Visible Light-Responsive Sulfone-Based Covalent Organic Framework as Metal-Free Nanoenzyme for Visual Colorimetric Determination of Uranium

Abstract

Covalent organic framework (COF) has been attracting considerable attention as a novel crystalline material owing to its extended π-electron conjugation and excellent spectral behavior. In this study, we present an imine-linked two-dimensional (2D) crystalline sulfone-based covalent organic framework (TAS-COF) synthesized by 2,4,6-triformylphloroglucinol (Tp) and 3,7-diaminodibenzo[b,d]thiophene (DAS) via a Schiff base condensation reaction. The benzothiophene sulfone endows the as-synthesized TAS-COF with excellent oxidase-like activity under visible light irradiation, ascribed to the generation of superoxide radicals (O₂^•−) by photo-generated electron transfer. TAS-COF can efficiently oxidase the colorless substrate 3,3′,5,5′-tetramethylbenzydine (TMB) into blue oxidized TMB (oxTMB) when exposed to visible light, and the presence of uranium (UO₂²⁺) leads to clear color fading due to the coordination between the imine of oxTMB and UO₂²⁺. A colorimetric strategy is thus developed for UO₂²⁺ determination with a detection limit of 0.07 μmol L⁻¹. Moreover, a paper-based visual sensing platform is also constructed to offer simple and fast UO₂²⁺ content evaluation in water samples. The present study not only provides a promising strategy to prepare visible light-triggered COF-based metal-free nanoenzymes but also extends the applications of COF material in radionuclide detection.

1. Introduction

It is of great importance to utilize renewable resources urgently for sustainable development, and the high density of nuclear power and its low emission of greenhouse gases have made the issue extremely attractive [1]. In the nuclear industry, UO₂²⁺ is the most widely used fuel to generate nuclear power and even if is beneficial for sustainable development [2], there has been concern about uranium leaching due to the massive mining and processing of uranium, improper handling of nuclear waste, and undesirable nuclear accidents in recent years [3]. UO₂²⁺ can reside in the environment for a long time due to its stability, posing a serious risk to human and animal health, such as cancer and irreversible renal injury [4,5]. Among the methodologies for monitoring the UO₂²⁺ level in the environment, the conventional techniques such as inductively coupled plasma mass spectrometry (ICP-MS) [6], Raman spectroscopy [7], and atomic absorption spectroscopy (AAS) [8] have been demonstrated to be quite powerful in achieving sensitive determination. However, the expensive instrumentation, tedious sample preparation process, and the requirement of professional manipulation restrict their application in quick field investigations. At this point, colorimetry has become a preferred tool owing to its ease of operation, rapid observation with the naked eye, favorable sensitivity, and attractive flexibility. Lu et al., reported the colorimetric determination of UO₂²⁺ by DNAzyme-modified gold nanoparticles (AuNPs) with excellent selectivity and high sensitivity, based on the uranyl induced disassembly of AuNP aggregates [9]. Liu et al., designed a visual colorimetric of a UO₂²⁺ detection protocol using o-phosphoryl ethanolamine-functionalized gold nanoparticles as the spectral probe, based on the ability of UO₂²⁺ to induce the aggregation of AuNPs [10]. He et al., found that UO₂²⁺ showed specific artificial peroxidase activity towards substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) and proposed a facile and reliable colorimetric protocol for UO₂²⁺ assay with a detection limit of 0.5 μM [11]. Though these colorimetric processes provide favorable sensitivity for UO₂²⁺ monitoring, they are usually troubled by complex labeling processes or surface functionalization procedures, and the instability of the spectral probes.

