# Electrochemiluminescence Drug Sensor for Acetaminophen using Boron Nitride Quantum Dots as Ru(bpy)32+  Coreactant for Acetaminophen

Authors: Xiyuan Tong; Fang Fang; Zhuozhe Li; Kun Qian; Yao Xu; Yifeng E
Journal: Journal of Nanomaterials
 (2023)

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## Abstract

Acetaminophen (AP) is a commonly used drug that has been detected in groundwater systems in many countries, and has received much attention from researchers in recent years due to its potential environmental impact. In this research, uniformly distributed boron nitride quantum dots (BNQDs) were prepared by a simple ultrasound-solvothermal method. Electrochemical luminescence (ECL) spectroscopy confirmed that BNQDs can act as an effective coreactant to create excellent efficiency in amplifying the ECL intensity of ruthenium-based ECL system. Based on the excited state ofRubpy32+∗ and the energy transfer quenching of AP oxidation products in the luminescent system, an AP concentration-quenched drug sensor was successfully constructed. For this sensor, a wide linear dynamic range and low detection limits (5.0 × 10−7−1.0 × 10−5 mol/L and 4.8 × 10−9 mol/L, respectively) were achieved. This ECL drug sensor has excellent performance in the accurate determination of AP content, relieving the stress of the previous AP detection process, and has good reproducibility and recovery in actual sample measurements.


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## Body

## 1. Introduction

Electrochemiluminescence (ECL) refers to a process in which the luminescent substance forms a high-energy excited state after electrochemical and chemical reactions on the electrode surface, and then relaxes to generate photon emission [1]. The unique luminescence mechanism enables it to combine the high sensitivity and wide dynamic range of chemiluminescence with the controllability, stability, and convenience of electrochemical analysis [2]. According to the response relationship between the test substance and the ECL signal, the quantitative detection of the substance can be realized [3]. Currently, Ru(bpy)32+ is widely used as the luminescent agent in ECL systems due to its good solubility and chemical stability, excellent photophysical, and redox properties [4]. How to obtain the stable and amplified ECL signal of Ru(bpy)32+ luminescence system has always been a research hotspot. Traditional coreactants are consumables of the ECL reaction process, and the ideal ECL luminescence intensity cannot be obtained at low concentration. On the other hand, the highly reactive side-reactants produced during the reaction are also dangerous to humans and other organisms [5].Due to near-zero optical background and photobleaching, ECL sensors are beginning to make their mark in ultrasensitive bioassays [6]. In recent studies, semiconductor polymers have attracted extensive attention from photocatalysis to recent biosensing due to their unique defect-tolerant optoelectronic properties as well as being metal-free, inexpensive, and highly stable [7]. The development of diverse ECL emitters is now critical to unlock their versatility and performance, but remains a formidable challenge due to the stringent requirements of ECL [8]. Quantum dots are size-dependent nanomaterials with excellent physicochemical stability and optical properties [9]. Its outstanding catalytic performance, good chemical stability, easy labeling, low toxicity, and other significant advantages have good application prospects in biomolecule detection, and it has been considered as an ideal coreactant for future ECL research [10–14]. Boron nitride (BN) forms a layered structure through covalent bonding of atoms, which is easily stripped by external forces [15]. Its electronic band structure is relatively sensitive to quantum size, and the synergy of edge effects and defect centers enables boron nitride quantum dots (BNQDs) to exhibit excellent fluorescence properties and good dispersibility [16, 17]. On the basis of fully studying the outstanding optical properties of BNQDs, BNQDs sensors have attracted the attention of many researchers [18, 19].Over the past two decades, the significant impact of agricultural and industrial activities on the environment and human health has attracted worldwide attention. The prevalence of drugs in the environment can affect water quality and can negatively impact human health and the viability of ecosystems. Drugs with high water solubility and low degradability may also escape water treatment and filtration steps, thereby posing a risk to the safety of drinking water. Acetaminophen (AP) is an organic contaminant that is continuously introduced into the water environment from various routes including hospital waste, human metabolism, disposal, and manufacturing facilities [20]. The Minnesota Department of Health (MDH) has established a guideline value of 200 ppb (200,000 ng/L) for acetaminophen in drinking water [21]. The liver is the organ most sensitive to acetaminophen exposure, therefore, the continued environmental introduction and high conversion rates of AP in the aqueous environment may cause long-term deleterious effects in humans.At present, AP has been found in sewage treatment plants and groundwater in several countries, and drug residues may threaten human health. The common methods for detecting AP measurement include titration, ultraviolet–visible (UV–vis) spectroscopy, high performance liquid chromatography (HPLC), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), fluorescence, and capillary electrophoresis (CE), and gas chromatography–mass spectrometry (GC–MS) [22–24]. These techniques basically meet the feasibility and sensitivity requirements of AP content determination. However, many detection methods require cumbersome sample pretreatment process, complicated and expensive operating instruments, and long analysis time, which are not desirable in quality control [25–27]. Considering the impact of drug residues on the environment and physical safety, it is very important to find a sensitive, simple, accurate, and reliable AP detection method as soon as possible.In this study, the use of BNQDs as the coreactant of Ru(bpy)32+ can enhance the ECL luminescence intensity of the system. Subsequently, we constructed an AP-Ru(bpy)32+/BNQDs-ECL drug sensor based on the linear relationship between the natural logarithmic concentration (lnC) of AP and the quenching intensity (ΔI) of the ECL signal. Compared with phenolic drug ECL sensors prepared by highly toxic, highly volatile and lowly soluble organic substances such as TPrA and DBAE for coreactants, BNQDs ECL sensor have the advantages of low cost, nontoxicity, and biocompatibility, which can be used in a wider field of analysis. Compared to other AP detection methods, no sample pretreatment process, modifiable buffer solution type and pH value, and high selectivity are the advantages of this ECL drug sensor. This sensor is easy to operate and has higher sensitivity. In the future, such stable, convenient, and highly sensitive ECL drug sensor could become a potentially powerful tool in analytical applications.


