replication inhibition, favoring the tumor cell death [5]. Monitoring of gemcitabine levels in the bloodstream of patients is crucial due to its cytotoxic effects on normal cells [6]. Gemcitabine has been analytically determined using chromatographic [7,8,9] and electrochemical methods [10, 11]. While chromatographic techniques offer selectivity and sensitivity, they suffer from drawbacks such as complex instrumentation, the requirement for highly skilled personnel, extensive pretreatment steps, high costs, and time consumption [12, 13]. On the other hand, electrochemical methods lack selectivity due to electrode fouling [14, 15]. Hence, there is a critical need to develop simple, rapid, selective, and sensitive detection methods for gemcitabine in real samples. In contrast, optical methods, such as fluorometric techniques, emerge as relevant tools for measuring molecules and biomolecules due to their selectivity, rapidity, simplicity, sensitivity, and low-cost [16,17,18,19,20,21].

Metal nanoclusters are widely applied in the sensing area due to their excellent water dispersion, low toxicity, biocompatibility, stability, and photoluminescence properties [22,23,24,25]. Various fabrication methods, including electrochemical, chemical reduction, etching, microwave-assisted, and template approaches, are employed to produce metal nanoclusters [26,27,28,29]. Among the widely utilized nanoclusters are Cu NCs, Au NCs, and Ag NCs. Silver nanoclusters (Ag NCs) are preferred over gold and copper counterparts due to their lower cost compared to Au NCs and superior oxidation stability compared to Cu NCs [30, 31]. The stabilization of Ag NCs was achieved using various compounds such as bovine serum albumin [32], dihydrolipoic acid [33], and polyethyleneimine [34]. Among these, tryptophan (TRP) is particularly employed for stabilizing blue emissive nanoclusters due to its distinctive optical properties and inherent blue fluorescence after excitation in the range of 275–290 nm [35, 36]. Furthermore, when TRP is surface-modified onto nanoparticles, its fluorescence diminishes, thereby enhancing the exciton lifetime on the nanoparticles [37, 38]. In literature, tryptophan-functionalized silver nanoclusters (TRP@Ag NCs) were employed to determine Cu (II) [39] and Fe (III) [40].

Herein, we synthesized a new fluorescent probe comprising tryptophan-functionalized silver nanoclusters (TRP@Ag NCs) for detecting gemcitabine. The addition of cationic gemcitabine led to the quenching of negatively charged TRP@Ag NCs due to aggregation. This interaction between positively-charged gemcitabine and negatively-charged TRP@Ag NCs involves many types of interaction such as non-covalent hydrogen bonding, Van der Waals, and electrostatic force.

Experimental

Materials and Reagents

Gemcitabine (98.8%), Sodium borohydride (NaBH4, 98.8%), dopamine (97.7%), ascorbic acid (96.8%), uric acid (98.3%), glutathione (97.9%), cysteine (98.6%), glucose (98.8%), glycine (97.5%), glutamine (98.7%), urea (97.6%), maltose (96.7%), and lysine (97.6%) were procured from Sigma Aldrich. Silver nitrate (99.8%), tryptophan (TRP, 97.8%), NaH2PO4, Na2HPO4, HCl, and NaOH were procured from Merck. GEMZAR® 200 mg injection was obtained from local markets.

Instruments and Quantum Yield (φ)

Description of instruments and calculation of φ (includes Fig. S1) were listed in supporting information.

Preparation of TRP@ Ag NCs

All glassware must underwent thorough rinsing with aqua regia (a mixture of HNO3 and HCl, in a 1:3 v/v ratio) prior to being washed with ultrapure water. Subsequently, a solution consisting of 1.2 mL of 0.05 M AgNO3, 1.8 mL of 0.5 M NaBH4, and 1.5 mL of 0.05 M TRP was prepared, and the volume was adjusted to 8.0 mL using ultrapure water. The resulting mixture was stirred for 10 min at room temperature (RT). Following this, the pH of the mixture was adjusted to 5.0 before it was heated at 60 °C for 6 h. The resulting yellow-colored solution was then centrifuged at 3000 rpm for 20 min. The TRP@Ag NCs probe obtained in this manner was stored at 4 °C until further use (Scheme 1).

Determination of Gemcitabine

400 µL of the TRP@Ag NCs prepared earlier was combined with 600 µL of various concentrations of gemcitabine. The mixture was then allowed to incubate at RT for 2 min. After that, ultrapure water was used to complete the volume to 1.5 mL. Finally, the fluorescence spectra were then measured at 460 nm following excitation at 320 nm (Scheme 1).