To date, numerous nanomaterials have been explored with favorable mimic enzyme activity, such as Q-graphene [12], V₂O₅ nanobelts [13], metal–organic frameworks [14], noble metal nanoparticles [15], and alloy nanoparticles [16]. By virtue of their high stability, low cost, facial synthesis, and tunable catalytic activity, these inorganic nanomaterial-based artificial enzymes can effectively avoid the inherent limitations of natural enzymes and have been widely used in the field of biomedicine and sensing. Most of the reported nanoenzymes are made of nanoscale metals, metal oxides, or metal-based frameworks, and few metal-free nanozymes are reported except for some carbon-based nanomaterials (graphene, carbon dots) and amorphous polymers with poor stability and limited enzyme activities [17,18,19].

Covalent organic frameworks (COFs) are emerging porous crystalline polymers linked via covalent bonding and merited with excellent stability, tunable functionality, low skeleton density, and regular periodic pore structures. Some COF materials show attractive light-responsive prosperities relying on their π-π array framework and high crystallinity [20]. Qiu et al., found that the two-dimensional COF (Tph-BDP) prepared by 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin and 5,5-difluoro-2,8-diformyl-1,3,7,9-tetramethyl-10-phenyl-5H-dipyrrolo [1,2-c:2′,1′-f][1,3,2]diazabori-nin-4-ium-5-uide exhibited favorable simulated oxidase activity under 635 nm laser irradiation, due to its low energy level bandgap [21]. Chen and co-workers reported the preparation of a three-dimensional COF (COF-300-AR) by terephthaldehyde and tetrakis (4-aminophenyl) methane. COF-300-AR has shown excellent oxidase-mimicking activity under purple light stimulation and has been successfully applied in the sensitive detection of glutathione (GSH) [22].

Herein, a visible light-responsive sulfone-based COF (TAS-COF) was fabricated with electron-rich aromatic C₃-symmetric 2,4,6-triformylphloroglucinol and sulfone substituted phenyl edges of C₂-symmetric 3,7-diaminodibenzo[b,d]thiophene sulfone as the building blocks via a Schiff base condensation reaction. It is interesting to note that the TAS-COF exhibited favorable oxidase-like activity under visible light irradiation, which could oxidize the colorless 3,3′,5,5′-tetramethylbenzydine into blue oxTMB when exposed under a white-light LED. Due to the ability of UO₂²⁺ to coordinate with the imines of oxTMB, the addition of UO₂²⁺ induces clear color fading of the sensing system. As illustrated in Scheme 1, a colorimetric sensing platform was thus constructed for the sensitive and selective detection of UO₂²⁺ content based on the visible light-triggered oxidase-like activity of TAS-COF. In addition, a paper-based detection platform is also constructed by visual monitoring of the color variations with targeted UO₂²⁺ at different concentrations, demonstrating the great potential of this sensing system in on-the-spot evaluation of UO₂²⁺ content in real water samples.

2. Materials and Methods

2.1. Materials

2,4,6-triformylphloroglucinol (Tp) and 3,7-diaminodibenzo[b,d]thiophene sulfone (DAS) were purchased from Bide Pharmatech Ltd. (Shanghai, China). Acetone, tetrahydrofuran (THF), o-dichlorobenzene (o-DCB), n-butanol (n-BuOH), acetic acid (HAC), sodium acetate, 3,3′,5,5′-tetramethylbenzydine (TMB), o-Phenylenediamine (OPD), and other reagents were purchased from Aladdin Industrial Inc. (Shanghai, China). Ultrapure water was prepared from the Millipore system. All reagents were analytical grade and used without further purification.

Ultraviolet-visible (UV-vis) absorption spectra were obtained on a U-3900 UV-vis spectrophotometer (Hitachi, Tokyo, Japan). Powder X-ray diffraction (PXRD) patterns were collected on a Maxima XRD-7000 diffractometer (Shimadzu, Kyoto, Japan). Scanning electron microscopy (SEM) images were collected on a SU8010 field-emission electron microscope at a voltage of 5.0 kV (Hitachi, Tokyo, Japan). Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet-6700 FT-IR spectrophotometer (Thermo Scientific, Massachusetts State, USA). The concentrations of UO₂²⁺ in practical samples were determined using an iCAP Q inductively coupled plasma mass spectrometer (ICP-MS, Thermo Fisher Scientific, USA). Visible light was generated by a white-light LED lamp (λ ≥ 420 nm, power density 62 mW·cm⁻², Guanyu Lighting Co., Ltd., Guangzhou, China).

