Introduction

Fluoro-sensors for detecting physiologically active metal ions and their applications in material, environmental, and biological sciences are continually expanding. The growth of fluorescence sensors for the qualitative and quantitative detection of biospecies has piqued the interest of supramolecular and analytical chemists due to the several benefits, including high selectivity, sensitivity, cost-effectiveness, ease of handling, low detection limit, and on-site real-time monitoring without the use of sophisticated equipments [1,2,3,4,5]. Fluorescent chemosensors comprise mainly two major components, i.e., a light-emitting group (fluorophore) and a recognition site. Due to mechanisms such as photo-induced electron transfer, excimer formation, fluorescence resonance energy transfer, internal charge transfer, C = N isomerization, and others, when the target analyte selectively interacts with the recognition site, the optical property of the fluorophore is disturbed, leading to a shift, quenching, or amplification in the initial fluorescence signal of the sensor (6–7). In the last few decades, a variety of fluorescent sensors have been reported using organic dyads and fluorescent nanomaterials to detect metal ions, anions, and neutral molecules.

Transition metal cations like Zn2+, Fe3+/2+, Co2+, Cu2+, etc., are essential for the normal functioning of metabolic processes. However, they are detrimental at large concentrations and pose a hazard to human health via cellular toxicity, liver damage, and neurological illnesses. (8–9) Like other transition metal ions, Zn2+ has received significant interest due to its biological role. Zinc is the second most prevalent and vital transition element in the human body. It regulates enzymes, acts as a physical cofactor in metalloproteins, transmits neural activity, and is responsible for regulating gene expression and plenty of other biological functions. Zn2+ mediated bio compounds are widely used in medical treatment as tumor photosensitizers, antimicrobial, antioxidant agents, and radioprotective agents [10,11,12,13]. On the other side, excess Zn2+ ion leads cellular processes to become unbalanced, resulting in neurological disorders including Alzheimer’s disease, Menkes and Wilson diseases, Parkinson’s disease, prostate cancer, and diabetes [14,15,16]. In addition, high levels of zinc in the environment may diminish soil microbial activity, resulting in phytotoxicity. As a result, there is significant research on developing fluorescence sensors for rapid and selective detection of Zn2+ in biological and environmental systems, especially in the presence of interfering metal ions with similar coordination behaviour like Cd2+. Also, Zn2+ is diamagnetic in nature, and therefore it does not produce any magnetic or spectroscopic signals, making it impossible to detect in biological processes using standard analytical techniques like electron paramagnetic resonance (EPR) spectroscopy or nuclear magnetic resonance (NMR), Mossbauer spectroscopy. [17,18,19,20,21,22] So, fluorescence spectroscopy has been chosen as an ideal and convenient approach for detecting Zn2+ in biomolecules at the authentic site.

Vitamin B6 (VB6) cofactors comprise a set of six water-soluble chemically related molecules with a pyridine ring at its centre, i.e., pyridoxamine (PYOA), pyridoxal (PL), pyridoxine (PN), and their monophosphorylated derivatives. [23] Pyridoxal-5’-phosphate (PLP) is the most active form of VB6 cofactors. These cofactors are essential in various biochemical activities, such as decarboxylations of aminoacid [24], racemization [25], and enzymatic and non-enzymatic transformations. [26,27,28] In animals and plants, VB6 aids in the manufacture of fatty acids, the breakdown of some storage compounds, and the biosynthesis of plant neurotransmitters, hormones, and organelle-specific chemicals like chlorophyll [29,30,31]. VB6 deficiency is frequently associated with dermatitis, microcytic anemia, or electroencephalographic abnormalities. Immune system dysfunction, convulsive seizures, depression, and confusion have also been reported (32–33). The United States Food and Nutrition Board of the Institute of Medicine recommends dietary allowances (RDA) for VB6 ranging from 1.3 mg (young adults) to 1.7 mg (adult males), with lactating women up to 2 mg. Nonetheless, a few case studies have been published in which high doses of the vitamin cause neurological disorders because VB6 participates in so many biochemical pathways, and also abnormal levels of VB6 cofactors, particularly PLP and pyridoxal, can be harmful to human health [34,35,36,37,38]. As a result, there is much interest in developing new methods for detecting and monitoring the VB6 cofactor PLP.

