Dual recognition strategy for ultra-sensitive fluorescent detection of Hg²⁺ at femto-molar level based on aptamer functionalized sulfur quantum dots

2.1 Reagents and apparatus

Sublimated sulfur powder, NaOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxy succinimide (NHS) and Hg²⁺ were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Hydrogen peroxide (H₂O₂, V/V 30%) and other metal ions (Ni²⁺, Mn²⁺, Fe³⁺, Zn²⁺, Co²⁺, Cd²⁺, Pb²⁺, Cr³⁺, Cu²⁺, Al³⁺, Mg²⁺ and Fe²⁺) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Tianjin, China). NH₂-polyethylene glycol-400-NH₂ (NH₂-PEG-400-NH₂) was synthesized by Huawei Ruike Chemical Co., Ltd. (Beijing, China). Dialysis membranes (1000 Da) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All chemicals were of analytical reagent grade. All the solutions used in this experiment were prepared with ultrapure water (18.2 MΩ cm). The aptamer used in this study was obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The base sequence (5́-3́) of the oligonucleotide was 5́–HOOC-(CH₂)₆-TTTTTTTTTTTT-3́.

High-resolution transmission electron microscopy (HR-TEM) images were captured using a JEM-2100F transmission electron microscope at an accelerating voltage of 200 kV (JEOL Ltd, Japan). A Nicolet 5700 Fourier transform infrared (FT-IR) spectrometer (Thermo Electron Corporation, USA) was used to obtain FT-IR spectra. Ultraviolet–visible absorption spectra (UV–vis) were recorded using an A560 ultraviolet–visible spectrometer (AOE Instruments, Shanghai) at a wavelength interval of 2 nm. X-ray photoelectron spectra (XPS) were measured using a Thermo ESCALAB-250 instrument (Thermo Fisher Scientific, USA). The fluorescence emission spectrum was obtained using an F-4700 fluorescence spectrophotometer (HITACHI, Japan). X-ray diffraction spectra (XRD) were measured using a SmartLab3 instrument (Rigaku Corporation, Japan).

2.2 Synthesis of NH₂-SQDs

The NH₂-SQDs was prepared according to a previous report (Wang et al., 2019). Briefly, 3 mL of NH₂-PEG-400-NH₂ and 50 mL of the prepared NaOH solution (0.08 g mL⁻¹) were added to 1.4 g of sublimated sulfur powder and stirred in an oil bath at 70 °C for 24 h. During this period, the color of the solution gradually changed from dark yellow to light yellow, and then 3 mL of H₂O₂ were added to it for etching to prepare NH₂-SQDs with good fluorescence performance. NH₂-SQDs were poured into dialysis membranes (with a molecular weight of 1000 Da) to remove unreacted small molecules, and dialysis was performed for 72 h with a water change every 12 h. Afterward, a light yellow solid was obtained by freeze-drying at −20 °C for 24 h. The synthesized solid NH₂-SQDs were stored at 4 °C until use in subsequent experiments.

2.3 Synthesis of Apt-SQDs

The Apt-SQDs was accomplished using a previously published method (Pasanphan and Chirachanchai, 2008). First, aptamers modified with —COOH (Apt-COOH, 10 mL) were activated by EDC (250 mg) and NHS (120 mg) with stirring for 1.5 h at room temperature to promote a better interaction with NH₂-SQDs. NH₂-SQDs was added and allowed to fully react with the activated —COOH for 2 h at room temperature to obtain Apt-SQDs, which were stored at 4 °C until use in subsequent experiments.