2.2. Synthesis of TAS-COF

A 10-mL Pyrex tube was charged with Tp (16.8 mg, 0.08 mmol), DAS (29.6 mg, 0.12 mmol), o-DCB (1 mL), n-BuOH (1 mL), and HAC (6 mol L⁻¹, 0.2 mL). This mixture was homogenized under sonication for 10 min and degassed by three freeze-pump-thaw cycles. Then the Pyrex tube was sealed off and heated at 120 °C for 72 h. The precipitate was collected via centrifugation and washed with 10 mL anhydrous THF and 10 mL acetone, in turn, three times at room temperature. The obtained powder was dried at 80 °C under vacuum for 12 h, giving the product of TAS-COF a yield of 83%

2.3. Steady-State Kinetic Studies of TAS-COF as Oxidase Mimic

The steady-state kinetic studies were performed in 300 μL reaction buffer (0.2 mol L⁻¹ HAC-NaAc, pH 3.5) comprising 0.2 mg mL⁻¹ TAS-COF and varied concentrations of TMB under irradiation by a white-light LED lamp (λ ≥ 420 nm) for 10 min. All reactions were carried out three times and the related spectra were recorded. The rate of enzymatic catalytic reaction was described by the following Michaelis–Menten equation [23]:

$${V=V}_{\max }{\left[S\right] / (K}_m+\left[S\right])$$

where V and V_max were the relating reaction rate, and the maximum rate achieved by the system. [S] was TMB concentration. K_m was the Michaelis–Menten constant.

2.4. Colorimetric Detection of UO₂²⁺

Typically, 100 μL TAS-COF solution (0.2 mg mL⁻¹) was mixed with HAc-NaAc buffer (200 μL, pH 3.5) at room temperature, then 100 μL TMB solution (0.5 mmol L⁻¹) and 100 μL UO₂²⁺ solution were added sequentially. The resultant mixture was exposed to a white-light LED lamp (λ ≥ 420 nm, 63 mW cm⁻²) to trigger the catalysis activity of TAS-COF. After irradiation for 10 min, the absorbance of this system at 650 nm (A) was recorded. The blank signal of the sensing system (A₀) was achieved without the addition of UO₂²⁺, and the absorbance difference (A₀–A) was exploited for the fabrication of the calibration curve.

For selectivity investigations, 100 μL of different potential interferents (10 mmol L⁻¹) including Na⁺, K⁺, Mn²⁺, Cu²⁺, Ni²⁺, Ca²⁺, Mg²⁺, Pb²⁺, Cd²⁺, Zn²⁺, and Cr³⁺ instead of UO₂²⁺ were added into the sensing system, and the spectral signal was recorded accordingly.

2.5. Analysis of UO₂²⁺ Content in Real Samples

Lake water samples collected from Nanhu (Shenyang, China) were centrifuged (8000 r min⁻¹, 10 min) and filtered with 0.22 µm membranes to remove large particles. Tap water samples were acquired from the laboratory. The UO₂²⁺ content in the water samples was monitored following the procedure depicted above.

2.6. Visual Inspection

Typically, 20 μL of 50 mg L⁻¹ TAS-COF (prepared in 0.2 mol L⁻¹ HAc-NaAc buffer) was dropped on a paper strip. An amount of 10 μL of TMB solution (30 mmol L⁻¹) and 10 μL UO₂²⁺ of different concentrations (0.5 mmol L⁻¹–10 mmol L⁻¹) were sequentially added to the above point where the TAS-COF was located. The paper strip was then exposed to visible light irradiation from a LED for 10 min, and the color changes were recorded.