## 2. Materials and Methods

### 2.1. Reagents and Chemicals

Tris(2,2′-bipyridyl) dichlororuthenium (II) hexahydrate (Ru(bpy)3Cl2·6H2O, 98.0%) was obtained from Aladdin Chemistry Co., Ltd., BN powder was purchased from Mclin Biochemical Technology Co., Ltd., acetaminophen (AP, 99%) was purchased from Shanghai Civic Chemical Technology Co., Ltd., N,N-dimethyl formamide (DMF) was purchased from Tianjin Tianli Chemical Reagent Co., Ltd., NaH2PO4·2H2O and Na2HPO4·12H2O were purchased from Tianjin Yongsheng Superfine Chemical Industry Co., Ltd., NaOH was purchased from Tianjin Beichen District Fangzheng Reagent Factory. All analytical grade reagents were used as received without further purification. All aqueous solutions were prepared with deionized water.


### 2.2. Apparatus

ECL signals were recorded by ECL analyzer (MPI-E II, Xian Remex Analysis Instrument Co., Ltd.) with a conventional three-electrode system. The cyclic voltammetry (CV) process was performed by the electrochemistry workstation (CHI 650D, Shanghai Chenhua Instrument Co., Ltd.). Atomic force microscope (FM-Nanoview 6800, Suzhou Flyingman Precision Instruments Co., Ltd.) was utilized for the characterization and size of BNQDs prepared under different conditions. Structural characteristic of BNQDs material is shown by high-resolution transmission electron microscopy (HRTEM) images taken by JEOL JEM-2100 F TEM. The fluorescence spectrum was obtained from the fluorescence (FL) spectrometer (F97Pro, Shanghai Zhong Yong Diagnostic Equipment Co., Ltd.). The UV–vis spectrometer (UV-1600, Shanghai Precision Scientific Instrument Co., Ltd.) was performed to record absorption spectra. Nitrogen generator (QPN-300 II, Shanghai Quanpu Scientific Instrument Co., Ltd.) was used to supply N2. The ultrasonic machine (DR-MS07, Dongguan Tec-Rich Engineering Co., Ltd.), rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory), and dialysis bag (3500 Da, Biosharp Technology Co., Ltd.) were used to obtain uniformly distributed water-soluble BNQDs.


### 2.3. Preparation of BNQDs

BNQDs dispersed in deionized water were obtained on the basis of Li et al. [28]. The specific steps are as follows: 0.5 g BN powder and 50 mL DMF are placed in a beaker and continuously ultrasound for 10 hr. After purging N2 for 30 min, the solution was poured into a polytetrafluoroethylene-lined high-pressure reaction kettle. The filling factor is 2/3, and the solvent heat process is carried out in the oven for several hours at different synthesis temperatures. After being cooled to room temperature, the obtained solution was centrifuged at 10,000 r/min for 15 min, and the obtained supernatant was BNQDs solution. Different from Li et al. [28], BNQDs dispersed in deionized water were obtained by dialysis. Change the water every 4 hr and measure the conductivity of the solution with a conductivity meter during dialysis, until the conductivity of the BNQDs solution is almost the same as that of deionized water. At this time, BNQDs dispersed in deionized water were obtained. The solution was further concentrated by a rotary evaporator to remove excess solvent and stored at 4°C protected from light.


### 2.4. Preparation of Ru(bpy)32+/BNQDs−ECL System

ECL experiments were performed on an MPI-E II ECL analyzer using a three-electrode system with the photomultiplier tube (PMT) voltage of 900 V. The CV process was recorded by a CHI 650D electrochemical workstation. A platinum (Pt) electrode (2 mm in diameter) was used as the working electrode, a Pt wire was used as the auxiliary electrode, and an Ag/AgCl reference electrode was used for all measurements. Before the experiment, the Pt electrode was carefully polished with 0.3 and 0.05μM Al2O3 powders on deerskin, respectively. Al2O3 residues should be removed by ultrasound in deionized water and then dried under N2 flow. Added different concentrations of BNQDs solutions to 0.1 M PB buffer solutions containing 0.1 mM Ru(bpy)32+ in the pH range of 5.5–8.5, and the ECL signal was cyclically scanned in the range of 0 to +1.5 V under different gas conditions. Kept the total volume of electrolyte always at 5 mL. The optimal luminescence conditions of Ru(bpy)32+/BNQDs−ECL system can be determined according to the measured ECL signal intensity.


### 2.5. Construction of the AP–Ru(bpy)32+/BNQDs–ECL Sensor

Twenty microliter of AP solutions (prepared from 0.1 M PB buffer solution at pH 8.0) at different concentrations were separately added to 0.1 M PB buffer solution (pH 8.0) containing 0.1 mM Ru(bpy)32+ and 15 mg/mL BNQDs; purged with N2 for 20 min before each measurement. The ECL signals were recorded by cyclic scanning over the range of 0 to +1.5 V. Based on the response of the ECL intensity of the Ru(bpy)32+/BNQDs system to AP concentration, the quantitative detection of AP was realized.


## 2.1. Reagents and Chemicals

Tris(2,2′-bipyridyl) dichlororuthenium (II) hexahydrate (Ru(bpy)3Cl2·6H2O, 98.0%) was obtained from Aladdin Chemistry Co., Ltd., BN powder was purchased from Mclin Biochemical Technology Co., Ltd., acetaminophen (AP, 99%) was purchased from Shanghai Civic Chemical Technology Co., Ltd., N,N-dimethyl formamide (DMF) was purchased from Tianjin Tianli Chemical Reagent Co., Ltd., NaH2PO4·2H2O and Na2HPO4·12H2O were purchased from Tianjin Yongsheng Superfine Chemical Industry Co., Ltd., NaOH was purchased from Tianjin Beichen District Fangzheng Reagent Factory. All analytical grade reagents were used as received without further purification. All aqueous solutions were prepared with deionized water.


## 2.2. Apparatus

ECL signals were recorded by ECL analyzer (MPI-E II, Xian Remex Analysis Instrument Co., Ltd.) with a conventional three-electrode system. The cyclic voltammetry (CV) process was performed by the electrochemistry workstation (CHI 650D, Shanghai Chenhua Instrument Co., Ltd.). Atomic force microscope (FM-Nanoview 6800, Suzhou Flyingman Precision Instruments Co., Ltd.) was utilized for the characterization and size of BNQDs prepared under different conditions. Structural characteristic of BNQDs material is shown by high-resolution transmission electron microscopy (HRTEM) images taken by JEOL JEM-2100 F TEM. The fluorescence spectrum was obtained from the fluorescence (FL) spectrometer (F97Pro, Shanghai Zhong Yong Diagnostic Equipment Co., Ltd.). The UV–vis spectrometer (UV-1600, Shanghai Precision Scientific Instrument Co., Ltd.) was performed to record absorption spectra. Nitrogen generator (QPN-300 II, Shanghai Quanpu Scientific Instrument Co., Ltd.) was used to supply N2. The ultrasonic machine (DR-MS07, Dongguan Tec-Rich Engineering Co., Ltd.), rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory), and dialysis bag (3500 Da, Biosharp Technology Co., Ltd.) were used to obtain uniformly distributed water-soluble BNQDs.


