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Abstract

In this study, a new sensor for the fluorescence detection of guanosine and Mg²⁺ ions using a “turn-off–on” mechanism was successfully constructed using 2-mercapto-5-nitrobenzimidazole (MNB) modified bimetallic molybdenum–gold nanoclusters (Mo-AuNCs). The method has minimal cost, excellent sensitivity, good selectivity, simplicity, and speed. After being excited at 390 nm, the MNB-Mo-AuNCs fluorescence emission peak was obtained at 531 nm. The addition of guanosine selectively reduces the fluorescence intensity of MNB-Mo-AuNCs and the turn-on mechanism recovers the quenched fluorescence in the presence of Mg²⁺. The limits of detection (LODs) for the guanosine and Mg²⁺ ions using this approach were 1.43 and 0.10 µM, respectively. The fluorescence sensing technique based on MNB-Mo-AuNCs demonstrated exceptional performance for guanosine detection in biological samples. The method showed great reproducibility (relative standard deviation < 2%) and recovery ranging from 96.3% to 99.21% in plasma and serum samples indicating the method considerable potential for real-world applications.

Graphical abstract

This is a visual representation of the abstract.

Keywords

2-Mercapto-5-nitrobenzimidazole MNB modified bimetallic molybdenum–gold nanoclusters Mo-AuNC guanosine off–on sensor fluorescence spectroscopy biological samples

Introduction

Purines derived from guanine are conventionally characterized as intracellular controllers in the brain. Guanosine, a purine nucleoside and guanosine triphosphate (GTP) metabolite, has been proposed as a neuromodulator and neuroprotective in the central nervous system.¹ Additionally, guanosine mediates the RNA splicing process and provides protection during brain ischemia.² Increased levels of extracellular guanosine are also linked to post-stroke neuronal survival and rescue.³ Guanosine has garnered significant attention as a molecular marker of central nervous system disorders and oxidative stress, including Parkinson's, Alzheimer's, and Segawa illnesses. Moreover, the chemical has been connected with systemic and airborne metal exposure in welders. Guanosine levels in normal human body fluids are 390 ± 240 µM in urine and 1.10 ± 0.06 µM in blood plasma.⁴

The Mg²⁺ ion is a mineral that is plentiful in the body and can be found in many foods, nutritional supplements and medications including laxatives and antacids.⁵ Additionally, Mg²⁺ is essential for both cell division and cell death. Mg²⁺ plays a key role in the control of hundreds of distinct enzymes and molecules which in turn affects the great majority of metabolic events and everyday bodily functions.⁶ An adult human needs 300 mg of magnesium per day, which can be found in whole grains, seafood, green vegetables, and dairy products.⁷ The human body with an abnormal Mg²⁺ content is linked to a number of illnesses. For instance, a lack of Mg²⁺ ions can cause a number of chronic conditions, including diabetes, hypertension, coronary heart disease, and osteoporosis.⁸ A lack of magnesium in the diet is a contributing factor to a number of illnesses.⁹ Thus, it is crucial to identify and eliminate the polluting Mg²⁺ ions in a practical and effective way.

Numerous methods, such as electrochemical,^4,10 colorimetry,¹¹ high-performance liquid chromatography,¹² voltammetry,¹³ and capillary electrophoresis¹⁴ have been developed for the detection of guanosine and Mg²⁺ ions. However, because of difficulties with sample preparation, time-consuming analysis, expensive reagents, and a complicated mechanism, these approaches are not very applicable for point-of-care evaluation and on-site monitoring. Meanwhile, because of fluorescence's low cost, robustness, convenience of use, quick measurement, high sensitivity, broad detection range, and real-time monitoring, the fluorescence approach provides a straightforward and useful solution for the detection of guanosine and Mg²⁺ ions.^15,16 It is very interesting to use a single chemical entity to determine these two analytes.

In order to monitor target analytes, metal nanoclusters (NCs) with fluorescent features that are shielded by ligands with a large number of binding sites are required due of their molecular-like traits, atomic arrangements, and ultra-small (<3 nm) size.^17–20 Recently, fluorescence sensors based on bimetallic NCs were developed for biomolecule fluorescence detection. In the context of bimetallic NCs, a synergistic effect is the result of combining two atom types with different characteristics, which creates NCs that are both geometrically and electrically stable.²¹ Their enhanced quantum yield, stability, electrical, optical, and catalytic qualities results from synergistic interactions between the two distinct metals.^22,23 The introduction of a secondary metal modifies the electronic density of states, resulting in changes in fluorescence properties and optical band gap.²⁴ This makes them superior than monometallic NCs. Biological applications have made use of light-emitting AuNCs due to their superior stability and biocompatibility. Recent years have also seen the synthesis of bimetallic nanoclusters composed of Au and other noble metals, such as Au/Cu^25,26 where Cu has been introduced into AuNCs, influencing the aggregation-induced emission²⁶ and Au/Au.^27,28 For instance, a red-emitting BSA capped Au/Ag NCs were reported. According to the comparing data with monometallic NCs, the fluorescence intensity may be considerably increased by adding silver to the reaction solution. The developed system used as a probe for the sensing of folic acid with a limit of detection (LOD) of 0.47 nM.²⁷ The most common bimetallic NCs are composed of Ag/Au because they have molecular like behavior since their size is comparable to an electron Fermi wavelength.^28–33 From these studies it can be inferred that incorporation of Ag enhances the fluorescence intensity of NCs. Aside from this, there has not been much research done on Au/Mo bimetallic NCs. In this context, lysozyme-capped Au-MoNCs for bilirubin detection have been documented up to this point.³⁴ When excited at 490 nm, the Au-MoNCs exhibited an emission of 642 nm. Further the synthesized Au-MoNCs were applied as a probe for the selective sensing of bilirubin in the urine samples of jaundice patients.

