Characterisation of Ag NPs@N/GQDs
The transmission electron microscopy (TEM) images showed a uniform distribution and relatively consistent size of the synthesised N/GQDs, indicating excellent dispersion (Fig. 2A). The particle size distribution plot in Fig. 2B shows that the synthesised N/GQDs ranged from 3 to 10 nm, with an average size of approximately 7 nm. When Ag NPs were generated by reducing the surface area of the N/GQDs, nucleoshell spherical nanoparticles formed with a core composed of N/GQDs (Fig. 2C). These particles were uniformly dispersed, and their particle size distribution ranged from 10 to 20 nm (Fig. 2D), with an average particle size of approximately 15 nm. The structure of the synthesised Ag NPs@N/GQDs was characterised and analysed, as depicted in Fig. 2E. Therefore, the XRD pattern that the synthesised Ag NPs@N/GQDs exhibit a distinct peak at 2θ=24.6°, which is low and narrow, indicating a significant particle size corresponding to the (002) crystal plane in the graphitic carbon structure[28]. Sharp peaks observed at 2θ=37.9°, 44.1°, 64.6°, and 77.1° correspond to the (111), (200), (220), and (311) crystal faces of Ag NPs respectively[29]. Furthermore, an elemental composition analysis of the Ag NPs@N/GQDs was conducted using the XPS data (Fig. 2F). Peaks C(1s) and N(1s) appear at 284.08 eV and 394.08 eV, respectively, originating from GQDs[30]; whereas the peaks of Ag(3d3/2) and Ag(3d5/2) appeared at 368.08 eV and 374.08 eV, which were both from AgNPs[31]. Therefore, the Ag NPs@N/GQDs were successfully synthesised.
MIP characterisation
The MIP on the probe was characterised using XPS and Fourier-transform infrared (FTIR) spectroscopy. Fig. 3A shows a significant reduction in the C(1s) peak at 284.08 eV, O(1s) at 394.08 eV, and N(1s) peak at 401.08 eV in the MIP after eluting noradrenaline, because it mainly comprises the elements C, H, O, and N. Further characterisation of the MIP was performed using FTIR spectroscopy. As shown in Fig. 2B, absorption peaks corresponding to the amino stretching vibration regions were observed for noradrenaline at 3448 cm–1 and 3418 cm–1,while the characteristic absorption peaks of the benzene ring skeleton were observed at 960 cm–1, 943 cm–1, 638 cm–1, and 599 cm–1. These peaks were also present in Ag NPs@N/GQDs/MIP but disappeared upon the removal of noradrenaline through elution from Ag NPs@N/GQDs/MIP. Therefore, the MIP was successfully synthesised.
Fluorescence properties of Ag NPs@N/GQDs/MIP probes
The optical properties of the fluorescent probes were examined using UV-Vis absorption and fluorescence spectroscopy. As shown in Fig.S1A (Supplementary Material), the Ag NPs@N/GQDs/MIP aptamer exhibited strong absorption at 495 nm. Additionally, Fig.S1B shows that the material has a maximum excitation wavelength near 480 nm (curve a). This wavelength was used to excite the probe, which resulted in a maximum emission wavelength of approximately 533 nm (curve b). The larger Stokes shift effectively prevented overlap between the excitation and emission spectra. Furthermore, the emission spectrum of the probe exhibited high intensity, symmetry, and a narrow peak shape, indicating excellent fluorescence performance. The quantum yield of a fluorescent substance is a crucial parameter that affects fluorescent probes. On the basis of the integrated fluorescence intensity (i.e. the area included in the corrected fluorescence spectrum) of the two dilute solutions of the fluorescence sample to be measured, the reference fluorescence reference material with known quantum yield under the same excitation conditions, and the absorbance of the incident light (UV–visible light) with the same excitation wavelength, the quantum yield of the tested fluorescent specimen can be calculated as[32]
Here, Φu and Φs represent the fluorescence quantum yields of the substance under measurement and the reference standard, respectively; Fu and Fs denote the integrated fluorescence intensities of the substance being measured and the reference substance; Au and As indicate the absorbance of incident light at the excitation wavelengths of the substance under measurement and the reference substance (A=εbc). In this study, Rhodamine B (RhB) solution was utilised as a reference fluorescence standard. According to the literature, RhB has a reported quantum yield of 0.89[33]; the relevant parameters shown in Table S1.