## 2.3. Preparation of BNQDs

BNQDs dispersed in deionized water were obtained on the basis of Li et al. [28]. The specific steps are as follows: 0.5 g BN powder and 50 mL DMF are placed in a beaker and continuously ultrasound for 10 hr. After purging N2 for 30 min, the solution was poured into a polytetrafluoroethylene-lined high-pressure reaction kettle. The filling factor is 2/3, and the solvent heat process is carried out in the oven for several hours at different synthesis temperatures. After being cooled to room temperature, the obtained solution was centrifuged at 10,000 r/min for 15 min, and the obtained supernatant was BNQDs solution. Different from Li et al. [28], BNQDs dispersed in deionized water were obtained by dialysis. Change the water every 4 hr and measure the conductivity of the solution with a conductivity meter during dialysis, until the conductivity of the BNQDs solution is almost the same as that of deionized water. At this time, BNQDs dispersed in deionized water were obtained. The solution was further concentrated by a rotary evaporator to remove excess solvent and stored at 4°C protected from light.


## 2.4. Preparation of Ru(bpy)32+/BNQDs−ECL System

ECL experiments were performed on an MPI-E II ECL analyzer using a three-electrode system with the photomultiplier tube (PMT) voltage of 900 V. The CV process was recorded by a CHI 650D electrochemical workstation. A platinum (Pt) electrode (2 mm in diameter) was used as the working electrode, a Pt wire was used as the auxiliary electrode, and an Ag/AgCl reference electrode was used for all measurements. Before the experiment, the Pt electrode was carefully polished with 0.3 and 0.05μM Al2O3 powders on deerskin, respectively. Al2O3 residues should be removed by ultrasound in deionized water and then dried under N2 flow. Added different concentrations of BNQDs solutions to 0.1 M PB buffer solutions containing 0.1 mM Ru(bpy)32+ in the pH range of 5.5–8.5, and the ECL signal was cyclically scanned in the range of 0 to +1.5 V under different gas conditions. Kept the total volume of electrolyte always at 5 mL. The optimal luminescence conditions of Ru(bpy)32+/BNQDs−ECL system can be determined according to the measured ECL signal intensity.


## 2.5. Construction of the AP–Ru(bpy)32+/BNQDs–ECL Sensor

Twenty microliter of AP solutions (prepared from 0.1 M PB buffer solution at pH 8.0) at different concentrations were separately added to 0.1 M PB buffer solution (pH 8.0) containing 0.1 mM Ru(bpy)32+ and 15 mg/mL BNQDs; purged with N2 for 20 min before each measurement. The ECL signals were recorded by cyclic scanning over the range of 0 to +1.5 V. Based on the response of the ECL intensity of the Ru(bpy)32+/BNQDs system to AP concentration, the quantitative detection of AP was realized.