Motivated by these intriguing studies, the current work is based on the MNB capped synthesis of bimetallic Mo-AuNCs to address the sensitive detection of guanosine and Mg²⁺ ion by a “turn-off–on” mechanism. When excited at 390 nm, the MNB-Mo-AuNCs showed a significant emission at 531 nm. As shown in Scheme 1, guanosine significantly lowers the fluorescence intensity of MNB-Mo-AuNCs. Additionally, the fluorescence intensity of MNB-Mo-AuNCs is recovered upon the addition of Mg²⁺ ion which may be the consequence of stable complexation products between guanosine and Mg²⁺ ion. This causes guanosine to separate from the MNB-Mo-AuNCs surface, restoring the fluorescence intensity. The suggested approach has demonstrated the practical applicability by proving to be a suitable platform for guanosine detection in human serum and plasma samples.

Scheme 1.

Illustration for fabrication of bimetallic MNB-Mo-AuNCs as a nanosensor for guanosine and Mg²⁺ ion detection by turn “off–on” sensing.

Table I.

Comparison between the fluorescence method based on MNB-Mo-AuNCs and the previously published guanosine detection techniques.

Analytical method	Material	Linear range (μM)	LOD (μM)	Reference
Electrochemical sensor	Carbon fiber microelectrode	67–1000	2	4
Fast-scan cyclic voltammetry	Carbon fiber microelectrode	–	0.03 ± 0.01	1
Electrochemical sensor	Gold nanoparticles, polypyrrole, and graphitic carbon nitride (Au-PPy@g-C₃N₄)	0.18–706.11	0.09	47
Fluorescence sensor	Citrate/Tb	0.28–37.67	0.18	48
Electrochemical sensor	Au nanocages	0.3–600	0.1	49
Electrochemical sensor	Poly(3-aminophenylboronic acid)	1–120 μM	0.14	50
Fluorescence sensor	MNB-Mo-AuNCs	5–10	1.43	Present method

Table II.

Comparison between the fluorescence method based on MNB-Mo-AuNCs and the previously published methods for detecting Mg²⁺.

Analytical method	Material	Linear range (μM)	LOD (μM)	Reference
Colorimetry and fluorescence sensor	1,10-phenanthroline and 8- hydroxyquinoline, 5-(1H-imidazole [4,5-f] [1,10]phenanthrolin-2-yl)quinolin-8-ol (PHQ)	–	0.03	5
Fluorescence sensor	N-doped carbon dots	–	60	51
Fluorescence sensor	8-hydroxyquinoline	0–5	0.06	52
Colorimetry sensor	L-tryptophan functionalized AgNPs	1–200	3	53
Colorimetry sensor	Tryptophan-functionalized AuNPs	1–40	0.2	11
Fluorescence sensor	8-hydroxyquinoline-5-benzothiazole	0–100	0.439	54
Fluorescence sensor	MNB-Mo-AuNCs	0.125–5	0.10	Present method

Experimental

Materials and Methods

MoCl₅ (≥99% purity) was purchased from TCI Chemicals. Gold chloride (HAuCl₄·xH₂O) of purity 99.9% was purchased from Sigma-Aldrich. MNB (97% purity) used in this research was procured from Alfa Aesar chemicals. The biomarkers used such as sodium selenite (≥98% purity), Se–methylselenocysteine (≥95% purity), creatinine (≥98% purity), and methyl nicotinate (≥99% purity) were procured from Sigma Aldrich. Other biomarkers like dehydroepiandrosterone (>99% purity), cortisone (>97% purity), hypoxanthine (99% purity), and serotonin hydrochloride (95% purity) were purchased from TCI Chemicals (India), and GLR Innovations. A Milli-Q water purification system for the purification of water used in the whole experiment. All chemicals and solvents used in the whole analysis were of analytical grade.

Instrumentation

Absorbance and fluorescence techniques were performed by employing a Maya Pro 2000 spectrophotometer from Ocean Optics, and an Agilent Technologies Cary Eclipse fluorescent spectrometer. The Fourier transform infrared (FT-IR) study of Mo-CuNCs was obtained using a Bruker Alpha II FT-IR (Germany). Using high-resolution transmission electron microscopy (HR-TEM; JEM 2100, JEOL, Japan), the size and structure of the MNB-Mo-AuNCs were observed. The surface composition of MNB-Mo-AuNCs was recorded using X-ray spectrometry (XPS), K-alpha + from Thermo Fisher Scientific (USA). An X-ray diffraction (XRD) analyzer (Bruker-AXS New D8-Advance) was used for the identification of crystal structure. The zeta potential and hydrodynamic diameter of Mo-CuNCs were determined by dynamic light scattering spectrometry (DLS) (Horiba, SZ-100).