3. Results

3.1. Synthesis and Characterization of TAS-COF

The TAS-COF was synthesized with electron-rich aromatic C₃-symmetric Tp and sulfone substituted phenyl edges of C₂-symmetric DAS as the building blocks via a Schiff base condensation reaction and underwent irreversible keto-enol tautomerism (Figure 1A). The as-synthesized TAS-COF formed flower-like nanosheets (Figure 1B). As shown in Figure 1C, the EDS analysis results indicated that there were C, N, O, and S elements present in the TAS-COF material, and the elements were evenly distributed.

Figure 1D shows the FT-IR spectrum of the TAS-COF material. The bands centered at ~3476 cm⁻¹ and ~3372 cm⁻¹ were attributed to the asymmetrical and symmetric stretching vibrations of aromatic amines of DAS, respectively. The peak at ~1639 cm⁻¹ was allocated to the asymmetrical stretching vibrations of –C=O of Tp. The new band at ~1577 cm⁻¹ that appeared in the TAS-COF material was the characteristic peak of −C=N stretching vibrations, suggesting the occurrence of a Schiff base condensation reaction between the monomers and the formation of an imine bond in the final COF product. The crystallinity of TAS-COF was then determined by powder X-ray diffraction (PXRD) and shown in Figure 1E. A set of intensive peaks at 2θ = 3.46° and 7.29° were observed in the PXRD pattern of TAS-COF, indicating that the obtained TAS-COF presents good crystallinity. Meanwhile, the as-synthesized TAS-COF adopted an AA stacking mode with an interlayer distance of 3.70 Å, and the weak π-π stacking between the vertically stacked layers contributed to the appearance of a diffraction peak at 2θ = 26° [24]. The AA stacking mode provides a 1D channel, which was beneficial for quick electron transfer [25]. The above results indicated the successful preparation of the TAS-COF material with favorable crystallinity.

3.2. Visible Light Stimulated Oxidase-Like Activity of TAS-COF

To evaluate the oxidase-like activity of TAS-COF, three typical chromogenic substrates TMB, APTS, or OPD prepared in HAc-NaAc buffer (0.2 mol L⁻¹) were mixed with TAS-COF (0.2 mg mL⁻¹) and exposed to the irradiation from a white-light LED lamp. As shown in Figure 2A, after 10 min irradiation, obvious color changes (blue for TMB, green for APTS, yellow for OPD) were observed for the substrates, and the absorption maxima were 650 nm, 410 nm, and 445 nm, respectively, corresponding to the characteristic absorption of oxTMB, oxAPTS, and oxOPD, respectively. These suggest the excellent oxidase-like activity of TAS-COF under visible light irradiation, and the ability of TAS-COF to catalyze the oxidation of the above colorless substrates under visible light stimulation. Figure 2B depicts the band alignment of TAS-COF. The DFT calculation results indicate that the LUMO position of TAS-COF (−1.32 V vs. Ag/AgCl) was lower than the oxygen’s reduction potential (−0.33 V vs. Ag/AgCl), indicating that the electrons in the LUMO of TAS-COF could theoretically reduce O₂ to generate O₂^·− [26]. Meanwhile, the absence of oxygen (N₂-saturated experiment) caused a significant absorbance signal decrease in oxTMB (Figure 2C), suggesting oxygen dissolved in the water played an important role in the process of oxidation [27]. In order to prove this presumption, electron paramagnetic resonance (EPR) spectroscopy was conducted with 5,5-dimethyl-1-pyrroline-noxide (DMPO) as the O₂^·− radical trapping agent. As depicted in Figure 2D, TAS-COF exhibited a significantly O₂^·− signal under the while-light LED lamp irradiation. Meanwhile, the oxidase-like activity of TAS-COF to TMB presented a staircase-like behavior with the successive “on-off” of light irradiation, demonstrating its light-triggered catalytic activity (Figure 2E).