Mechanism of Ag NPs@N/GQDs/MIP probe signal reduction by norepinephrine
With the addition of different concentrations of noradrenaline, the probe captured increasing amounts of noradrenaline using the MIP. As shown in Fig. 5A, the fluorescence intensity of the Ag NPs@N/GQDs/MIP decreased, indicating that noradrenaline effectively suppressed the fluorescence intensity of the probe. When the template molecule norepinephrine was readded, the Ag NPs@N/GQDs/MIP was able to interact with it, leading to fluorescence quenching. We suggest that an electron transfer between the N/GQDs and norepinephrine is responsible for this phenomenon. The mechanism of fluorescence quenching was studied through UV-visible and fluorescence spectroscopy. As shown in Fig. 5B, according to previous reports, the fluorescence quenching mechanism may mainly involve electron or energy transfer from Ag NPs@N/GQDs/MIP to norepinephrine. The maximum norepinephrine adsorption was 453 nm (curve a), which was close to the band gap of the Ag NPs@N/GQDs/MIP at an excitation wavelength of 480 nm (curve b). These results indicate that the electrons in the conduction bands of the Ag NPs@N/GQDs/MIP could be transferred to the lowest unoccupied orbital of norepinephrine. In addition, the propanil absorption peaks exhibited evident red shifts after APTES addition (curve c). This indicates that analytes may act as electron acceptors to trigger a non-radiative decay electron process[34]. The maximum emission of the Ag NPs@N/GQDs/MIP occurred at 533 nm, exhibiting a narrow and symmetrical line width (curve e). There is no spectral overlap between the emission spectrum of the Ag NPs@N/GQDs/MIP and the absorption spectra of norepinephrine. Hence, we do not think that the fluorescence quenching is caused by the energy transfer mechanism.
These results also imply that the electron transfer from the Ag NPs@N/GQDs/MIP to norepinephrine could be the main optosensing turn-off mechanism[35]. During the fluorescence response, the electrons in the N/GQDs were excited by the UV spectrum energy. Subsequently, when the excited electrons returned to their ground state, the N/GQDs emitted fluorescence. Correspondingly, after norepinephrine was added, it was specifically adsorbed onto the imprinting cavities. The Meisenheimer complex was formed through the strong interactions between the –NH2 of the functional monomer APTES and –OH of norepinephrine. Therefore, the electrons of the N/GQDs were transferred to the complex, resulting in fluorescence quenching.
In addition, the fluorescence quenching mechanism can be explained by molecular orbital theory. As shown in Fig. S2A, an electron of the N/GQDs is excited from the valence band (ground state) to the conduction band after accepting the UV photon. Afterward, the excited electron returns to the valence band, and the N/GQDs produce the fluorescence signal. In addition, in the presence of the template norepinephrine, a hydrogen bonding interaction exists between the amino groups of the N/GQDs and norepinephrine. The interaction force is so strong that it can cause the electron to be transferred between the norepinephrine and N/GQDs. The excited electron is able to transition directly into the LUMO of the complex. It would then return to the ground state, generating no fluorescence signals, because the energy level of the complex would be higher than that of the N/GQDs[36]. This explains the phenomenon of fluorescence quenching (Fig. S2B).
Experimental optimisation conditions
The effects of elution time, buffer pH and reaction time on the detection of noradrenaline by the Ag NPs@N/GQDs/MIP aptamer probes were investigated. As shown in Fig. S3A, as the elution time increased, increasing amounts of noradrenaline were was removed from the MIP, the fluorescence intensity of probe was continuously enhanced. After 5 min, the probe fluorescence increased to the maximum and remained unchanged thereafter, indicating that the probe had been completely eluted. As shown in Fig. S3B, the ratio of the decrease in the fluorescence value △IF (△IF=F0–F1, where F0 represents the fluorescence intensity before adding 1.0 nM noradrenaline and F1 represents the fluorescence intensity after) exhibited different changes as the pH increased from 6.4, but reached its maximum at pH=7.4. Therefore, PBS (pH 7.4) was selected as the optimal buffer. Additionally, the effect of the reaction time of noradrenaline with the fluorescent probe on the quenching effect was investigated. As shown in Fig. S3C, as the reaction proceeded, an increasing amount of noradrenaline was adsorbed onto the probe by MIP, and the probe fluorescence intensity continuously decreased until the reaction progressed for a minimum of 5 min and remained constant thereafter. Therefore, a reaction time of 5 min was selected as the optimal reaction time.