Synthesis Procedure of MNB-Mo-AuNCs

The synthesis of MNB-Mo-AuNCs was achieved by using MoCl₅ and HAuCl₄ as a metal precursor and MNB as a ligand. Initially, 5.0 mL of 15.0 mM MoCl₅ was added to 5.0 mL of 5 mM of HAuCl₄ and stirred the mixture for 2 min. Further, 5.0 mL of MNB (30 mM) was added to this solution followed by the introduction of 0.5 mL NaOH (1.0 M). The obtained solution was stirred for 1 h at room temperature followed by microwave for 5 min at 180 W. The resulting pale yellow solution exhibits yellow fluorescence at 365 nm under ultraviolet (UV) light, confirming the development of MNB-Mo-AuNCs. The obtained MNB-Mo-AuNCs were kept at 4 °C for future use. The synthesis of MNB–Mo–AuNCs has been proposed to occur through the coordination of Au³⁺ and Mo⁵⁺ ions with the thiol and benzimidazole nitrogen donor sites of MNB, subsequently followed by alkaline reduction and formation under microwave condition. Similarly, ligand-mediated, rapid, simple bimetallic NC synthesis have been demonstrated for Au/Ag system.³⁵

Quantum Yield (QY) Calculation

The QY of MNB-Mo-AuNCs was calculated using reference as rhodamine B employing the formula:

Q Y_{N C s} = Q Y_{R} \times (I_{N C s} / I_{R}) \times (η_{N C s}^{2} / η_{R}^{2}) \times (A_{R} / A_{N C s})

(1)where QY_NCs = QY (MNB-Mo-AuNCs), QY_R = QY (reference) (QY = 0.31), I_NCs = fluorescence emission area of MNB-Mo-AuNCs (30163.651), I_R = fluorescence emission area of reference (1088.780), A_NCs = absorbance (MNB-Mo-AuNCs) (324.75), A_R = absorbance (reference) (552.35), η_NCs² = refractive index of MNB-Mo-AuNCs, and η_R² = refractive index of water for reference. Therefore, the obtained QY of NCs obtained was 14.60%.

Detection of Guanosine and Mg²⁺ Ion Using MNB-Mo-AuNCs as a Sensor

Detection of guanosine and Mg²⁺ ion by “turn-off–on” was carried out with MNB-Mo-AuNCs as a fluorescence sensor. To detect guanosine, 0.5 mL (1 mM) of guanosine was mixed with 1 mL of MNB-Mo-AuNCs solution. The emission spectra of MNB-Mo-AuNCs at 531 nm demonstrated that guanosine successfully reduced the fluorescence intensity of MNB-Mo-AuNCs. Additionally, MNB-Mo-AuNCs–guanosine was mixed with 0.5 mL of Mg²⁺ (1 mM) to restore MNB-Mo-AuNCs fluorescence. After that, the mixture was vortexed for around 120 s. Notably, the addition of Mg²⁺ significantly restored the quenched emission intensity of MNB-Mo-AuNCs by guanosine at λ_Ex 531 nm, indicating that MNB-Mo-AuNCs can be used as a nanosensor for the parallel recognition of Mg²⁺ and guanosine using a turn-off–on fluorescence system.

Optimization and Characterization of MNB-Mo-AuNCs

During the synthesis of NCs, MNB acts as a reducing agent since it contains NH and thiol groups. Thus, MNB was selected as a reducing and capping agent for this work. The MNB was used as a template to create water-soluble luminous bimetallic MNB-Mo-AuNCs (Scheme 1). Initially, monometallic MNB-MoNCs and MNB-AuNCs were synthesized and Figure S1a (Supplemental Material) shows the corresponding emission wavelength spectra. From the spectra it can be observed that MNB-Mo-AuNCs showed highest emission intensity at 531 nm when excited at 390 nm. Compared to monometallic MNB-MoNCs and MNB-AuNCs, bimetallic MNB-Mo-AuNCs exhibited noticeably stronger yellow emission intensity under UV light. Further, Figure S1b (Supplemental Material) displays the emission wavelength spectra of various molar ratios of Mo⁵⁺ and Au³⁺ ions (1:1, 1:2, 1:3, 1:4, 2:1, 3:1, and 4:1, mM:mM). Only after employing the 3:1 ratio of Mo⁵⁺ to Au³⁺ ions, MNB-Mo-AuNCs exhibit a noticeable emission peak at 531 nm (Figure S1b, Supplemental Material). The emergence of two centers of emission in the MNB-Mo-AuNCs spectra was maybe because of the varying wavelength (and quanta) of incident photons may have enough energy to get absorbed and often generate transitions from another internuclear separation distance and vibrational energy level.³⁶ These studies confirmed that at a molar ratio of 3:1, Mo⁵⁺: Au³⁺ ions exhibit the maximum emission intensity.