The steady-state kinetics of TAS-COF was then investigated to evaluate their catalytic activity and affinity to the substrate TMB. As illustrated in Figure 2F, the absorbance increased gradually with the TMB concentration. Based on the relationship between the corresponding initial reaction rates and the TMB concentration, the Lineweaver–Burk plot was obtained in Figure 2F. According to Michaelis–Menten equation [28], the Michaelis–Menten constant (K_m) and the maximum initial velocity (V_max) of TAS-COF were calculated to be 4.88 μM and 1.8 × 10⁻⁴ M min⁻¹, respectively. Compared with the natural enzyme horseradish peroxidase (HRP) (K_m = 0.434 mM, V_max = 6 × 10⁻⁶ M min⁻¹), TAS-COF showed a lower K_m and a higher V_max value, suggesting that TAS-COF as a metal-free nanoenzyme possessed superior affinity toward TMB and faster reaction rate [29].

3.3. Parameters Optimization for UO₂²⁺ Detection

As described above, TAS-COF exhibited favorable oxidase-like activity and could oxide the colorless TMB into blue oxTMB. The oxTMB contained imines that could coordinate with UO₂²⁺ to produce a colorless complex [21]. Figure 3A illustrated the spectra of the TAS-COF + TMB system with the presence of UO₂²⁺. A clear color change from blue to colorless was observed with the presence of UO₂²⁺, providing the solid base for UO₂²⁺ detection.

In order to achieve high sensitivity for the colorimetric monitoring of UO₂²⁺ based on the light-triggered oxidase-like activity of TAS-COF, the key detection parameters, including the reaction pH, irradiation time, TAS-COF dosage, and TMB concentration were optimized. It can be seen from Figure 3B that TAS-COF exhibited the best oxidase-like activity under pH 3.5, and the largest signal difference was achieved under the same pH condition. As shown in Figure 3C, the results indicate that the absorbance signal firstly increased obviously with the irradiation time, and then leveled off when the time was longer than 10 min. Meanwhile, it was found that the signal change increased with TAS-COF concentration up to 0.2 mg mL⁻¹, and the further increase in TAS-COF dosage made no contribution to the signal (Figure 3D). At the same time, a similar trend was observed for TMB concentration when the concentration increased to 0.5 mmol L⁻¹ (Figure 3E), and the further increase in TMB concentration caused no obvious change in the absorbance signals due to the limited TAS-COF content. Therefore, the following optimal experimental conditions were adopted for UO₂²⁺ detection: a TAS-COF concentration of 0.2 mg mL⁻¹, a TMB concentration of 0.5 mmol L⁻¹, a reaction pH of 3.5, and 10 min irradiation under a white-light LED lamp (λ ≥ 420 nm).

3.4. Analytical Performance for UO₂²⁺ Detection

The analytical performance for UO₂²⁺ detection was investigated by acquiring the absorbance signal after the addition of different concentrations of UO₂²⁺ under the optimal conditions. As depicted in Figure 4A, the absorbance of the TAS-COF + TMB sensing system changed obviously with UO₂²⁺ content. The absorbance signal difference (A₀–A, A_0, and A represented the absorbance at 650 nm with/without the presence of UO₂²⁺) showed a good linear relationship with UO₂²⁺ concentration in the range of 0.25–25 μmol L⁻¹ (R² = 0.98) (Figure 4B). The limit of detection (LOD, 3σ/k) was deduced to be 0.07 μmol L⁻¹ (27.6 µg·L⁻¹), which was below the maximum pollution level of UO₂²⁺ in drinking water stipulated by the World Health Organization (30 µg·L⁻¹) [30]. Compared with the reported sensing system, the present colorimetric strategy also offered superior sensitivity toward UO₂²⁺ detection (Figure 4C) [31,32,33,34,35,36,37], contributed by the efficient light-triggered oxidase-like activity of TAS-COF.