2.4 Fabrication of ultrasensitive fluorescent aptasensors

A series of experiments was designed to explore the sensitivity of the fluorescence sensing platform based on NH₂-SQDs. First, the fluorescence properties of Apt-SQDs were fully measured and compared with those of pure SQDs under the same conditions. Next, different concentrations of Hg²⁺ solutions (10⁻¹⁵ to 10⁻⁷ M) were prepared. Dilute 10 µL of the original Hg²⁺ solution (10⁻³ M) to 100 times (10⁻⁵ M) by adding 990 µL of ultrapure water. According to these steps, the different concentrations of Hg²⁺ that we want could be obtained. But we should ensure that the number of dilutions should be as small as possible to ensure the accuracy of the result. Then, different concentrations of Hg²⁺ solutions were added to the Apt-SQDs in a cuvette, and a fluorescence spectrophotometer was used to record the fluorescence quenching of Apt-SQDs by Hg²⁺.

2.5 Quantum yield (QY) measurement

The QY of SQDs, NH₂-SQDs and Apt-SQDs were determined with reference to quinine sulfate (in 0.1 M H₂SO₄). The calculation of QY is according to the following equation: $$Q=Q_R\frac{I{A}_R}{I_{R}A}{\left(\frac{\eta }{{\eta }_R}\right)}^2$$ where ‘Q’ refers to the quantum yield, ‘I’ and ‘A’ stand for emission intensity and absorbance, respectively. ‘η’ is the refractive index of the solvent. The ‘R’ subscripts represent reference standard (Q_R = 0.54).

2.6 Stern-Volmer plot

For fluorescence quenching analysis, a Stern–Volmer plot (F0/F and [Q] relationship) was used according to the following equation: $$\frac{F_0}{F}=1+Ks\left[Q\right]$$ where F₀ and F are the fluorescence intensities in the absence and presence of quencher, respectively, and [Q] is the quencher concentration. The Stern–Volmer constant, K_SV, provides a direct measure of the quenching efficiencies and is determined from the linear portion of the Stern–Volmer plot.

2.7 Determination of Hg²⁺ in real samples

Water samples were collected from three different areas in Yantai City to evaluate the performance of the proposed fluorescence sensing platform. The collected samples were incubated for 2 h and then filtered through a 0.45 μm membrane to remove large particles of impurities and suspended solids. After treatment, the samples were stored at 4 °C to prevent bacterial growth. After the aforementioned treatment, Hg²⁺ was detected using the ultrasensitive fluorescence sensing platform, and different concentrations of Hg²⁺ were added to the real sample to evaluate the detectability of the fluorescence sensing platform in real samples.

2.8 Preparation of test papers

Because Hg²⁺ exhibits a high level of biological toxicity, an effective and convenient method for monitoring Hg²⁺ concentrations in environmental water samples must be developed. The test paper based on the Apt-SQDs quickly detected trace Hg²⁺ in environmental water samples through colorimetry. According to previous reports (Guo et al., 2020), the filter paper was cut into 1 × 1.5 cm pieces, which were immersed into the Apt-SQD solution. Then, the filter papers were dried at room temperature. Finally, filter papers were treated with different concentrations of Hg²⁺ and reacted for 5 min. The color changes were recorded by irradiation with a 365 nm UV lamp.

3.1 Characterization of NH₂-SQDs and Apt-SQDs

The morphology and particle size distribution of the prepared SQDs, NH₂-SQDs and Apt-SQDs were characterized using HR-TEM. Fig. 1A-C and the insets show the TEM and HR-TEM images of SQDs, NH₂-SQDs and Apt-SQDs, respectively. The morphologies of the three preparations were highly similar and all of them were spherical with good dispersion, suggesting that the original structure of SQDs was not damaged in the process of functionalization. However, in Fig. 1D-F, SQDs had the smallest particle size (∼2.5 nm), followed by NH₂-SQDs (∼4 nm), and Apt-SQDs had the largest particle size (∼6 nm), which may be caused by the gradual increase in SQDs functionalization. Fig. S1 showed the dynamic light scattering (DLS) spectra of SQDs, indicating that the average particle size of prepared SQDs was about 4 nm.

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Fig. 1

TEM images of (A) SQDs, (B) NH₂-SQDs and (C) Apt-SQDs. Insets show HR-TEM images of SQDs, NH₂-SQDs and Apt-SQDs at 5 nm. Histogram showing the size distribution of (D) SQDs, (E) NH₂-SQDs and (F) Apt-SQDs.