Furthermore, the emission and absorption spectra of MNB-Mo-AuNCs as well as with the spectra of the precursors used (MNB, MoCl₅, and HAuCl₄) shown in Figure S2 (Supplemental Material). The as-synthesized MNB-Mo-AuNCs showed an absorption band at 325 nm. When compared to the used precursors, the bimetallic Mo-AuNCs clearly showed different absorption characteristics (Figure S2a, Supplemental Material). The synthesis of MNB-Mo-AuNCs was further demonstrated by the fact that only MNB-Mo-AuNCs displayed a noticeable fluorescence peak at 531 nm with a higher emission intensity compared to other solutions (MNB, MoCl₅, and HAuCl₄) as displayed in Figure S2b (Supplemental Material). Also, a weak blue fluorescence peak observed around 450 nm could be attributed to the emission of MNB, as evidenced by the emission spectra of pure MNB in Figure S2b (Supplemental Material). The peak around 490 nm was maybe due to the presence of Mo metal in the solution which can be evidenced by the emission spectra of MNB-MoNCs in Figure S1a (Supplemental Material). This peak maybe the resultant of ligand to metal charge transfer (LMCT) which arises from transfer of charges from ligand to metal.³⁷ The dominant emission at 531 nm was due to the Au metal as can be explained by the fluorescence spectra of MNB-AuNCs in Figure S1a (Supplemental Material). This can also be explained by the energy transfer phenomena from the higher energy ligand state to lower energy metal centered or ligand to metal–metal charge transfer (LMMCT).³⁸ Additionally, by examining the emission spectra of Mo-AuNCs, the impact of MNB concentration (5 mM–60 mM) on the synthesis of MNB-Mo-AuNCs was assessed (Figure S3a, Supplemental Material). The concentration of 30 mM MNB was found to have the maximum emission wavelength intensity of Mo-AuNCs, suggesting that this is the ideal concentration for Mo-AuNCs synthesis. The emission intensity of MNB-Mo-AuNCs was measured using a microwave at 180 W at various reaction intervals (1, 2, 3, 4, 5, and 6 min) in order to get the optimal reaction time (Figure S3b, Supplemental Material). These spectral data demonstrated that 5 min was the ideal duration for the fabrication of MNB-Mo-AuNCs. The synthesized NCs when excited at 390 nm, the optimum emission intensity was observed at 531 nm, whereas the absorption band was noticed at 325 nm (Figure 1). The as-synthesized MNB-Mo-AuNCs show strong yellow fluorescence under UV light at 365 nm, appearing pale yellow in the daylight (Figure 1). MNB-Mo-AuNCs were excited at several wavelengths ranging from 370 to 420 nm to investigate their emission properties and identify the maximum emission wavelength (Figure S4, Supplemental Material). The maximum emission intensity was found to occur at λ_Em 531 nm with excitation at λ_Ex 390 nm. The quantum yield (QY) of bimetallic MNB-Mo-AuNCs obtained was 14.60% as calculated by using rhodamine B as the reference chemical.

Figure 1.

The MNB-Mo-AuNCs excitation, emission, and absorbance spectra.

The FT-IR properties of MNB and MNB-Mo-AuNCs were examined in order to identify the functional groups that are present on their surface (Figure S5, Supplemental Material). The characteristic absorption peak of N–H stretching at 3128 cm^–1 of MNB shifted to the broad band at 3449 cm^–1 representing –OH/N–H stretching in MNB-Mo-AuNCs. The band corresponds to –NH bending at 1624 cm^–1 in MNB shifted to 1632 cm^–1 with a decrease in intensity in MNB-Mo-AuNCs. Moreover, there was a significant change in the spectra of MNB-Mo-AuNCs with two peaks at 1395 cm^–1 and 1337 cm^–1 as compared to MNB displaying –OH bending. The decrease and shifting in the absorption peaks of MNB-Mo-AuNCs to 1523 cm^–1 and 1339 cm^–1 corresponds to N–O stretching and C–H bending. The characteristic band corresponds to thiol (–SH) at 2554 cm^–1 was not observed in the formed MNB-Mo-AuNCs. These results indicate the successful formation of MNB-Mo-AuNCs.