## 3. Results and Discussion

### 3.1. Characterization and Optical Properties of BNQDs

Uniformly dispersed BNQDs were prepared by ultrasonic-solvothermal method. Highly polar solvents can overcome van der Waals forces, resulting in higher stripping efficiencies. In practical applications, the dispersibility of the material in the solvent and the stripping ability of the solvent need to be comprehensively selected. In our study, DMF, a strong polar solvent with strong polarity and good dispersibility, was selected as the dispersant. As shown in Figure1, the bulk BN dispersed in DMF was first peeled into layered BN by continuous ultrasound for 10 hr. DMF had appropriate surface energy to overcome the van der Waals force between bulk BN layers and peeled them to obtain a large number of BN nanosheets with surface and edge defects. Subsequently, the obtained BN nanosheets were prepared into BNQDs with smaller diameters by the solvothermal reaction for 12 hr. During this process, the high temperature made the DMF attached to the surface of BN nanosheets interact strongly with them, resulting in defects on the surface of the nanosheets, and finally nanosheets were cleaved into smaller fragments until BNQDs were obtained. At this time, the DMF was also considered as a chemical reaction site. Compared with bulk BN, the surface of BNQDs will be in completely different chemical states under the impact shear of the solvent, making them highly soluble in various solvents and facilitating further integration into the polymer matrix.Figure 1
Ultrasonic-solvothermal synthesis process of BNQDs.The synthesis temperature and reaction time of the solvothermal process are important parameters affecting the size of quantum dots. In this study, atomic force microscopy (AFM) was used to investigate and characterize the effect of different reaction conditions on the morphology of BNQDs. The reaction solvent and filling factor are always consistent. The surface morphologies and size distributions of BNQDs prepared at different synthesis temperatures for 12 hr are shown in Figure2. As shown in Figure 2(a), the distribution of the prepared BNQDs is inconsistent when the synthesis temperature is low (140°C), and the height distribution of particle size in Figure 2(d) similarly indicates that the products obtained under this condition are mostly particles with larger diameters. High temperature can steeply increase the pressure in the autoclave, which will speed-up the frequency of movement of molecules and strengthen the interaction between solvent and BN. In this way, it is easier to obtain BNQDs with small particle size and uniform distribution. The results shown in Figures 2(b) and 2(e) also verify this process. The particle size of BNQDs was more uniform after the temperature was increased to 160°C. The particle height shown in Figure 2(e) (corresponding to the white underline in Figure 2(b)) is significantly lower compared to the low temperature condition (140°C). However, bright agglomerated particles could still be observed in Figure 2(b), so we increased the reaction temperature again. When the synthesis temperature was 180°C, as shown in Figure 2(c), they have good physical dispersion and uniform transverse size, the height of the delineated particles is between 1 and 2 nm in Figure 2(f), corresponding to 1 and 2 layers of BNQDs.Figure 2
AFM morphology images (a–c) of BNQDs prepared at 140, 160, and 180°C for 12 hr in high-pressure reactor, respectively, and the height profiles (d–f) performed along the white line plotted in (a–c) AFM images.
(a)(b)(c)(d)(e)(f)Figure3 shows the effect of solvothermal reaction time on the surface morphology and size distribution of BNQDs. As shown in Figure 3(a), the distribution of particles obtained after 6 hr of solvothermal reaction is not uniform, and many highlights also indicate that the particles are relatively bulky at this time. Figure 3(d) shows the particle size height of the scribed portion in Figure 3(a), and the result shows that most of the particles are still over 1 μM in diameter. The extension of the solvothermal reaction time can increase the boiling time of the solvent in the autoclave, and the long-term shearing action can make the BN surface decompose more thoroughly. In Figure 3(b), the BNQDs obtained by the solvothermal reaction for 9 hr basically present at uniform particle size. As shown in Figure 3(e), the height of the particles has been substantially reduced to below 10 nm, and the diameter has also been significantly reduced to around 200 nm. Figure 3(c) shows the morphologies of BNQDs prepared at 180°C for 12 hr, here we present 3D-AFM images of the prepared BNQDs. The height distribution range in Figure 3(f) shows that the uniformly distributed BNQDs are highly concentrated around 1 nm with a maximum value not exceeding 4.1 nm, which meets the size requirement of QDs. HR-TEM image was carried out for the characterization of the synthesized BNQDs, which is shown in Figure S1. By treating the blank solvent under the same conditions, we confirmed that the solution is indeed BNQDs, rather than simple carbon quantum dots (CQDs). The fact that quantum dots were not observed in AFM may be because the participation of oxygen is crucial for the production of CQDs. Under our current experimental conditions, in order to avoid the formation of carbon quantum dots, N2 has been used to remove oxygen in the pretreatment, so it can be preliminarily considered that the obtained QDs are indeed BNQDs.Figure 3
AFM morphology images (a, b) and 3D-AFM image (c) of BNQDs prepared at 180°C for 6, 9, and 12 hr in high-pressure reactor, respectively, and the height profiles (d, e) performed along the white line plotted in (a) and (b) AFM images. (f) shows the height distribution range of BNQDs in image (c).
(a)(b)(c)(d)(e)(f)The optical properties of BNQDs prepared at 180°C for 12 hr were studied by UV–vis spectroscopy and FL spectroscopy. The characteristic ultraviolet absorption peak of BNQDs dispersed in deionized water is observed at 300 nm in Figure4(a), extending along the whole UV–vis region. In Figure 4(b), BNQDs have two sharp excitation peaks at 210 and 290 nm, with Stokes displacements of 165 and 110 nm, respectively, which are significantly higher than the widely used CdSe/ZnS nanocrystalline (20 nm) [34]. Figure 4(c) records the fluorescence emission of BNQDs at different excitation wavelengths. Consistent with most nonheavy metal quantum dots, the prepared BNQDs exhibit typical excitation wavelength-dependent fluorescence behavior, which is attributed to the synergistic effect of size, surface chemistry, and edge effects. In the range of 270–290 nm, the fluorescence intensity increases with the increase of excitation wavelength but decreases gradually beyond 290 nm (290–370 nm). Meanwhile, the position of the fluorescence emission peak is red-shifted with increasing excitation wavelength, which is also consistent with the previously reported fluorescence analysis results of BNQDs [30]. At the excitation wavelength of 290 nm, the maximum emission peak of BNQDs appears at 400 nm. This property indicates that the fluorescence properties of BNQDs depend on the uniform surface state and chemical environment, which is consistent with the characteristics of quantum dots aggregation luminescence. Based on the fact that the preparation method and fluorescence properties of BNQDs have been thoroughly investigated by previous authors, and supported by the HRTEM characterization result of BNQDs prepared under optimized reaction conditions (Supplementary Figure S1), we can assume that the experimental results in the manuscript have basically confirmed that the prepared materials are indeed BNQDs.Figure 4
UV–vis absorption of BNQDs dispersed in deionized water: (a) excitation and emission fluorescence behavior of BNQDs dispersed in deionized water, (b) and the excitation-dependent fluorescence emission behavior of BNQDs dispersed in deionized water, excited at different wavelengths from 210 to 370 nm (c).
(a)(b)(c)The quantum yield of BNQDs is calculated on the basis of the previously established program. It is determined with quinine sulfate (∅ = 0.54) in 0.1 M sulfuric acid (refractive index η = 1.33) as the standard. Under the excitation wavelength of 350 nm, the quantum yield of BNQDs dissolved in DMF (refractive index η = 1.43) is calculated as follows:(1)∅X=∅STIXISTηX2ηST2ASTAX.Here,I is the measured integrated fluorescence emission intensity, η is the refractive index of the solvent, and A is the optical density. X represents BNQDs and ST represents the standard reference of quinine sulfate. According to the formula, the quantum fluorescence yield of BNQDs is 17.4%.The fluorescence stability of the prepared BNQDs is shown in Figure5, where I0 and I represent the original fluorescence intensity and measured fluorescence intensity, respectively. The result shows that the fluorescence intensity of the prepared BNQDs has no obvious change after 15 days at room temperature, indicating that they have good optical stability. From the above results, it can be considered that this synthesis method opens up a prospect for the preparation of heavy metal-free quantum dots with controllable quantum size and strong fluorescence intensity.Figure 5
Fifteen-days photostability of BNQDs prepared at 180°C for 12 hr.