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Fig. 2A shows the UV–Vis spectra of SQDs (black curve), NH₂-SQDs (red curve) and Apt-SQDs (blue curve). At 303 nm, all three nanomaterials produced obvious absorption peaks, potentially due to the adsorption of S₂²⁻ on the surface of the SQDs 33. The spectrum (red curve) of NH₂-SQDs was similar to that of SQDs (black curve). However, at approximately 350 nm, NH₂-SQDs produced a small and not obvious absorption peak, which may indicate that other S_x²⁻ molecules were adsorbed on the surface of SQDs during functionalization, similar to the situation reported in the literature ³³. However, Apt-SQDs (blue curve) generated a weak absorption peak at 370 nm due to the weak absorption of the carbonyl group of the amide bond and the conjugation of N, and thus we estimated that aptamer had successfully combined with SQDs to generate Apt-SQDs. And the UV–Vis spectra of aptamer was shown in Fig. S2. An obvious absorption peak was observed at 260 nm, which was characteristic of aptamer. In addition, FT-IR spectra of NH₂-SQDs (black curve) and Apt-SQDs (red curve) were recorded to further confirm the successful preparation of Apt-SQDs, as shown in Fig. 2B. Both NH₂-SQDs and Apt-SQDs produced characteristic peaks at 3400 and 2870 cm⁻¹ of SQDs, similar to previous reports (Wang et al., 2019). However, the two nanomaterials showed N-H stretching vibration absorption peaks and N-H bending vibration peaks at 3300 and 1500 cm⁻¹, respectively, while Apt-SQDs had C⚌O stretching vibration peaks at 1700 cm⁻¹. Therefore, Apt-SQDs was successfully synthesized and were applied in the next experiment. FT-IR spectra were analyzed to further verify our conclusions, as shown in Fig. S3. Compared with Fig. 2B, characteristic peaks were located at 3300, 2870, 1680 and 1500 cm⁻¹, and characteristic peaks of thymine were located at 3509, 1870, 1196 and 549 cm⁻¹, which was also consistent with a previous report (Bader, 1991), proving that the binding mode of Apt-SQDs to Hg²⁺ was mainly attributed to the T-Hg²⁺-T structure. Fig. 2C and 2D further reveal the composition of Apt-SQDs analyzed using the XPS approach. Fig. 2C shows the presence of mainly four different peaks in the spectrum, indicating that Apt-SQDs mainly contain C, O, N and S. The S peak was selected for further study, and the amplified peak is shown in Fig. 2D. As shown in the figure, the S peak was subdivided into five peaks in the high-resolution XPS spectrum at 161.72, 162.86, 166.86, 168.01 and 169.06 eV. The peaks at 161.72 and 162.86 eV were attributed to S, while the peaks at 166.86 and 168.01 eV were attributed to SO₃²⁻ (2p_2/3), and the peak at 169.06 eV was attributed to SO₃²⁻ (2p_1/2). Therefore, abundant groups were present on the surface of Apt-SQDs (Wang et al., 2019). The XRD pattern of SQDs presented many sharp peaks, as is shown in Fig. S4, indicating its better crystalline properties, which is similar to previous reports (Chen et al., 2016). The QY of SQDs and Apt-SQDs were 5.3% and 3.4%, respectively, which proves that functionalization has no great impact on SQDs performance. When Hg²⁺ was present, QY of SQDs and Apt-SQDs decreased to 1.4% and 1.1%, respectively, indicating that the prepared material is responsive to Hg²⁺. The fluorescence lifetime of SQDs and Apt-SQDs were shown in Fig. S5, exhibiting fluorescence lifetime with value of 6.4 ns, which was superior to existing organic fluorophores (generally less than 5 ns). Zeta potential of SQDs, NH₂-SQDs and Apt-SQDs were shown in Fig. S6. It can be seen that the surface of SQDs have many negative groups. Due to –NH₂ is the electron-donating group, NH₂-SQDs has a more negative charge on its surface, so the zeta potential of NH₂-SQDs was more negative than SQDs. As for Apt-SQDs, the aptamer carries more negative charge, which is consistent with the data obtained in Fig. S5 (white column). However, after the addition of Hg²⁺, the HgS and T-Hg²⁺-T structure reduces the negative charge on the surface of SQDs, NH₂-SQDs and Apt-SQDs, so the Zeta potential increases (gray column).