The size, form, and fluorescence lifespan of MNB-Mo-AuNCs were described using the HR-TEM and fluorescence methods. The spherical shape and uniform distribution of the as-prepared MNB-Mo-AuNCs with a mean size of 2.72 ± 0.41 nm was demonstrated in Figures 2a–d. The MNB-Mo-AuNCs crystal structure is revealed by the X-ray diffraction (XRD) spectrum (Figure S6, Supplemental Material). The 110 plane of Mo is represented by the diffraction peaks (2θ) at 31.7°. The peak observed at 45.4° corresponds to the 200 plane of Au. Other diffraction peaks (2θ) at 24.4°, 25.7°, 27.3° and 38.4° corresponds to the 011, 040, 021 planes of Mo and 111 plane of Au.^39–41 The crystalline character of MNB-Mo-AuNCs is evident from the presence of the peak diffraction at the 45.4° (111) plane of Au and the 31.7° (110) plane of Mo.³⁴ The fluorescence lifetime was studied to instigate the MNB-Mo-AuNCs decay time revealing the lifetime of 2.67 ns (Figure S7, Supplemental Material). The zeta potential was used to calculate the surface charge of the MNB-Mo-AuNCs (Figure S8a, Supplemental Material). The resultant bimetallic NCs showed a charge of −30.62 mV, demonstrating the stability of MNB-Mo-AuNCs. Further, X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental configuration of MNB-Mo-AuNCs as illustrated in Figure S9 (Supplemental Material). The XPS survey spectrum verified the presence of Au 4f, Mo 3d, S 2p, C 1 s, N 1 s, and O 1 s in MNB-Mo-AuNCs (Figure S9a, Supplemental Material). The deconvoluted Mo 3d core level spectra included notable peaks at 233.2 (Mo⁴⁺ 3d_3/2) and 236.3 eV that were attributed to MoO₃ (Figure S9b, Supplemental Material).⁴² It was found that Au 4f_5/2 and Au 4f_7/2 have binding energies of 87.3 and 83.7 eV, respectively (Figure S9c, Supplemental Material), and this demonstrates that stable MNB-Mo-AuNCs are formed, with the majority of gold atoms at an Au⁰ oxidation state.⁴³ Concurrently, Figure S9d (Supplemental Material) shows two peaks in the C 1 s band at 235.2 and 288.3 eV, which are ascribed to the different C = O, C–N, and C–S carbon state compositions. Figure S9e (Supplemental Material) displays the S 2p peak fitting results. A peak that appears at 166.7 eV indicates that the surface of bimetallic Mo-CuNCs has a thiol group bond. A distinctive peak at 402.1 eV can be seen in the high-resolution image of the N 1 s (Figure S9f, Supplemental Material), which may be caused by the C–NH–C group.⁴⁴ As shown in Figure S9 g (Supplemental Material), the oxygen spectra was separated into two peaks with the binding energies of 531.9 and 534.0 eV, which represent the bond lengths of Mo–O and oxygen in the adsorbed water composition.⁴⁵ The emission spectra at various time intervals were examined in order to verify the stability of MNB-Mo-AuNCs (Figure S10, Supplemental Material). These findings show that, up until 60 days, there was not much change in the emission wavelength intensity of MNB-Mo-AuNCs; following that, there was a minor decrease. This indicates that 60 days stability of the formed MNB-Mo-AuNCs.

Figure 2.

(a) HR-TEM of MNB-Mo-AuNCs at 20 nm scale bar, (b) at 10 nm scale bar, (c) at 5 nm scale bar. (d) Histogram showing MNB-Mo-AuNCs average size. (e) MNB-Mo-AuNCs–guanosine HR-TEM at 10 nm scale bar. An inset histogram showing average size of complex MNB-Mo-AuNCs–guanosine. (f) HR-TEM of MNB-Mo-AuNCs–guanosine after the addition of Mg²⁺ ion. Following the addition of the Mg²⁺ ion, the mean size of MNB-Mo-AuNCs–guanosine is shown in the inset histogram.

Fluorescence “Turn-Off–On” Response and Detection Mechanism of MNB-Mo-AuNCs Toward Guanosine and Mg²⁺

The detection ability of MNB-Mo-AuNCs was investigated by taking several biomarkers (hypoxanthine, creatinine, Se–methylselenocysteine, serotonin, dehydroepiandrosterone, cortisone, selenite methyl nicotinate and guanosine, 1 mM) of volume 0.5 mL each which was added separately to 1 mL of NCs. The obtained mixtures were vortexed for around 120 s at room temperature and their fluorescent emission spectra are displayed in Figure 3a. Based on these fluorescent responses, it can be concluded that MNB-Mo-AuNCs are selective for guanosine because no appreciable fluorescence quenching was observed when additional bioactive molecules were added. Further, as shown in Figure 3b different 0.5 mL metal cations (Mn²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Zn²⁺, Al³⁺ and Mg²⁺, 1 mM) were added separately to the 1.5 mL of MNB-Mo-AuNCs–guanosine in order to restore the emission of MNB-Mo-AuNCs. The above solutions were vortexed for around 120 s at room temperature. The addition of Mg²⁺ significantly increased the fluorescence intensity of MNB-Mo-AuNCs at 531 nm, suggesting that MNB-Mo-AuNCs might be used as a fluorescence “turn-off–on” sensor to detect Mg²⁺ and guanosine. Further, the UV–visible (UV–Vis) study of MNB-Mo-AuNCs was performed with all the interferents taken as shown in Figure 3c, demonstrating selectivity of MNB-Mo-AuNCs towards guanosine.

Figure 3.

MNB-Mo-AuNCs emission spectrum data with (a) several biomarkers (hypoxanthine, creatinine, Se–methylselenocysteine, serotonin, dehydroepiandrosterone, cortisone, methyl nicotinate, selenite, and guanosine, 1 mM). Inset: image showing the addition of various biomarkers effect on the fluorescence intensity in MNB-Mo-AuNCs: (1) MNB-Mo-AuNCs, (2) hypoxanthine, (3) creatinine, (4) Se–methylselenocysteine, (5) serotonin, (6) dehydroepiandrosterone, (7) cortisone, (8) methyl nicotinate, (9) selenite, and (10) guanosine under UV light. (b) MNB-Mo-AuNCs emission spectrum data with the addition of different metal cations (Mn²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Zn²⁺, Al³⁺ and Mg²⁺, 1 mM). Inset: Image showing the addition of various metal cations effect on the fluorescence intensity in MNB-Mo-AuNCs– guanosine (1) MNB-Mo-AuNCs, (2) Mn²⁺, (3) Ca²⁺, (4) Sr²⁺, (5) Ba²⁺, (6) Zn²⁺, (7) Al³⁺, and (8) Mg²⁺ under UV light. (c) UV–visual study of MNB-Mo-AuNCs with guanosine, hypoxanthine, creatinine, Se–methylselenocysteine, serotonin, dehydroepiandrosterone, cortisone, methyl nicotinate, and selenite 1 mM.