### 3.2. ECL Behavior of the BNQDs/Ru(bpy)32+ System

The anode ECL and CV behaviors of 15 mg/mL BNQDs, 0.1 mM Ru(bpy)32+, and their mixtures on the surface of Pt electrodes were tested in PB buffer solution at pH 8.0, respectively. As shown in Figure 6(a), there is almost no ECL signal of BNQDs. In the absence of coreactant, the ECL signal of 0.1 mM Ru(bpy)32+ is extremely weak, which is consistent with the previous work [5, 31]. Surprisingly, a strong ECL emission signal can be observed in the mixed solution of the two, which is about 50 times stronger than Ru(bpy)32+ of the same shape and position. The ECL curve shows that Ru(bpy)32+ starts to produce a strong ECL signal peak at +1.05 V and then peaks at +1.28 V with an intensity of 650 a.u. The absence of ECL emission signal of BNQDs also indicates that Ru(bpy)32+ is the luminophore in the BNQDs/Ru(bpy)32+ system, and BNQDs can sensitize the ECL emission of Ru(bpy)32+. The sensitization effect of BNQDs on ECL can also be demonstrated from the CV curves in Figure 6(b). After adding 15 mg/mL BNQDs to the system, the characteristic oxidation peak current of Ru(bpy)32+ at +1.33 V increased significantly. At this time, some reducing species are formed in the system to enhance the ECL signal. The above results suggest that BNQDs can act as coreactants of Ru(bpy)32+ to enhance the anode ECL signal.Figure 6
ECL (a) and CV (b) behaviors of 15 mg/mL BNQDs, 0.1 mM Ru(bpy)32+, and Ru(bpy)32+/BNQDs system, respectively. The electrolyte is 0.1 M pH 8.0 PB buffer with the scanning rate of 0.1 V/s.
(a)(b)The ECL signal intensity plots obtained from BNQDs, BN powder precursors, and TPA as core coreactants under the same reaction conditions are shown in Figure7(a), where the ECL signal intensity of BNQDs/Ru(bpy)32+/Pt is significantly enhanced compared to the other two coreactants. In the inset in Figure 7(a), the ECL signal obtained from BN precursors/Ru(bpy)32+/Pt is almost negligible. Furthermore, we compared the values of the increased ECL intensity induced by BNQDs and TPA as synergists of Ru(bpy)32+ in Figure 7(b). The enhancement ratio due to 15 mg/mL BNQDs was found to be equal to that obtained from 1.66 mg/mL TPA.Figure 7
The ECL response of: (a) BNQDs, BN powder precursors and TPA as core reactants of Ru(bpy)32+, and (b) Ru(bpy)32+ with 1.66 mg/mL TPA (red line) and 15 mg/mL BNQDs. The electrolyte is 0.1 M pH 8.0 PB buffer with the scanning rate of 0.1 V/s.
(a)(b)The edges of BNQDs in different surface states are rich in amino groups [32]. We speculate that the mechanism of BNQDs enhancing the ECL luminescence intensity of Ru(bpy)32+ is shown in Figure 8. On the electrode surface, amino functional group-rich BNQDs and Ru(bpy)32+ were electrochemically oxidized to form BNQDs NH+• and Ru(bpy)33+, respectively. Subsequently, under alkaline conditions, the reduced intermediate BNQDs–N• formed after deprotonation reacts with Ru(bpy)33+ to form the excited state of Rubpy32+∗, which can emit an ECL signal when it returns to the ground state. The specific process is as follows:(2)Rubpy32+−e−⟶Rubpy33++1.33V,(3)BNQDs−NH−e−⟶BNQDs−NH+•+productchemical oxidation,(4)BNQDs−NH+•−H+⟶BNQDs−N•deprotonation,(5)BNQDs−N•+Rubpy33+⟶Rubpy32+∗+product,(6)Rubpy32+∗⟶Rubpy32++hv.Figure 8
Reaction mechanism of BNQDs as the coreactant of Ru(bpy)32+ to enhance anode ECL signal intensity.


### 3.3. Optimization of Ru(bpy)32+/BNQDs ECL System

As shown in Figure9(a), after purging with N2 for 20 min, the ECL signal intensity of the Ru(bpy)32+/BNQDs system is higher than that under atmospheric environment and O2 conditions. In the presence of dissolved oxygen, the reducing intermediates BNQDs–N+• and Ru(bpy)32+ will interact with O2−•, respectively, resulting in insufficient excited state substance produced by the reaction of BNQDs–N• and Ru(bpy)33+, and eventually, the ECL signal will decrease. Therefore, all experiments thereafter were performed after 20 min of continuous N2 purging. Furthermore, the concentration of the coreactant has a significant effect on ECL signal. As shown in Figure 9(b), in the range of 0.5–30 mg/mL, the intensity of ECL varies with the concentration of BNQDs. In the range of 0.5–15 mg/mL, ECL intensity is linearly related to the concentration of BNQDs, R2 = 0.998. With further increase in the concentration of BNQDs (above 15 mg/mL), the ECL signal no longer changes. At this time, the content of Ru(bpy)32+ in the Ru(bpy)32+/BNQDs system is not enough to form more excited state of Rubpy32+∗. Therefore, 15 mg/mL was selected as the concentration of the coreactant in the luminescence system.Figure 9
Effects of gas conditions: (a) BNQDs concentration, (b) and pH, (c) on ECL signal intensity of Ru(bpy)32+/BNQDs system, and the stability of Ru(bpy)32+/BNQDs–ECL drug sensor (d).
(a)(b)(c)(d)The effect of pH on the ECL signal intensity of Ru(bpy)32+/BNQDs system indicates that the deprotonation progress of BNQDs also participates in ECL luminescence process. As shown in Figure 9(c), the ECL signal gradually increases with increasing alkalinity of the solution when the pH value is in between 5.5 and 8.0. Alkaline conditions contribute to the deprotonation process of BNQDs–NH+•, and the generated Rubpy32+∗ will also increase. However, when the pH value of the solution is higher than 8.0, the excess OH− in the system will react with Ru(bpy)33+ after the deprotonation process, which will lead to the consumption of Ru(bpy)33+, and finally, cause the decline of ECL signal. Therefore, 8.0 was considered to be the optimum pH value of Ru(bpy)32+/BNQDs–ECL system. Under optimal conditions, the stability of Ru(bpy)32+/BNQDs–ECL system was tested. The result is shown in Figure 9(d), there is no significant change in ECL intensity of this system under 40 cycles, indicating that this Ru(bpy)32+/BNQDs–ECL system has high stability and good potential for future applications.