Preparation of Samples

The injection sample was initially diluted with ultrapure water. Following this, 600 µL was combined with 400 µL of TRP@Ag NCs and allowed at RT for 2 min. Then, 1.5 mL of ultrapure water was added. For human serum and urine samples, they underwent centrifugation at 4000 rpm to eliminate suspended particles, after which they were diluted to 10 mL with ultrapure water. Next, 600 µL of the serum or urine was mixed with 400 µL of TRP@Ag NCs and incubated at RT for 2 min. Subsequently, 1.5 mL of ultrapure water was added. All samples were measured before and after addition of gemcitabine.

Scheme 1
scheme 1

Preparation of fluorescent TRP@ Ag NCs and determination of gemcitabine

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Results and Discussion

Characterization

The surface morphology of the probe was examined using TEM, as illustrated in Fig. 1A. The analysis revealed a spherical and well-dispersed pattern. Figure 1B presents the size distribution of the nanoclusters, indicating an average diameter of 1.8 nm ± 0.02 nm (n = 80). In Fig. 1C, the XRD pattern of the Ag NCs is depicted, with a diffraction peak observed at 38.9° and low intensity, indicating the formation of Ag NCs [41, 42]. Figure 1D showcases the modification of Ag NCs with TRP using FT-IR spectroscopy. In Fig. 1D(a), the FT-IR spectrum of TRP displays main absorption bands at 3455 cm− 1, 3306 cm− 1, 1686 cm− 1, and 1228 cm− 1, corresponding to stretching vibrations of OH, NH, CH2, C = O, and COO−, respectively, while bands at 1613 cm− 1 and 678 cm− 1 represent bending vibrations of NH/OH and –NH-, respectively [43, 44]. Figure 1D(b) presents the FT-IR spectrum of TRP@Ag NCs, revealing a shift of fundamental bands to lower wavenumbers, which indicates successful modification of Ag NCs with TRP amino acid. Additionally, the absence of the bending vibration at 678 cm− 1 confirms the chemical interaction between Ag NCs and TRP via –NH- functionality [45, 46]. Fig. S2A illustrates the binding energies of various elements, including C 1s, Ag 3d, N1s, and O 1s. It was observed that the binding energies for C 1s, Ag 3d, N1s, and O 1s were located at 293.6 eV, 369.8 eV, 398.7 eV, and 524.8 eV, respectively [47]. Fig. S2B presents the high-resolution XPS of Ag 3d, with binding energies at 368.5 eV and 371.7 eV, corresponding to Ag 3d5/2 and Ag 3d3/2, respectively [48]. These findings suggested that Ag (I) was successfully reduced to Ag (0) [49, 50].

Fig. 1
figure 1

TEM (A), size distribution (B), and XRD (C) of TRP@ Ag NCs while FT-IR spectra (D) of TRP (a) and TRP@ Ag NCs (b)

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In Figure 2A, the absorption spectrum of TRP@Ag NCs is depicted, showing peak absorption at 342 nm, potentially indicating the formation of nanoclusters [51]. The lack of surface Plasmon in the range from 400 nm to 500 nm confirms the formation of nanoclusters rather than nanoparticles [52]. Figure 2B presents the excitation/emission spectra of TRP@Ag NCs, with an emission wavelength of 460 nm observed following excitation at 320 nm. Figure 2C illustrates the relationship between emission wavelengths and excitation wavelengths. It was found that the emission wavelengths were red-shifted with increasing the excitation wavelengths, which may be attributed to the variation of size and/or surface functional groups on the surface of TRP@ Ag NCs [53]. Fig. S3 demonstrates the effect of hydrogen peroxide (H2O2) etching on the fluorescence response of TRP@Ag NCs. The addition of 10 µM H2O2 caused a slight decrease in the fluorescence emission of TRP@Ag NCs, which may be attributed to breaking down Ag NCs into Ag+ [54].

To investigate the potential applications of TRP@ Ag NCs, we delved into their stability, as shown in Fig. S4. It was observed that the initially prepared probe displayed a remarkable level of stability even in high ionic strength conditions (Fig. S4a). Fig. S4b illustrates the exceptional stability of TRP@ Ag NCs across various pH values, which can be attributed to the protonation/deprotonation of surface functional groups. Notably, continuous irradiation of TRP@ Ag NCs for 150 min showed no discernible impact on the fluorescence readings of the probe, underscoring its robust stability (Fig. S4c). Furthermore, exposure of the probe to different temperatures (ranging from 25 to 45 °C) did not induce significant alterations in the fluorescence responses (Fig. S4d). The quantum photoluminescence yield was quantified at 9.78%.