The fluorescent data, zeta potential, fluorescence lifespan, HR-TEM, and FT-IR of MNB-Mo-AuNCs were examined with and without guanosine and Mg²⁺. This was done to hypothesize the cause for the fluorescent quenching of MNB-Mo-AuNCs by guanosine and the subsequent restoration of quenched fluorescent with Mg²⁺. The MNB ligand serve as surface functionality on NCs (via Au–S and Mo–N bonds), facilitating sites of interaction for guanosine via hydrogen bonding, resulting in NCs aggregation. The “turn-off” detection of guanosine using MNB-Mo-AuNCs maybe assisted by inner filter effect (IFE). As the absorption maxima of guanosine at 321 nm is completely overlapped with the excitation spectra of MNB-Mo-AuNCs (Figure S11, Supplemental Material). The HR-TEM image shows that the addition of guanosine causes MNB-Mo-AuNCs to aggregate. The HR-TEM showed that the average size of MNB-Mo-AuNCs had increased to 10.14 ± 1.06 nm (Figure 2e), verifying that guanosine interacts with MNB-Mo-AuNCs. The dynamic light scattering (DLS) display the increase in the hydrodynamic diameter of MNB-Mo-AuNCs from 4.97 nm to 16.84 nm. This study also confirms the aggregates formation after the introduction of guanosine to the NCs (Figure S12b, Supplemental Material). The lifetime of MNB-Mo-AuNCs was decreased to 0.17 ns as soon as guanosine was added, indicating that guanosine was detected by dynamic quenching (Figure S7, Supplemental Material). FT-IR and zeta potential were performed to support the suggested binding between guanosine and MNB-Mo-AuNCs. The characteristic OH stretching peak was observed at 3141 cm^–1 in FT-IR spectra of guanosine. The narrow peak at 1692 cm^–1 representing C = N stretching/NH bending in guanosine is shifted to 1634 cm^–1 in the absorption band of MNB-Mo-AuNCs–guanosine. The FT-IR spectrum of MNB-Mo-AuNCs-guanosine shows the shift in the characteristic band of –OH/–NH stretching to 3460 cm^–1. The disappearance of two peaks (–NO stretching and C–H bending) with the appearance of one narrow peak of OH bending in MNB-Mo-AuNCs–guanosine, confirms the detection of guanosine (Figure S13, Supplemental Material). The formed NCs exhibited a highly negative zeta potential of −30.62 mV which is due to the presence of nitro and thiolates groups on its surface. As depicted in Figure S8b (Supplemental Material), guanosine alone displays a zeta potential of −16.59 mV showing its slightly negative nature. Moreover, after the addition of guanosine, the zeta potential of MNB-Mo-AuNCs shifted to −17.98 mV (Figure S8c, Supplemental Material), signifying decrease in the surface negativity. This shift to less negative value maybe due to the guanosine adsorption onto the MNB-Mo-AuNCs surface through electrostatic interaction and hydrogen bonding between hydroxyl, amino groups of guanosine and nitro group of MNB-Mo-AuNCs. Partial neutralization of surface charge due to surface adsorption of guanosine confirms the interaction of guanosine with MNB-Mo-AuNCs.

The zeta potential of Mg²⁺ alone is 7.30 mV indicating the positively charged surface. However, the introduction of Mg²⁺ to the formed MNB-Mo-AuNCs leads to the reduction in the zeta potential of MNB-Mo-AuNCs to −8.39 mV (Figure S8d,e, Supplemental Material). Interestingly, the addition of Mg²⁺ effectively increases the zeta potential of MNB-Mo-AuNCs–guanosine to −4.76 mV (Figure S8f, Supplemental Material). This implies the effective interaction of Mg²⁺ with the probe. HR-TEM, FT-IR, fluorescence lifespan, and zeta potential were used to examine the mechanism behind the restoration of bimetallic fluorescence both with and without Mg²⁺. The average size of 3.46 ± 0.36 nm was maintained by dispersing the aggregated MNB-Mo-AuNCs–guanosine system following the addition of Mg²⁺ (Figure 2f). This is confirmed by the DLS of MNB-Mo-AuNCs–guanosine that when Mg²⁺ was added to the above system the hydrodynamic diameter was decreased to 6.34 nm (Figure S12c, Supplemental Material). After adding Mg²⁺, the lifespan of MNB-Mo-AuNCs–guanosine was miraculously restored to 2.14 ns (Figure S7, Supplemental Material). These findings demonstrated that when Mg²⁺ was added, guanosine could be separated from the MNB-Mo-AuNCs surface, confirming the formation of a new guanosine–Mg²⁺ complex that supports the retaining of the MNB-Mo-AuNCs dispersion state. The tri-coordinated complex is the most stable complex for Mg²⁺ and guanosine, which supports the aforementioned observations.⁴⁶ A nucleoside hydrogen bonding may be significantly impacted or broken by metal–cation interaction, leading to conformational changes and the development of this tri-coordinated complex.⁴⁶ Additionally, Figure S13 (Supplemental Material) displays the FT-IR data of MNB-Mo-AuNCs–guanosine after addition of Mg²⁺. The shift of –OH/–NH stretching band to 3419 cm^–1 and absence of two peaks (–NO stretching and C–H bending) with the emergence of one –OH bending band in MNB-Mo-AuNCs–guanosine–Mg²⁺ indicates the sensing of Mg²⁺.