Due to their discrete energy levels, noble metal nanoclusters (NCs) have unique photophysical and chemical properties that make them useful for a variety of applications, including catalysts, sensing, and cell labelling (39–40). In these domains, NCs based on gold (Au) and silver (Ag) have been widely used. Gold nanoclusters, which are made up of a few hundred to several thousand Au atoms, have remarkable optical characteristics, [41] such as strong fluorescence, good photostability, and biocompatibility [42, 43]. They also show intense fluorescence. The primary determinant of the fluorescence characteristics of gold nanoclusters is the Au core, which experiences a radiative transition due to metal-metal interaction. Furthermore, the surface ligands are important because they influence the fluorescence properties via mechanisms including ligand-to-metal charge transfer (LMCT) and/or ligand-to-metal-metal charge transfer (LMMCT) [44]. Here, a fluorescent gold nanocluster stabilised by papain that emits red light (pap-AuNCs) was synthesized and post-functionalized with PLP to create the nanoprobe PLP_pap-AuNCs (Scheme 1). The development of PLP_pap-AuNCs led to the modification in fluorescent color from red to orange, which allowed the detection of PLP using pap-AuNCs as a nanoprobe. Subsequently, the orange-emitting PLP_pap-AuNCs were used for the fluorescent turn-on sensing of Zn2+ in an aqueous medium.

Scheme 1
scheme 1

Schematic representation for the change in fluorescence of Papain-AuNCs for the relay detection of PLP and Zn2+

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Experimental

Reagents and Instruments

The analytical grade NaOH and metal salts such as CaCl2, HgCl2, Fe(NO3)3⋅9H2O, ZnCl2, MgCl2⋅6H2O, Cr(NO3)3⋅9H2O, MnCl2⋅4H2O, CdCl2.2H2O, NiCl2⋅6H2O, PbCl2, FeSO4⋅7H2O, Al(NO3)3⋅9H2O were provided by Rankem and Finar Pvt. Ltd, India. HAuCl4.4H2O, papain (pap), and various VB6 cofactors (PY, PN, PLP, and PYOA) were procured from Sigma-Aldrich. Stock solutions of various analytes (1 mM) and EDTA (1 mM) were made with double-distilled water and utilised following appropriate dilution.

An Agilent Technologies Cary Eclipse fluorescence spectrophotometer was used to perform fluorescence spectral measurements with both the excitation and emission slit widths set at 10 nm, and the solution containing the nanoclusters was excited at a wavelength of 380 nm. UV-visible absorption spectra were obtained using the Varian Cary 50 spectrophotometer. A quartz cuvette with a 1 cm path length was used for recording the spectra. With a HORIBA SZ-100, dynamic light scattering (DLS) data was obtained.

Synthesis of pap-AuNCs

The reported method was adopted to prepare AuNCs stabilized with papain (pap-AuNCs) [45]. Initially, 2.5 mL of HAuCl4 solution (10 mM) and 2.5 mL of papain solution (50 mg/mL) were thoroughly mixed at room temperature via vigorous mixing. Subsequently, the mixture was incubated at 90 °C for 5 min. Following this, 0.8 mL of NaOH solution (1.0 M) was added to the sample solution and further incubated for an additional 15 min. The color of the mixture changed to yellow, indicating the formation of pap-AuNCs. The purified pap-AuNCs solution via dialysis was kept at 4 °C for later studies.

Sensing Procedure

From the stock, 100 µL of pap-AuNCs was diluted to 2 mL with water to prepare the nanoclusters’ working solution. In order to examine the interactions with VB6 cofactors, 100 µL of pap-AuNCs was mixed with 100 µL of 1 mM VB6 cofactors, and then diluted to 2 mL using water. Significant changes in fluorescence emission were seen when PLP was added to pap-AuNCs. An emission band appeared at 525 nm, and concomitantly quenched at 700 nm. These alterations were indicative of the development of PLP_pap-AuNCs. The PLP_pap-AuNCs fluorescence emission spectra were evaluated by adding several metal ions (100 µL, 1.0 mM), including Zn2+, Mg2+, Pb2+, Ni2+, Mn2+, Hg2+, Ca2+, Al3+, Cd2+, Fe3+, Fe2+, and Cr3+. The competitive experiments were carried out by recording the fluorescence of PLP_pap-AuNCs in the presence of Zn2+ (100 µL, 1.0 mM) and other interfering metal ions at equimolar concentrations. The method used for fluorescence titration involved successive addition of Zn2+ ions to the nanoprobe incrementally. The limit of detection (LOD) was computed using the slope of the calibration curve using the IUPAC-approved formula, which is LOD = 3σ/slope, where σ is the relative standard deviation (RSD) of 10 blank measurements.