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Fig. 2

(A) UV–Vis spectra of SQDs (black curve), NH₂-SQDs (red curve) and Apt-SQDs (blue curve). (B) FT-IR spectra of NH₂-SQDs (black curve) and Apt-SQDs (red curve). (C) XPS survey spectrum and (D) high-resolution S 2p XPS spectrum of NH₂-SQDs.

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3.2 Quenching mechanism of dual recognition strategy for Hg²⁺

XPS spectra were recorded to further study the modes of NH₂-SQDs and Apt-SQDs binding to Hg²⁺, as shown in Fig. 3. As shown in Fig. 3A-H, the binding modes of NH₂-SQDs and Apt-SQDs with Hg²⁺ were thoroughly studied. The elemental analysis is shown in Fig. 3A and E, and the SQDs contained Hg, S, C, N and O elements, as expected. Fig. 3B-D shows Hg 4f, S 2p and N 1s spectra, respectively. Fig. 3B shows the XPS spectrum of Hg 4f electrons to reveal the binding modes of NH₂-SQDs with Hg²⁺. The peak at 100.9 eV was attributed to HgS. This result indicated that HgS was the main form of Hg binding to NH₂-SQDs. Fig. 3C shows the spectrum of S 2p electrons. The peak at 161.5 eV was also attributed to HgS, which also proved the conclusion listed above that Hg and NH₂-SQDs interacted through HgS. Fig. 3F shows the XPS spectrum of Hg 4f electrons to reveal the binding modes of Apt-SQDs with Hg²⁺. The peak at 100.9 eV in Fig. 3F was much smaller than that in Fig. 3B, which proved that the binding mode of HgS still existed in the interaction of Apt-SQDs and Hg²⁺, but it was not the main mode. A similar conclusion was drawn from the comparison between the data presented in Fig. 3G and Fig. 3C. The comparison between Fig. 3H and Fig. 3D shows that the peak at 403.4 eV shifted to 401.9 eV, suggesting that the binding mode of Apt-SQDs and Hg²⁺ was indeed different from that of NH₂-SQDs and Hg²⁺, which may be caused by the T-Hg²⁺-T structure.

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Fig. 3

XPS results for (A) NH₂-SQDs + Hg²⁺ and high-resolution (B) Hg 4f, (C) S 2p, (D) N 1 s spectra. XPS results for (E) Apt-SQDs + Hg²⁺ and high-resolution (F) Hg 4f, (G) S 2p, (H) N 1 s spectra.

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In order to further proving the quenching mechanism of dual recognition strategy for Hg²⁺, the FT-IR of aptamer was measured in Fig. S7. Comparing with Fig. S3, it can be seen clearly that it also included N-H stretching vibration absorption peaks, N-H bending vibration peaks and C⚌O stretching vibration peaks, indicating the adaptor structure was not destroyed during the process of functionalization. However, when the solution exists Hg²⁺, characteristic peak of aptamer would produce blue shift, attributed to T-Hg²⁺-T structure.