Sensitivity Study of MNB-Mo-AuNCs

With the introduction of guanosine (5–1000 µM), the emission spectra of MNB-Mo-AuNCs were recorded in order to examine the sensitivity of MNB-Mo-AuNCs. It was found that the emission intensity of the MNB-Mo-AuNCs gradually decreased as the guanosine concentration increased from 5 µM to 1000 µM (Figure 4a). Thus, as illustrated in Figure 4b, a good linearity between the emission intensity and guanosine concentration from 5 to 10 µM was discovered (regression equation y = 0.065x + 0.768) (R² = 0.997). The LOD is determined to be 1.43 µM using 3 s/slope, where “s” is the blank standard deviation. Further, after adding different concentrations of Mg²⁺ (0.125–1000 µM), the emission intensity of MNB-Mo-AuNCs–guanosine was examined under similar conditions (Figure 4c). Figure 4d illustrates the emission intensity (MNB-Mo-AuNCs–guanosine) at 531 nm increases linearly with increasing Mg²⁺ concentrations in the 0.125–5 µM concentration range. The LOD of 0.10 µM for Mg²⁺ was obtained with the correlation coefficient (R²) as R² = 0.991. As indicated in Tables I and II listed below, the analytical performance of the MNB-Mo-AuNCs based fluorescent sensor is contrasted with the reported approaches^47–54 for guanosine and Mg²⁺ detection. The method offers one step synthesis of MNB-Mo-AuNCs instead of the materials which require multiple steps or having tedious sample preparation. The synthesis requires shorter reaction time than the materials proposed previously. Furthermore, the approach exhibits lower sensitivity for Mg²⁺ detection than the reported methods.

Figure 4.

(a) changes in the emission intensity of MNB-Mo-AuNCs with the increasing guanosine concentrations ranging from 5 to 1000 µM. (b) Curve plotted between I₀/I ratio of MNB-Mo-AuNCs at 390 nm with log of concentration of guanosine increasing in range of 5–1000 µM. Inset of calibration graph plotted between I₀/I ratio of MNB-Mo-AuNCs and concentration of guanosine (5–10 µM). (c) Variations in the emission spectra of MNB-Mo-AuNCs–guanosine complex at various Mg²⁺ concentrations (0.125–1000 µM). (d) Curve plotted between I/I₀ ratio of MNB-Mo-AuNCs–guanosine at 390 nm with concentration of Mg²⁺ increasing in range of 0.125–1000 µM. Inset of calibration graph plotted between I/I₀ ratio of MNB-Mo-AuNCs and log of concentration of Mg²⁺ (0.125–5 µM).

Selectivity Study of MNB-Mo-AuNCs for Guanosine and Mg²⁺

The fluorescence emission of MNB-Mo-AuNCs was investigated in relation to a number of chemically interfering agents such as inosine, cytidine, and adenosine monophosphate (1 mM), a mixture of cations (Na⁺, K⁺, Ca²⁺, Cu²⁺, Mn²⁺, and Fe³⁺, 1 mM), and anions (PO₄^3–, Br^–, SO₄^2–, I^–, Cl^–, CH₃COO^–, and NO₃^–, 1 mM). The fluorescence intensity of MNB-Mo-AuNCs does not change even after the intervening species is introduced as shown in Figure S14a (Supplemental Material). Furthermore, the emission intensity of MNB-Mo-AuNCs sharply declined when guanosine was added with the stated interferents (Figure S14a, Supplemental Material). Nevertheless, it was recovered by introducing Mg²⁺ (Figure S14b, Supplemental Material) demonstrating the MNB-Mo-AuNCs probe selectivity for guanosine and Mg²⁺ recognition via a turn “off–on” mechanism, respectively. Moreover, selectivity of MNB-Mo-AuNCs towards various metal ions (K⁺, Cd²⁺, Ni²⁺, Mn²⁺, Hg²⁺, Pb²⁺, Co²⁺, Cu²⁺, and Fe³⁺, 1 mM) was also studied (Figure S15a, Supplemental Material), showed not much change in the fluorescence spectra of MNB-Mo-AuNCs even after introduction of these interferents. This data confirms the selective nature of MNB-Mo-AuNCs. Also, as shown in Figure S15b (Supplemental Material) UV–Vis spectra reveal no increase in wavelength and no change in peak position, indicating that Cu²⁺and Fe³⁺ ions do not cause the probe to aggregate. These findings support that Cu²⁺and Fe³⁺ ions do not interact with the MNB-Mo-AuNCs.

pH Study and Detection of Guanosine in Real Samples

The emission spectra of MNB-Mo-AuNCs were examined at different phosphate-buffered saline (PBS) pHs ranging from 2.0 to 12.0 without guanosine and Mg²⁺ in order to study the influence of pH (Figure S16a, Supplemental Material). It was observed that the pH of PBS had no effect on the emission intensity of MNB-Mo-AuNCs. Correspondingly, MNB-Mo-AuNCs emission intensity was quenched at all pH levels when guanosine was added to them at PBS pH (2.0–12.0) (Figure S16b, Supplemental Material). Even without pH adjustment using PBS, the emission intensity of MNB-Mo-AuNCs was significantly quenched by guanosine, indicating MNB-Mo-AuNCs serves as a biosensor for guanosine detection. The fluorescent intensity of MNB-Mo-AuNCs at 531 nm was dramatically recovered at all pHs from 2.0 to 12.0 upon adding Mg²⁺ to the solutions that had previously been quenched by guanosine (Figure S16c, Supplemental Material), demonstrating the “turn-off–on” capability for guanosine and Mg²⁺. According to these findings, MNB-Mo-AuNCs were used as a fluorescent probe in “turn-off–on” analysis of guanosine and Mg²⁺ without the addition of PBS pH.