Fig. 2
figure 2

Absorption spectra (A), excitation/emission spectra (B), and dependency of emission wavelength on the excitation wavelength of TRP@ Ag NCs (C)

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Optimization of Detection Conditions

Factors influencing the interaction between TRP@Ag NCs and gemcitabine were carefully optimized. The effect of incubation time was investigated, revealing an optimal value of 2.0 min (Fig. S5a). Beyond this duration, the fluorescence reading stabilized, indicating saturation. Hence, 2.0 min was determined as the optimal incubation time for the interaction between TRP@Ag NCs and gemcitabine. Moreover, the pH value of the diluting solvent was explored using different solvents (Fig. S5b). It was observed that both H2O and various buffers at pH 7.0 yielded similar outcomes. Consequently, H2O was used as the diluting solvent for the entire study, ensuring consistency in the experimental conditions. Additionally, the effect of different pH values was tested using phosphate buffer in the range of 5.0 to 9.0 (Fig. S5c). The results indicated that pH 7.0 provided the best response. Therefore, pH 7.0 was selected as the optimal pH for subsequent determinations. Fig. S5d shows the effect of TRP@Ag NCs amounts on the sensing of gemcitabine. It was evident that 400 µL of TRP@Ag NCs was the optimal amount. Below this value, the fluorescence ratio remained constant.

Detection of Gemcitabine Using TRP@ Ag NCs

The newly developed fluorescent probe was utilized to assess various concentrations of gemcitabine under optimal conditions. Figure 3 illustrates the decrease in fluorescence responses of TRP@ Ag NCs upon addition of gemcitabine within the range of 0.005-60 µM, attributable to aggregation-induced quenching. The plot depicting the relationship between the fluorescence ratio (F°/F, where F and F° represent fluorescence readings after and before gemcitabine addition, respectively) and gemcitabine concentration demonstrates a linear correlation with a high determination coefficient (R2) of 0.9982 and a regression equation of F°/F = 1.005 + 0.0167 C gemcitabine. The limit of detection (LOD) was calculated based on signal to noise = 3:1 and calculated as 1.7 nM. Comparative analysis of the analytical parameters between the proposed probe and other methods detailed in the literature for gemcitabine determination is presented in Table 1. It was concluded that the TRP@ Ag NCs probe exhibits the lowest LOD and a wide-dynamic linear range for the drug determination.

Fig. 3
figure 3

The emission spectra of TRP@ Ag NCs diminish upon the addition of various amounts of gemcitabine (ranging from 0.005 to 60 µM) under optimal conditions. Inset displays the calibration plot. Diluting solvent used is water

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Table 1 Different analytical methods for the determination of gemcitabine
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Anti-interference Study

The study investigated the impact of common interfering species on the fluorescence readings of TRP@Ag NCs (Fig. 4). It was found that 600 µM of Na+, K+, Ca2+, Mg2+, Cl−, CO32−, and PO43−; 500 µM of ascorbic acid, dopamine, uric acid, and glycine; 350 µM of urea, glucose, and maltose; and 300 µM of glutathione, cysteine, and glutamine did not significantly influence the fluorescence signal of the fluorescent probe. Moreover, the selectivity of the proposed probe was investigated in the presence of co-administered anti-cancer drugs used with gemcitabine. It was found that 300 µM anti-cancer drugs including, cisplatin, oxaliplatin, carboplatin, 5-fluorouracil, and imatinib did not remarkably affect the fluorescence response of the probe. These results suggest the probe has good selectivity.

Fig. 4
figure 4

The selectivity of the proposed TRP@Ag NCs fluorescent probe for detection of gemcitabine

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Applications

Table 2 Determination of gemcitabine in real samples (n=5)
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The newly prepared TRP@AgNCs fluorescent probe was employed for the estimation of gemcitabine in various real samples, including injections, serum, and urine. The standard addition method was utilized to quantify the drug in these matrices. Table 2 illustrates that the recovery percentages ranged between 97.0% and 102.0%, with RSD% falling within the range of 2.67–3.87%. Moreover, the results of the fluorescent probe were compared with a standard HPLC/UV method [61] for determining gemcitabine in real samples. These findings demonstrate the robustness and reliability of the method for detecting gemcitabine in these matrices.