### 3.4. Application of ECL Sensor for Acetaminophen

AP is a commonly used antipyretic analgesic that can be rapidly metabolized in the body. It is worth noting that more than safe doses of AP metabolites can accumulate in the body, which will lead to some toxic side effects and may even be life-threatening. Considering its significant impact on the environment and human health, a large number of researchers are working to study high-sensitivity detection methods for AP. The Ru(bpy)32+/BNQDs–ECL sensor has good stability and simple operation, and we believe that it is expected to have simple and effective applications in the field of analysis. The quantitative detection principle of AP by this sensor can be attributed to the energy transfer between the excited state of Rubpy32+∗ and AP radicals. The CV curves in Figure 10 show that after adding 10 mM AP to the Ru(bpy)32+/BNQDs luminescent system, AP shows a distinct oxidation peak at +0.4 V compared with the Ru(bpy)32+/BNQDs luminescent system. It means that the phenolic hydroxyl group of AP is oxidized to benzoquinone radical at this time, and the energy is transferred from the excited state of Rubpy32+∗ to the benzoquinone radical, which finally quenches the ECL signal.Figure 10
CV behavior of AP in Ru(bpy)32+/BNQDs system.We also investigated the relationship between AP concentration and Ru(bpy)32+/BNQDs–ECL sensor signal intensity. Twenty microliter of AP at different concentrations were added to the Ru(bpy)32+/BNQDs–ECL system, and the obtained ECL curves are shown in Figure 11(a). As the AP concentration increased from 500 nM to 10 mM, the ECL signal intensity of the Ru(bpy)32+/BNQDs–ECL sensor became weaker and eventually disappeared. According to the relationship between the natural logarithmic concentration (lnC) of AP and the quenching value ΔI (I0−I) of ECL signal, the content of AP in the system can be quantitatively detected. Here, I0 represents the ECL intensity without AP and I represents the ECL intensity with AP. In Figure 11(b), ΔI was linearly related to the lnC of AP when AP concentrations ranged from 1 to 1,000 μM. The regression equation is Y = 34.79 + 70.82 lnC (R2 = 0.991). S/N = 3. LOD is as low as 4.8 × 10−9 mol/L.Figure 11
ECL intensity with different concentrations of AP from 500 nM to 10 mM (a) and the linear relationship between lnC of AP in the range of 1–1,000μM and ΔI (I0–I) (b).
(a)(b)In order to evaluate the selectivity of Ru(bpy)32+/BNQDs–ECL sensor, 1 mM β-cyclodextrin, glucose, AP, sulfamethoxazole (SMZ), bisphenol A (BPA), catechol (CL), cations (Na+, Mg2+, Zn2+), and anions (SO42−, SO32−, HCO3−) were tested, respectively. Results as shown in Figure 12, it can be found that the addition of ions has no significant effect on ECL signal, while the compounds with phenolic hydroxyl or aniline structure are easily oxidized to form free radicals, resulting in energy transfer quenching effect. The quenching effect of these compounds on ECL signals has also been found in other luminescence systems [33–35]. Therefore, these interfering substances should not be ignored when they are contained in solution. In addition, no obvious response of the Ru(bpy)32+/BNQDs–ECL sensor to other substances such as L-cysteine, glucose, and β-cyclodextrin was observed.Figure 12
Selectivity of Ru(bpy)32+/BNQDs–ECL sensor to different substances. A: β-cyclodextrin, B: glucose, C: AP, D: SMZ, E: BPA, F: CL, G: Na+, H: Mg2+, I: Zn2+, J: SO42−, K: SO32−, L: HCO3−, 1 mM, respectively.The constructed AP–Ru(bpy)32+/BNQDs–ECL sensor was compared with the results of other AP detection methods and phenolic drug ECL sensors. As shown in Table 1, the LOD of the AP–ECL sensor is well comparable to other phenolic ECL sensors, and these studies provide a basis for further development of ECL drug sensors. HPLC is a powerful tool for the separation of multiple analytes and has become an important technique in the field of liquid phase separations. But its operation process is cumbersome and the sensitivity is relatively low. Enzyme-linked immunosorbent assay (ELISA) has high sensitivity and accuracy, but requires expensive equipment and complicated sample preparation procedures, which is not conducive to repeated analysis. In contrast to chromatographic techniques, electrochemical sensors can be used directly to provide real-time sample information. Many studies have improved the performance of analyte detection in this method by modifying electrode materials. Among various electrochemical techniques, CV and differential pulse voltammetry (DPV) have been mainly used for the high-sensitivity determination of paracetamol. Despite the simplicity of the electrochemical method, there is room for further research in terms of detection limit and sensitivity. Compared with other AP detection methods, the LOD of the AP–Ru(bpy)32+/BNQDs–ECL sensor is lower than that of other AP detection methods, and the advantages of simple operation, high sensitivity, and fast response make it a good application prospect in the field of drug analysis and detection.Table 1
Comparison of different detection methods for phenols.
SubstanceMethodSystemLinear concentration rangesLODRef.CatecholECLCDs/HKUST-15.0 × 10−9 − 2.5 × 10−5 mol/L3.8 × 10−9 mol/L[36]DopamineECLAu@SiO2–NPs/Fe–SAC/luminol1.0 × 10−12 − 1.0 × 10−9 mol/L1.0 × 10−10 mol/L[37]DopamineECLCNQDs/Ru(bpy)32+1.0 × 10−6 − 1.0 × 10−3 mol/L5.0 × 10−7 mol/L[38]HydroquinoneECLZnSe QDs/GCE/K2S2O81.0 × 10−9 − 1.0 × 10−6 mol/L3.4 × 10−10 mol/L[39]APFluorescence spectrometryThiol-capped core/shell quantum dot/millipore water3.0 × 10−9 − 1.0 × 10−7 mol/L1.6 × 10−9 mol/L[40]APHPLC–UVC18 column/deionized water/MeOH0.17 − 10.35 mg/L3.5 × 10−9 mol/L[25]APRP–HPLC–PDAC18 column/methanol/phosphate0.8 – 270µg/mL1.4 × 10−6 mol/L[26]APFIASPCE/NFG/tyrosinase-GTA/Nafion2.36 × 10−7 − 1.98 × 10−7 mol /L1.1 × 10−6 mol/L[27]APELISA3-O-3-BSA0.91 − 48.54 ng/mL2.3 × 10−9 mol/L[41]APDPVGraphitic-N-rich nitrogen-doped graphene2.0 × 10−6 − 5.0 × 10−5 mol /L3.8 × 10−7 mol/L[42]APDPVCeO2–SPE1.5 × 10−4 − 5.7 × 10−5 mol /L5.1 × 10−8 mol/L[43]APDPVCe0.75Bi0.25Ox NFs–SPE2.5 × 10−6 − 1.3 × 10−4 mol/L2.0 × 10−7 mol/L[44]APDPVP-RG0/GCE1.5 × 10−6 − 1.2 × 10−4 mol/L3.6 × 10−7 mol/L[45]APDPVGdTiO3 perovskite–graphene7.2 × 10−10 − 1.5 × 10−6 mol /L5.9 × 10−8 mol/L[46]APDPVSMCNTM–GPE2.0 × 10−5 − 3.5 × 10−4 mol /L4.1 × 10−8 mol/L[47]APECLPt–Ru(bpy)32+/BNQDs5.0 × 10−7 − 1.0 × 10−5 mol /L4.8 × 10−9 mol/LThis workUnder the same conditions, five AP–Ru(bpy)32+/BNQDs–ECL sensors were prepared to detect AP solution with the concentration of 1 × 10−6 mol/L. The ECL signal intensities are 78, 75, 86, 82, and 84, respectively; the relative standard deviation (RSD) is 4.47%, indicating that the AP–Ru(bpy)32+/BNQDs–ECL sensor has good reproducibility. To study the feasibility of Ru(bpy)32+/BNQDs–ECL sensor, tap water is selected in the experiment as the actual sample. As shown in Table 2, the standard addition recovery rate is between 92.3% and 108.0% and the RSD range is 3.74%–4.26%, indicating that the AP–Ru(bpy)32+/BNQDs–ECL sensor has a good application prospect.Table 2
Recoveries of AP spiked from tap water samples (n = 3).
SampleStandard value of AP (M)Found (M)Recovery (%)RSD (%,n = 5)Tap water1 × 10−69.23 × 10−7–9.87 × 10−792.3%–98.7%4.031 × 10−59.58 × 10−6–1.08 × 10−595.8%–108.0%3.745 × 10−55.08 × 10−5–5.32 × 10−5101.6%–106.4%4.26