The MNB-Mo-AuNCs were used to quantitatively detect guanosine by spiking different concentrations (5, 6, and 7 μM) into a healthy person serum and plasma in order to show their practical applicability (Table S1, Supplemental Material). The serum and urine samples were collected from the local lab. The samples were diluted 100-fold and further spiked with different three concentrations of guanosine (5, 6, and 7 μM). The amount of guanosine was examined by the above method and showed great recovery in the range of 96.30–99.21% with RSD < 2% in plasma samples and human serum, indicating that it could be used as a fluorescence material to detect guanosine in actual biological samples.

Conclusion

To summarize, MNB functionalized bimetallic Mo-AuNCs were used to develop a straightforward and sensitive fluorescence sensor for the turn “off–on” detection of guanosine and Mg²⁺. The as synthesized MNB-Mo-AuNCs exhibit yellow fluorescence at a wavelength of 365 nm under UV light with λ_Em at 531 nm at λ_Ex of 390 nm. The current study has uncovered some intriguing aspects of the Mo-AuNCs-analyte interaction phenomenon. The fluorescent emission intensity of MNB-Mo-AuNCs was quenched by guanosine but as soon as Mg²⁺ was introduced to the quenched system recovery in fluorescence was observed. This indicates that the probe can function as an appealing turn-off sensor for guanosine and a turn-on sensor for Mg²⁺. The detection limits obtained were 1.43 µM and 0.10 µM for guanosine and Mg²⁺, respectively. Additionally, this method offers a remarkable analytical framework for guanosine assaying in human plasma and serum samples.

Supplemental Material

sj-doc-1-app-10.1177_27551857261426354 - Supplemental material for Molybdenum–Gold Nanoclusters Using 2-Mercapto-5-nitrobenzimidazole as Ligand for Sensing Guanosine and Mg²⁺ Ions Through a Fluorescence Turn Off–On Mechanism

Supplemental material, sj-doc-1-app-10.1177_27551857261426354 for Molybdenum–Gold Nanoclusters Using 2-Mercapto-5-nitrobenzimidazole as Ligand for Sensing Guanosine and Mg²⁺ Ions Through a Fluorescence Turn Off–On Mechanism by Harshita, Hirakendu Basu, Tae Jung Park and Suresh Kumar Kailasa in Applied Spectroscopy Practica

Footnotes

Declaration of Conflicting Interests

Suresh Kumar Kailasa is a member of the Editorial Advisory Board of Applied Spectroscopy Practica. The authors declared no potential conflicts of interest with respect to the research, authorship, review, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Suresh Kumar Kailasa

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Molybdenum–Gold Nanoclusters Using 2-Mercapto-5-nitrobenzimidazole as Ligand for Sensing Guanosine and Mg 2+ Ions Through a Fluorescence Turn Off–On Mechanism

Abstract

Keywords

Introduction

Experimental

Materials and Methods

Instrumentation

Synthesis Procedure of MNB-Mo-AuNCs

Quantum Yield (QY) Calculation

Detection of Guanosine and Mg2+ Ion Using MNB-Mo-AuNCs as a Sensor

Optimization and Characterization of MNB-Mo-AuNCs

Fluorescence “Turn-Off–On” Response and Detection Mechanism of MNB-Mo-AuNCs Toward Guanosine and Mg2+

Sensitivity Study of MNB-Mo-AuNCs

Selectivity Study of MNB-Mo-AuNCs for Guanosine and Mg2+

pH Study and Detection of Guanosine in Real Samples

Conclusion

Supplemental Material

sj-doc-1-app-10.1177_27551857261426354 - Supplemental material for Molybdenum–Gold Nanoclusters Using 2-Mercapto-5-nitrobenzimidazole as Ligand for Sensing Guanosine and Mg2+ Ions Through a Fluorescence Turn Off–On Mechanism

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

Supplemental Material

References

Supplementary Material

Detection of Guanosine and Mg²⁺ Ion Using MNB-Mo-AuNCs as a Sensor

Fluorescence “Turn-Off–On” Response and Detection Mechanism of MNB-Mo-AuNCs Toward Guanosine and Mg²⁺

Selectivity Study of MNB-Mo-AuNCs for Guanosine and Mg²⁺

sj-doc-1-app-10.1177_27551857261426354 - Supplemental material for Molybdenum–Gold Nanoclusters Using 2-Mercapto-5-nitrobenzimidazole as Ligand for Sensing Guanosine and Mg²⁺ Ions Through a Fluorescence Turn Off–On Mechanism