To conduct recovery experiments in real samples, river water from the Tapi River, Surat, India, and tap water from the laboratory were collected and filtered using Whatman filter paper to eliminate any particulate matter. Initially, the samples of water were added directly to the nanoprobe solution. No discernible alterations in the fluorescence spectra of the probe were observed. Therefore, to assess the feasibility of the developed nanoprobe for practical applications, a known concentration of Zn2+ solution from its standard solution was poured into the water samples. The poured water samples were then added to the probe solution. The resulting change in the fluorescence spectra was used to estimate the concentration of Zn2+ in the real water samples. This approach allows for the evaluation of the nanoprobe performance in real-world scenarios and provides insight into its potential for practical applications in environmental monitoring or water quality assessment.

Results and Discussion

Synthesis of pap-AuNCs

Proteins are useful biocompatible stabilizers to obtain fluorescent metal nanoclusters. Papain, an endolytic cysteine protease is extracted from latex of Carica papaya. It is a single polypeptide chain with 212 amino acid residues, some of which are cysteine, histidine, and asparagine. Through vigorous stirring of a mixture of HAuCl4 and papain under a basic medium at 90 °C for 15 min, pap-AuNCs were synthesized facilely. [46] According to reports, the sulfhydryl group of cysteine residues is essential for promoting the Au-S bond-mediated pap-AuNC synthesis. Studies also revealed that certain amino acid residues were more likely to take part in hydrogen bonding and polar interactions to regulate the growth of pap-AuNCs. The optical properties of pap-AuNCs were characterized by UV-Vis absorption and fluorescence spectra. The surface plasmon resonance band is absent in the UV-Vis spectrum and intense emission in the visible region suggests the molecule-like properties of AuNCs and the lack of larger particles. [47] The developed nano-system exhibited the distinct characteristics fluorescence band at 670 nm and red fluorescent colour under UV light at 365 nm, indicating the successful formation of fluorescent gold nanoclusters (Fig. 1). Further, the DLS analysis of pap-AuNCs was performed to measure the nanocluster size. To measure DLS, 100 µL of pap-AuNCs from the stock was diluted to 2 mL with water. According to the DLS analysis, the average hydrodynamic size of pap-AuNCs was 17 nm (Fig. 2).

Fig. 1
figure 1

Fluorescence emission (λex = 380 nm), excitation (λem = 670 nm) and UV-Vis absorption spectra of pap-AuNCs (inset showed the fluorescent vial of pap-AuNCs under UV light)

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Fig. 2
figure 2

The DLS image of pap-AuNCs

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Interaction with VB6 Cofactors

Pap-AuNCs’ fluorescence response was investigated with various VB6 cofactors. For this study, the probe was prepared by diluting 100 µL of pap-AuNCs with 1900 µL of water, followed by the recorded fluorescence spectrum showed the red-emitting fluorescent band at ~ 670 nm (λex = 380 nm). The selectivity of pap-AuNCs was explored by adding various VB6 cofactors (PL, PN, PLP, and PYOA). To study the interaction, 100 µL of pap-AuNCs from the stock was taken and mixed with 100 µL of various VB6 cofactors (1 mM) followed by diluted to 2 mL with double distilled water. The vials were gently shaken, and then the fluorescence spectra were recorded (Fig. 3). The emission band of pap-AuNCs was quenched, and a new band appeared for PLP. However, no observation in the fluorescence spectra and color of the AuNCs were noted by other VB6 cofactors.

The addition of PLP altered the fluorescence emission of pap-AuNCs. Thus, fluorescence titration was carried out to assess the sensitivity. Incremental amounts of PLP (10 µL to 130 µL, 1 mM) were added to the pap-AuNC solution, and the spectra were recorded after each successive aliquot (10 µL) addition. The spectral changes showed the pap-AuNCs’ fluorescence intensity dropped at 670 nm, together with the simultaneous enhancement at 525 nm (Fig. 5a). Red-emitting pap-AuNCs was changed into orange-emitting PLP_pap-AuNCs as a result of PLP addition. The bond formed between PLP’s aldehyde group and the unbound amine groups of papain functionalized AuNCs resulted in the formation of a Schiff base over the nanoclusters surface, [48] which was responsible for the changes observed in the fluorescence spectra and color. The particles have a hydrodynamic size of approximately 59 nm, according to the DLS measurement of PLP_pap-AuNCs (Fig. 4). A plot was drawn by plotting of pap-AuNCs’ ratiometric fluorescence spectral changes (700 nm/525 nm) against the added PLP concentrations. The calibration plot displays a good linearity with R2 = 0.9977 between the concentration range of 10 µM to 65 µM (Fig. 5b). From the slope of the calibration plot, the detection limit was calculated as 0.59 µM.