## 3.1. Characterization and Optical Properties of BNQDs

Uniformly dispersed BNQDs were prepared by ultrasonic-solvothermal method. Highly polar solvents can overcome van der Waals forces, resulting in higher stripping efficiencies. In practical applications, the dispersibility of the material in the solvent and the stripping ability of the solvent need to be comprehensively selected. In our study, DMF, a strong polar solvent with strong polarity and good dispersibility, was selected as the dispersant. As shown in Figure1, the bulk BN dispersed in DMF was first peeled into layered BN by continuous ultrasound for 10 hr. DMF had appropriate surface energy to overcome the van der Waals force between bulk BN layers and peeled them to obtain a large number of BN nanosheets with surface and edge defects. Subsequently, the obtained BN nanosheets were prepared into BNQDs with smaller diameters by the solvothermal reaction for 12 hr. During this process, the high temperature made the DMF attached to the surface of BN nanosheets interact strongly with them, resulting in defects on the surface of the nanosheets, and finally nanosheets were cleaved into smaller fragments until BNQDs were obtained. At this time, the DMF was also considered as a chemical reaction site. Compared with bulk BN, the surface of BNQDs will be in completely different chemical states under the impact shear of the solvent, making them highly soluble in various solvents and facilitating further integration into the polymer matrix.Figure 1
Ultrasonic-solvothermal synthesis process of BNQDs.The synthesis temperature and reaction time of the solvothermal process are important parameters affecting the size of quantum dots. In this study, atomic force microscopy (AFM) was used to investigate and characterize the effect of different reaction conditions on the morphology of BNQDs. The reaction solvent and filling factor are always consistent. The surface morphologies and size distributions of BNQDs prepared at different synthesis temperatures for 12 hr are shown in Figure2. As shown in Figure 2(a), the distribution of the prepared BNQDs is inconsistent when the synthesis temperature is low (140°C), and the height distribution of particle size in Figure 2(d) similarly indicates that the products obtained under this condition are mostly particles with larger diameters. High temperature can steeply increase the pressure in the autoclave, which will speed-up the frequency of movement of molecules and strengthen the interaction between solvent and BN. In this way, it is easier to obtain BNQDs with small particle size and uniform distribution. The results shown in Figures 2(b) and 2(e) also verify this process. The particle size of BNQDs was more uniform after the temperature was increased to 160°C. The particle height shown in Figure 2(e) (corresponding to the white underline in Figure 2(b)) is significantly lower compared to the low temperature condition (140°C). However, bright agglomerated particles could still be observed in Figure 2(b), so we increased the reaction temperature again. When the synthesis temperature was 180°C, as shown in Figure 2(c), they have good physical dispersion and uniform transverse size, the height of the delineated particles is between 1 and 2 nm in Figure 2(f), corresponding to 1 and 2 layers of BNQDs.Figure 2
AFM morphology images (a–c) of BNQDs prepared at 140, 160, and 180°C for 12 hr in high-pressure reactor, respectively, and the height profiles (d–f) performed along the white line plotted in (a–c) AFM images.
(a)(b)(c)(d)(e)(f)Figure3 shows the effect of solvothermal reaction time on the surface morphology and size distribution of BNQDs. As shown in Figure 3(a), the distribution of particles obtained after 6 hr of solvothermal reaction is not uniform, and many highlights also indicate that the particles are relatively bulky at this time. Figure 3(d) shows the particle size height of the scribed portion in Figure 3(a), and the result shows that most of the particles are still over 1 μM in diameter. The extension of the solvothermal reaction time can increase the boiling time of the solvent in the autoclave, and the long-term shearing action can make the BN surface decompose more thoroughly. In Figure 3(b), the BNQDs obtained by the solvothermal reaction for 9 hr basically present at uniform particle size. As shown in Figure 3(e), the height of the particles has been substantially reduced to below 10 nm, and the diameter has also been significantly reduced to around 200 nm. Figure 3(c) shows the morphologies of BNQDs prepared at 180°C for 12 hr, here we present 3D-AFM images of the prepared BNQDs. The height distribution range in Figure 3(f) shows that the uniformly distributed BNQDs are highly concentrated around 1 nm with a maximum value not exceeding 4.1 nm, which meets the size requirement of QDs. HR-TEM image was carried out for the characterization of the synthesized BNQDs, which is shown in Figure S1. By treating the blank solvent under the same conditions, we confirmed that the solution is indeed BNQDs, rather than simple carbon quantum dots (CQDs). The fact that quantum dots were not observed in AFM may be because the participation of oxygen is crucial for the production of CQDs. Under our current experimental conditions, in order to avoid the formation of carbon quantum dots, N2 has been used to remove oxygen in the pretreatment, so it can be preliminarily considered that the obtained QDs are indeed BNQDs.Figure 3
AFM morphology images (a, b) and 3D-AFM image (c) of BNQDs prepared at 180°C for 6, 9, and 12 hr in high-pressure reactor, respectively, and the height profiles (d, e) performed along the white line plotted in (a) and (b) AFM images. (f) shows the height distribution range of BNQDs in image (c).
(a)(b)(c)(d)(e)(f)The optical properties of BNQDs prepared at 180°C for 12 hr were studied by UV–vis spectroscopy and FL spectroscopy. The characteristic ultraviolet absorption peak of BNQDs dispersed in deionized water is observed at 300 nm in Figure4(a), extending along the whole UV–vis region. In Figure 4(b), BNQDs have two sharp excitation peaks at 210 and 290 nm, with Stokes displacements of 165 and 110 nm, respectively, which are significantly higher than the widely used CdSe/ZnS nanocrystalline (20 nm) [34]. Figure 4(c) records the fluorescence emission of BNQDs at different excitation wavelengths. Consistent with most nonheavy metal quantum dots, the prepared BNQDs exhibit typical excitation wavelength-dependent fluorescence behavior, which is attributed to the synergistic effect of size, surface chemistry, and edge effects. In the range of 270–290 nm, the fluorescence intensity increases with the increase of excitation wavelength but decreases gradually beyond 290 nm (290–370 nm). Meanwhile, the position of the fluorescence emission peak is red-shifted with increasing excitation wavelength, which is also consistent with the previously reported fluorescence analysis results of BNQDs [30]. At the excitation wavelength of 290 nm, the maximum emission peak of BNQDs appears at 400 nm. This property indicates that the fluorescence properties of BNQDs depend on the uniform surface state and chemical environment, which is consistent with the characteristics of quantum dots aggregation luminescence. Based on the fact that the preparation method and fluorescence properties of BNQDs have been thoroughly investigated by previous authors, and supported by the HRTEM characterization result of BNQDs prepared under optimized reaction conditions (Supplementary Figure S1), we can assume that the experimental results in the manuscript have basically confirmed that the prepared materials are indeed BNQDs.Figure 4
UV–vis absorption of BNQDs dispersed in deionized water: (a) excitation and emission fluorescence behavior of BNQDs dispersed in deionized water, (b) and the excitation-dependent fluorescence emission behavior of BNQDs dispersed in deionized water, excited at different wavelengths from 210 to 370 nm (c).
(a)(b)(c)The quantum yield of BNQDs is calculated on the basis of the previously established program. It is determined with quinine sulfate (∅ = 0.54) in 0.1 M sulfuric acid (refractive index η = 1.33) as the standard. Under the excitation wavelength of 350 nm, the quantum yield of BNQDs dissolved in DMF (refractive index η = 1.43) is calculated as follows:(1)∅X=∅STIXISTηX2ηST2ASTAX.Here,I is the measured integrated fluorescence emission intensity, η is the refractive index of the solvent, and A is the optical density. X represents BNQDs and ST represents the standard reference of quinine sulfate. According to the formula, the quantum fluorescence yield of BNQDs is 17.4%.The fluorescence stability of the prepared BNQDs is shown in Figure5, where I0 and I represent the original fluorescence intensity and measured fluorescence intensity, respectively. The result shows that the fluorescence intensity of the prepared BNQDs has no obvious change after 15 days at room temperature, indicating that they have good optical stability. From the above results, it can be considered that this synthesis method opens up a prospect for the preparation of heavy metal-free quantum dots with controllable quantum size and strong fluorescence intensity.Figure 5
Fifteen-days photostability of BNQDs prepared at 180°C for 12 hr.