The sensing selectivity is of particular significance in practical applications. The selectivity of this sensing system for UO₂²⁺ detection was then explored. The absorbance signals after the addition of a series of aqueous solutions containing potential interfering ions of 5 equiv were recorded. As depicted in Figure 4D, negligible color change or absorbance variation was observed with the addition of interfering ions except for UO₂²⁺. These suggested the favorable specificity of the present colorimetric sensing system for UO₂²⁺ determination.

3.5. Colorimetric Detection of UO₂²⁺ Content in Real Water Samples

To demonstrate the feasibility of the TAS-COF-based colorimetric procedure in UO₂²⁺ detection, UO₂²⁺ content in tap water (T) and lake water (L) samples were determined. To verify the accuracy of determination, UO₂²⁺ contents in these samples were also determined by ICP-MS.

As shown in

Table 1. The colorimetric analysis results for UO₂²⁺ in real samples.

Samples	Added (μM)	Found (μM)	Recovery (%)	ICP-MS	p Value (t-Test)
T-1#	0.5	0.48 ± 0.03	96.00 ± 1.02	0.49 ± 0.02	0.456
T-2#	5	5.04 ± 0.06	100.80 ± 1.51	5.01 ± 0.05	0.378
T-3#	10	9.95 ± 0.18	99.50 ± 0.42	9.99 ± 0.06	0.832
L-1#	0.5	0.45 ± 0.03	90.00 ± 0.87	0.51 ± 0.04	0.668
L-2#	5	4.97 ± 0.05	99.40 ± 3.12	4.95 ± 0.03	0.609
L-3#	10	10.06 ± 0.26	100.60 ± 1.03	10.01 ± 0.12	0.629

, the recoveries ranged from 90.00% to 100.8% for the samples spiked with different levels of UO₂²⁺, and the relative standard deviation (RSD) was less than 3.12%. Figure 5 also suggested that there was no significant difference in the found values of the samples among colorimetry modes. Moreover, the determination results agreed well with those from ICP-MS (p > 0.05), demonstrating that the TAS-COF-based colorimetric sensing platform was potentially sensitive and accurate in monitoring UO₂²⁺ content in complex matrix samples.

3.6. Visual Colorimetric Detection of UO₂²⁺

The analytical performance of the paper-based visual colorimetric detection of UO₂²⁺ with TAS-COF was also inspected. The oxTMB product oxidized by TAS-COF presented a clear blue color in the absence of UO₂²⁺ and faded off gradually after the addition of UO₂²⁺ ranging from 0.5 mmol L⁻¹ to 10 mmol L⁻¹ (Figure 6). The color fading induced by the addition of UO₂²⁺ was readily observed by the naked eye. The visual detection of UO₂²⁺ contents in water samples was also performed. The color of the paper-based sensor after the addition of these spiked waters is similar to the UO₂²⁺ standard solution of UO₂²⁺, suggesting that this paper-based sensor could provide a feasible visual detection platform for simple and rapid UO₂²⁺ content evaluation, which might provide a convenient tool for on-the-spot radionuclide detection.

4. Conclusions

In summary, we have developed a photosensitive sulfone-based COFs material via a Schiff base condensation reaction. The obtained TAS-COF showed oxidase-like activity under visible light irradiation and can direct catalysis of colorless TMB into blue product oxTMB. The presence of UO₂²⁺ will induce clear color fading of this system due to the coordination between oxTMB and UO₂²⁺. A sensitive colorimetric strategy was thus built for the monitoring of UO₂²⁺ content. Meanwhile, the detection of UO₂²⁺ content on a paper-based sensor via visual inspection mode is also achieved, providing a powerful tool for the simple on-the-spot colorimetric UO₂²⁺ analysis in real water samples. This research not only provides a potential path for radionuclide quantitative assay but also expands the applications of COF in sensing by taking advantage of its unique structure and favorable photosensitive properties.