Fluorescence response of Ag NPs@N/GQDs/MIP probes to noradrenaline
The fluorescence intensities of the Ag NPs@N/GQDs/MIP probes were measured before and after adding different concentrations of noradrenaline under the optimal experimental conditions. The decrease in the fluorescence intensity value (△IF=F0–F1) was calculated, and a calibration curve was plotted. As shown in Fig. 5A, the fluorescence intensity of the probe increased continuously with increasing noradrenaline concentration.In the range of 0.5 –700 pM, there was a good linear relationship between the logarithm of noradrenaline concentration (ln(c)) and the logarithm of the decrease in fluorescence intensity (ln(△IF)). The linear regression equation was ln(△IF) = 0.505 ln(c) (pM) + 5.27 (Fig. 5B), with an r-value of 0.9991 and a detection limit of 0.154 pM (D.L.= KSb/a, K = 3). This method exhibited a higher sensitivity for detecting noradrenaline than previously reported methods ( Table S2).
Selectivity of Ag NPs@N/GQDs/MIP probes
The selectivity of the Ag NPs@N/GQDs/MIP probes was evaluated by studying their response in the presence of dopamine, epinephrine, catechol, vitamin B6, vitamin B2, chlorpyrifos, and methyl parathion. As shown in Fig. 6, because there are no molecularly imprinted recognition sites, the Ag NPs@N/GQDs/NIP cannot recognize norepinephrine and adsorb it to the surface of the probe, so it does not decrease the fluorescence intensity of the probe. Similarly, the aforementioned interfering substances are not recognized and adsorbed by the NIP, and the fluorescence intensity of probe does not decrease. In contrast, because the molecularly imprinted sites in the Ag NPs@N/GQDs/MIP can identify and adsorb norepinephrine, the fluorescence intensity of the probe is decreased. However, because the interfering substances cannot enter the imprinted holes or be recognized and adsorbed by the MIP, the fluorescence of the probe does not change. This result demonstrates that the probes were able to recognize norepinephrine with excellent specificity.
Reproducibility and stability of Ag NPs@N/GQDs/MIP probes
The reproducibility and stability of the Ag NPs@N/GQDs/MIP probes for noradrenaline detection were investigated. Five Ag NPs@N/GQDs/MIP probes were subjected to the addition of 1.0 nM noradrenaline under identical conditions, and the fluorescence intensities before and after the addition were measured to calculate ΔIF. The results demonstrated a relative standard deviation (RSD) of 2.73% for the ΔIF values from five experiments, indicating excellent reproducibility (Fig. S4). Furthermore, the prepared Ag NPs@N/GQDs/MIP fluorescent probe was exposed to 1.0 nM noradrenaline, and its fluorescence intensity was monitored at intervals of 5 min. As shown in Fig. S5, after a reaction time of 30 min,the fluorescence intensity of the probe reached 92.2% of its intensity after the completion of the reaction (5 min), demonstrating remarkable stability.
Real sample analysis
A fluorescent probe was used to detect the banana samples, and a spiked recovery test was conducted. Table 1 presents the results of the study. The method exhibited recoveries ranging from 93.6% to 112.0%, with an RSD valueless than 5.0%. Therefore, the current fluorescent probe method can be used effectively to detect actual samples and yield satisfactory results.
Table 1. Results of sample assay and recovery analysis.
Samples
|
This method
(nM, n=5)
|
RSD
%
|
Added
(nM)
|
Total found
(nM, n=5)
|
RSD
%
|
Recoveries
%
|
Banana Ⅰ
|
1.85
|
4.97
|
1.50
|
3.27
|
3.44
|
94.7
|
15.00
|
15.89
|
4.90
|
93.6
|
Banana Ⅱ
|
2.83
|
3.76
|
1.00
|
3.95
|
3.76
|
112.0
|
10.00
|
12.32
|
4.32
|
94.9
|