Fig. 3
figure 3

Fluorescence spectral changes of pap-AuNCs in the presence of various VB6 (inset shows the fluorescent colour changes of pap-AuNCs in the absence (i) and presence of PLP (ii), PY (iii), PN (iv), PYOA (v))

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Fig. 4
figure 4

The DLS image of 2 mL solution of PLP_pap-AuNCs in water containing 100 µL of pap-AuNCs from the stock and 100 µL of PLP (1 mM)

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Fig. 5
figure 5

(a) Fluorescence spectral changes of PLP-pap-AuNCs upon successive incremental addition of PLP (10 µL, 1 mM). (b) Calibration curves for the detection of PLP

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Detection of Zn2+

To detect metal ions, the PLP-conjugated nano-assembly PLP_pap-AuNCs was used as a nanoprobe. To create a 2 mL aqueous solution of the nanoprobe, 100 µL (1 mM) of PLP was combined with 100 µL of pap-AuNCs stock solution. Different metal ions (100 µL, 1 mM), such as Ni2+, Ca2+, Mg2+, Cd2+, Zn2+, Cr3+, Pb2+, Mn2+, Fe2+, Fe3+, Hg2+, and Al3+ were introduced in aqueous medium to test the selectivity of the nanoprobe. Interestingly, Zn2+ significantly altered the fluorescence colour and profile of PLP_pap-AuNCs (Fig. 6). A 3-fold enhancement in the fluorescent intensity of PLP_pap-AuNCs was observed at 477 nm. Addition of Mg2+ and Mn2+ induced a very negligible (~ 0.3-fold) fluorescence enhancement at 477 nm, but other metal ions failed to enhance the fluorescence intensity of PLP_pap-AuNCs at 477 nm. When PLP_pap-AuNCs interacted with Zn2+, their fluorescence showed a blue shift and increased at 477 nm. Additionally, PLP_pap-AuNCs’ fluorescence spectra were captured when Zn2+ and other interfering metal ions were present in equimolar concentrations (100 µL, 1 mM). Interestingly, the fluorescence enhancement at 477 nm of PLP_pap-AuNCs by Zn2+ alone was also observed in the co-presence of interfering metal ions. The recorded spectra in Fig. 7 revealed that a slight variation in the Zn2+ induced fluorescence enhancement of PLP_pap-AuNCs was observed when other tested interfering metal ions were present. These results validated the nanoprobe PLP_pap-AuNCs’s exceptional Zn2+ ion selectivity.

Fig. 6
figure 6

Fluorescence spectral changes of probe PLP-pap-AuNCs in the presence of different metal cations (50 µM)

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Further, by adding different quantities of Zn2+, fluorescence titration was used to test the sensitivity of PLP_pap-AuNCs as a Zn2+ nanoprobe (Fig. 8). Orange fluorescence emission was seen by the nanoprobe, with noticeable bands at 525 and 670 nm. As the Zn2+ amounts increased from 10 µL to 120 µL (1 mM), a gradual blue-shift from 525 nm and enhancement was observed at 477 nm. At 670 nm, no discernible alterations in the fluorescence spectrum were seen. Plotting the calibration curve against the added Zn2+ concentrations shows a good linear relationship for the fluorescence increase (Fig. 8b), with values ranging from 14.7 µM to 56.9 µM and the detection limit computed down to 0.14 µM.

Fig. 7
figure 7

The fluorescence spectral changes of PLP_pap-AuNCs (probe) in the presence of Zn2+ and equimolar amount of other interfering metal cations

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The average hydrodynamic diameter of PLP_pap-AuNCs increased from 59 nm to 97 nm following the addition of Zn2+(Fig. 9). The interaction between Zn2+ and PLP_pap-AuNCs is suggested from the increase in the hydrodynamic diameter. With two donor atoms (phenolic-OH and imine-N) on the surface of PLP_pap-AuNCs due to PLP conjugation offers an optimal coordination environment that facilitates Zn2+ recognition and modifies fluorescence as a result of internal charge transfer from ligand to metal (LMCT). EDTA, a potent chelating agent, was added to PLP_pap-AuNCs’ surface to verify the complexation of Zn2+. When EDTA was added, the increased fluorescence emission of PLP_pap-AuNCs at 477 nm caused by Zn2+ was reversed. This reversal happened as a result of the strong complexing activity of EDTA, which is predicted to decomplex Zn2+ and restore PLP_pap-AuNCs’ fluorescence emission (Fig. 10).