3.3 Optimizations of conditions

Some optimization experiments were performed to explore the optimal experimental conditions. As shown in Fig. S8A, when Hg²⁺ was added, the fluorescence intensity of Apt-SQDs decreased rapidly, and did not change further after 1 min. Based on this result, Apt-SQDs was useful for Hg²⁺ detection, their response speed to Hg²⁺ detection was extremely fast, and the detection was basically completed within 1 min. The inset showed the quenching of fluorescence intensity in the presence of Hg²⁺. Fig. S8B shows the results for the detection of Hg²⁺ with Apt-SQDs at different pH values. The fluorescence of Apt-SQDs showed excellent pH stability, and the fluorescence intensity changed by only 5% in the range of pH 4.0–12.0. When Hg²⁺ was added, SQDs showed a good response in the range of pH 6.0–8.0, and 7.0 was selected as the final pH. We speculate that the mechanism of high pH affecting Apt-SQDs fluorescence may be: when pH is too high, Hg²⁺ generates Hg(OH)₂ under alkaline conditions. Due to its low solubility, there is not enough Hg²⁺ in the solution to react with Apt-SQDs. Fig. S8C shows the fluorescence spectra of SQDs with different concentration (10⁻¹, 1, 2, 4, 6, 8 and 10 mg mL⁻¹). It can be seen that with the increase of SQDs concentration, fluorescence intensity increased first and then decreased, and reached the maximum at 1 mg mL⁻¹. The decrease of fluorescence intensity of SQDs at high concentration may be attributed to the fluorescence quenching caused by particle aggregation. The influence of ion strength on SQDs fluorescence intensity was explored, and the results were shown in Fig. S8D. Different concentrations of NaCl (10⁻⁴, 10⁻³, 10⁻² and 10⁻¹ M) was selected as testing reagent. The result shows that within a certain range of ionic strength, the fluorescence intensity of SQDs almost unchanged.

3.4 Performance of Apt-SQDs for Hg²⁺ detection

Fig. 4A shows the fluorescence spectra recorded after adding Hg²⁺ solutions with different concentrations (10⁻¹⁵ to 10⁻⁷ M) to Apt-SQDs (1 mg mL⁻¹, pH = 7.4 in PBS). The fluorescence signal of Apt-SQDs decreased gradually with the addition of increasing Hg²⁺ concentrations. The quantitative curves for different concentrations of Hg²⁺ were obtained under certain conditions. Corresponding standard curves were prepared according to the degree of fluorescence quenching, as shown in Fig. 4B. When the Hg²⁺ concentration ranged from 10⁻¹⁵ to 10⁻⁷ M, the linear equation was ΔI = 419.8 lgc(Hg²⁺) + 7268 (R² = 0.9956) with a low limit of detection (LOD) of 0.3 fM, indicating the effective detection of Hg²⁺ at ultralow concentrations (ΔI = F₀ - F, where F₀ represents the initial fluorescence intensity and F represents the fluorescence intensity after adding Hg²⁺). In order to further prove the sensitivity of Apt-SQDs to Hg²⁺, the fluorescence spectra for NH₂-SQDs detecting various concentrations of Hg²⁺ was supplemented, as shown in Fig. S9. By comparison with Fig. 4, it can be clearly seen that the sensitivity of Apt-SQDs is better than NH₂-SQDs. Meanwhile, we speculate that Apt-SQDs has better selectivity to Hg²⁺ than NH₂-SQDs. In addition, Table S1 summarizes the performance of several existing Hg²⁺ sensors and reported detection methods in terms of the LOD and detection range. Compared with other methods, the sensor platform had a lower LOD and a wider detection range, which met the increasingly rigorous requirements for real sample detection. Table S2 summarizes the detection methods of Hg²⁺ based on CQDs. It can be seen from the table that our material sensitivity and detection range are far superior to previous reports. To get insight into the fluorescence quenching mechanism involved, the fluorescence quenching data were analyzed by the Stern-Volmer equation and K_SV is the Stern-Volmer constant. As shown in Fig. S10, the Stern-Volmer plot does not fit a conventional linear Stern-Volmer equation, indicating both dynamic and static quenching processes occur in this sensor system.

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Fig. 4

(A) The signal response of the proposed fluorescent aptasensor for detecting various concentrations of Hg²⁺ and the corresponding concentrations of Hg²⁺ (10⁻¹⁵, 10⁻¹⁴, 10⁻¹³, 10⁻¹², 10^-11, 10⁻¹⁰, 10⁻⁹, 10⁻⁸ and 10⁻⁷ M, from top to bottom, n = 8). (B) Linear relationship between the fluorescence intensity and the logarithm of Hg²⁺ concentrations.