Mechanism

The fluorescence feature at the surface of nanoclusters/nanoparticles is closely linked to physical or chemical changes that occur during their interactions [62, 63]. Spectroscopic and microscopic analyses were conducted to elucidate the detection mechanism between TRP@Ag NCs and gemcitabine (Fig. 5). The lack of overlap between the UV/Vis of gemcitabine and the emission spectrum of TRP@Ag NCs eliminates the possibility of Förster resonance energy transfer (FRET) or inner-filter effect (IFE), as depicted in Fig. 5a. TEM imaging was performed after introducing gemcitabine (Fig. 5b), revealing aggregation and an increase in the particle size of nanoclusters, consequently leading to a reduction in fluorescence [64]. Dynamic light scattering (DLS) measurements were taken before and after introducing gemcitabine. The DLS of TRP@Ag NCs was found to be 108.5 ± 5.67 nm (Fig. 5c), whereas that of TRP@Ag NCs/gemcitabine was 248.8 ± 8.32 nm (Fig. 5d), confirming the aggregation of nanoclusters following the introduction of the drug, consistent with TEM observations. The zeta potentials of TRP@Ag NCs and TRP@Ag NCs/gemcitabine were measured as -32.45 mV and − 8.78 mV, respectively (Fig. 5e). Reduction in the surface negative charge of the initially prepared TRP@Ag NCs after the addition of gemcitabine can be attributed to the cationic nature of the drug, which diminishes the surface charge of the negative-charged nanoclusters. Figure 5f illustrates the fluorescence lifetimes of TRP@Ag NCs and TRP@Ag NCs/gemcitabine, which remained unchanged, indicating static quenching [65]. Figure 6A illustrates the effect of gemcitabine addition on the UV/Vis spectrum of TRP@Ag NCs. The addition of gemcitabine increases the absorption intensity of the nanoclusters, suggesting the static quenching between gemcitabine and TRP@Ag NCs. Figure 6B shows the Stern-Volmer plots for TRP@Ag NCs with varying concentrations of gemcitabine. The data indicate that the slope (Ksv) increases with rising temperature, suggesting static quenching [66, 67].

Fig. 5
figure 5

(a) UV/Vis absorption spectrum of gemcitabine (i) and fluorescence emission spectrum of TRP@ Ag NCs (ii); (b) TEM image of TRP@ Ag NCs after addition of gemcitabine; (c) DLS of TRP@ Ag NCs; (d) DLS of TRP@ Ag NCs after addition of gemcitabine; (e) Zeta potentials of TRP@ Ag NCs and TRP@ Ag NCs /gemcitabine : (f) Fluorescence life times of TRP@ Ag NCs in absence and presence of 100 µM gemcitabine

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Fig. 6
figure 6

(A) The UV/Vis absorption spectra of TRP@ Ag NCs and TRP@ Ag NCs/ gemcitabine. (B) Stern-Volmer calibration plots at 290 K, 300 K, 310 K, 320 K, and 330 K

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Binding Energies Analysis

Thermodynamic parameters, were estimated using the following equation to understand the nature of interactions between gemcitabine and TRP@Ag NCs:

$$\:\varvec{l}\varvec{n}\:\varvec{K}\varvec{b}=-\frac{\varvec{\varDelta\:}\varvec{H}}{\varvec{R}\varvec{T}}+\frac{\varvec{\varDelta\:}\varvec{S}}{\varvec{R}}$$

Here, Kb equals the Stern-Volmer constant (Ksv) and R is the universal gas constant [68]. By plotting the relationship between lnKb and 1/T (Fig. 7), the values of ∆H and ∆S can be obtained. The change in free energy (∆G) can be calculated using the following equation:

$$\varDelta\textbf{G}\,\left(\textbf{T}\right)=\varDelta\textbf{H}-\textbf{T}\varDelta\textbf{S}$$
Fig. 7
figure 7

Plot relationship between Ksv and 1/T

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The models of interaction between gemcitabine and TRP@Ag NCs can be categorized as follows: (i) if both ∆S and ∆H are positive, it suggests hydrophobic interactions. (ii) if both ∆S and ∆H are negative, it indicates hydrogen bonding and Van der Waals forces. (iii) if ∆S is positive and ∆H is negative, it suggests electrostatic interactions [69, 70]. Table S1 shows that both ∆S and ∆H are negative, indicating that the main interactions between gemcitabine and TRP@Ag NCs are hydrogen bonding, Van der Waals forces, and electrostatic interactions. Additionally, the negative sign of ∆G indicates that the interaction between them is spontaneous.

Conclusions

A novel blue-emitting “signal-off” probe comprising tryptophan-functionalized silver nanoclusters (TRP@Ag NCs) was synthesized to detect gemcitabine. Upon exposure to gemcitabine, the TRP@Ag NCs probe experienced quenching due to aggregation, facilitated by hydrogen bonding, π-π stacking, and electrostatic attraction. Optimization of factors influencing the detection sensitivity was conducted. The fluorescent probe demonstrated notable selectivity, rendering it suitable for practical applications. Successful application of the TRP@Ag NCs probe was demonstrated in detecting gemcitabine in pharmaceutical injections, human serum, and urine samples.