Fig. 8
figure 8

(a) The fluorescence spectral changes of PLP_pap-AuNCs (probe) upon incremental addition of Zn2+ (10 µL, 1 mM). (b) Calibration curves for the PLP_pap-AuNCs@Zn2+

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Fig. 9
figure 9

The DLS image of 2 mL solution of PLP_Pap-AuNCs@Zn2+ in water containing 100 µL of pap-AuNCs from the stock, PLP (100 µL, 1 mM) and Zn2+ (100 µL, 1 mM)

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Fig. 10
figure 10

Fluorescence spectra of PLP-pap-AuNCs in the absence and presence of Zn2+ and EDTA

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Overall, the detection of Zn2+ by PLP_pap-AuNCs is schematically presented in Scheme 1. The red-emitting pap-AuNCs interacted with PLP by forming an imine linkage and changed the fluorescent colour from red to orange. The Schiff base formation over the surface of the nanoclusters provide an ideal coordination environment to chelate Zn2+. When Zn2+ is added to the PLP_pap-AuNCs, the fluorescent emission enhanced at 477 nm due to the complexation-induced aggregation of PLP_pap-AuNCs. The aggregation of PLP_pap-AuNCs was supported by DLS, whereas the complexation-induced aggregation was complemented by reversibility experiment performed by adding strong chelating agent EDTA.

Real Sample Analysis

Zn2+ measurement was done in environmental water samples to show the applicability of PLP_pap-AuNCs. To get rid of big particles, the collected water samples were first filtered. These samples were then added to the PLP_pap-AuNCs solution and spiked with known concentrations of Zn2+. The calibration curve was used to calculate the Zn2+ concentration after the fluorescence spectra were recorded. Table 1 presents a summary of the acquired results, which indicate recoveries between 91% and 99%, with a maximum standard deviation (RSD) of roughly 3%. These results demonstrate the PLP_pap-AuNCs nanoprobe’s potential for measuring Zn2+ in actual environmental water samples.

The paper strips and cotton buds coated with pap-AuNCs and PLP_pap-AuNCs were prepared and shown in Fig. 11. The developed paper strips and cotton buds displayed the fluorescent colour of the respective nanoclusters, i.e., (i) pap-AuNCs, (ii) PLP_pap-AuNCs, and (iii) PLP_pap-AuNCs in the presence of Zn2+ ions. The nanoprobe PLP_pap-AuNCs paper strips and cotton buds also showed visual fluorescent colour changes in the presence of Zn2+. Thus, the created a nanoprobe PLP_pap-AuNCs can also be used to visually detect Zn2+ ions.

Fig. 11
figure 11

Paper strips and cotton buds coated with (i) pap-AuNCs, (ii) PLP_pap-AuNCs, and (iii) PLP_pap-AuNCs in the presence of Zn2+ ions

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Table 1 Results of real sample analysis of Zn2+ in tap water and river water
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Conclusions

In summary, the nanocluster emitting red-fluorescent pap-AuNCs was employed for the cascade detection of two important biospecies PLP and Zn2+ in a straightforward and cost-effective manner. The interaction between PLP and papain-functionalized AuNCs leads to a noticeable change in the nanocluster’s emission, which allowed the detection of vitamin B6 cofactor i.e. PLP down to 0.59 µM. The added PLP is expected to form imine linkage with the free –NH2 groups of papain stabilized over pap-AuNCs and form the PLP conjugated nanoclusters PLP_pap-AuNCs. Further addition of Zn2+ ions to the solution of PLP_pap-AuNCs resulted in a significant fluorescent enhancement at 477 nm due to the complexation-induced aggregation of PLP_pap-AuNCs. PLP_pap-AuNCs can be used to detect Zn2+ ions down to 0.14 µM. The Zn2+ ions detection conducted in actual water samples proved the analytical usefulness of PLP_pap-AuNCs.