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The good selectivity of the sensor platform based on Apt-SQDs was a prerequisite for practical application. The interference of other ions must be eliminated to accurately determine the content of Hg²⁺ in real samples. Fig. 5A shows the response of the sensing platform to Hg²⁺ and other heavy metal ions under the same conditions, and the degree of fluorescence quenching was recorded. With the exception of Hg²⁺, other heavy metal ions had little effect on the fluorescence intensity of the sensing platform, which was mainly attributed to the high selectivity of the T-Hg²⁺-T structure for Hg²⁺. Common anions were further tested for interference with the probe, as shown in Fig. S11, indicating that the prepared probe has good selectivity. Furthermore, reproducibility of the sensing platform was also important. Five groups of Apt-SQDs were prepared under the same conditions to construct the fluorescence sensing platform and test fluorescence performance (Fig. 5B). The fluorescence performance of the fluorescence sensing platform based on the five groups of Apt-SQDs was almost identical, and thus the reproducibility of the sensor platform was good. In addition, good stability was one of the important factors determining the feasibility of this sensing platform. Therefore, within two weeks, the same batch of sensing platforms constructed from Apt-SQDs was tested to determine the fluorescence stability under the same experimental conditions, and the results are shown in Fig. 5C. The experimental results indicated that the performance of the constructed sensing platform was very stable. Even after two weeks of placement, the fluorescence emission intensity of the sensor platform was approximately the same as that recorded at the beginning of the experiment. Without special instructions, all the above experiments were repeated eight times to obtain accurate results, that means, n = 8.

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Fig. 5

(A) Selectivity toward other interfering substances in the presence 1.0 × 10⁻⁸ M Hg²⁺: Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Zn²⁺, Co²⁺, Cd²⁺, Pb²⁺, Cr³⁺, Cu²⁺, Al³⁺, Ni²⁺ and Fe²⁺ (the concentration of each of the aforementioned interferents was 1.0 × 10⁻⁷ M). (B) Reproducibility of the five groups of Apt-SQDs. (C) The fluorescence stability of Apt-SQDs within two weeks (n = 8).

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Although our study can achieve sensitive detection of Hg²⁺, there are still many methods superior to our work due to the limitation of fluorescence detection technology and material (Zhang et al., 2020; Rathnakumar et al., 2021., Liu et al., 2021a,b,c; Hasanjani and Zarei, 2018; Babamiri et al., 2018; Zhang et al., 2015).

3.5 Detection of Hg²⁺ concentrations in real samples

With the development of society, the problem of heavy metal pollution has gradually attracted the attention of the public, especially the prblem of heavy metal pollution in water. Hg²⁺ was well recognized by the sensing platform. We randomly tested the local water source (Yantai City) as real samples. The concentration of Hg²⁺ was calculated from the standard curve, as shown in Table 1. The recovery rate of Hg²⁺ was 87.21–114.1%, and the relative standard deviation (RSD) was 1.1–3.8%. Therefore, the sensing platform had high accuracy, accuracy and selectivity and was useful for the detection of Hg²⁺ in water samples.

3.6 Portable test papers for Hg²⁺ detection

If Hg²⁺ enters the water environment, it will eventually endanger human health. Therefore, a portable and fast method to monitor Hg²⁺ in environmental water samples must be developed. Fig. S12 clearly shows the quenching of the fluorescence of the test papers to different degrees within 1 min in the presence of different concentrations of Hg²⁺. After an incubation with increasing Hg²⁺ concentrations, the degree of fluorescence quenching gradually increased, consistent with the result of Hg²⁺ detection by the fluorescent aptasensor. Based on this result, the test paper had ultrahigh sensitivity for Hg²⁺ detection, which may be a good qualitative method for Hg²⁺ and had potential application in the safety monitoring of Hg²⁺ in environmental water samples.