## 3.2. ECL Behavior of the BNQDs/Ru(bpy)32+ System

The anode ECL and CV behaviors of 15 mg/mL BNQDs, 0.1 mM Ru(bpy)32+, and their mixtures on the surface of Pt electrodes were tested in PB buffer solution at pH 8.0, respectively. As shown in Figure 6(a), there is almost no ECL signal of BNQDs. In the absence of coreactant, the ECL signal of 0.1 mM Ru(bpy)32+ is extremely weak, which is consistent with the previous work [5, 31]. Surprisingly, a strong ECL emission signal can be observed in the mixed solution of the two, which is about 50 times stronger than Ru(bpy)32+ of the same shape and position. The ECL curve shows that Ru(bpy)32+ starts to produce a strong ECL signal peak at +1.05 V and then peaks at +1.28 V with an intensity of 650 a.u. The absence of ECL emission signal of BNQDs also indicates that Ru(bpy)32+ is the luminophore in the BNQDs/Ru(bpy)32+ system, and BNQDs can sensitize the ECL emission of Ru(bpy)32+. The sensitization effect of BNQDs on ECL can also be demonstrated from the CV curves in Figure 6(b). After adding 15 mg/mL BNQDs to the system, the characteristic oxidation peak current of Ru(bpy)32+ at +1.33 V increased significantly. At this time, some reducing species are formed in the system to enhance the ECL signal. The above results suggest that BNQDs can act as coreactants of Ru(bpy)32+ to enhance the anode ECL signal.Figure 6
ECL (a) and CV (b) behaviors of 15 mg/mL BNQDs, 0.1 mM Ru(bpy)32+, and Ru(bpy)32+/BNQDs system, respectively. The electrolyte is 0.1 M pH 8.0 PB buffer with the scanning rate of 0.1 V/s.
(a)(b)The ECL signal intensity plots obtained from BNQDs, BN powder precursors, and TPA as core coreactants under the same reaction conditions are shown in Figure7(a), where the ECL signal intensity of BNQDs/Ru(bpy)32+/Pt is significantly enhanced compared to the other two coreactants. In the inset in Figure 7(a), the ECL signal obtained from BN precursors/Ru(bpy)32+/Pt is almost negligible. Furthermore, we compared the values of the increased ECL intensity induced by BNQDs and TPA as synergists of Ru(bpy)32+ in Figure 7(b). The enhancement ratio due to 15 mg/mL BNQDs was found to be equal to that obtained from 1.66 mg/mL TPA.Figure 7
The ECL response of: (a) BNQDs, BN powder precursors and TPA as core reactants of Ru(bpy)32+, and (b) Ru(bpy)32+ with 1.66 mg/mL TPA (red line) and 15 mg/mL BNQDs. The electrolyte is 0.1 M pH 8.0 PB buffer with the scanning rate of 0.1 V/s.
(a)(b)The edges of BNQDs in different surface states are rich in amino groups [32]. We speculate that the mechanism of BNQDs enhancing the ECL luminescence intensity of Ru(bpy)32+ is shown in Figure 8. On the electrode surface, amino functional group-rich BNQDs and Ru(bpy)32+ were electrochemically oxidized to form BNQDs NH+• and Ru(bpy)33+, respectively. Subsequently, under alkaline conditions, the reduced intermediate BNQDs–N• formed after deprotonation reacts with Ru(bpy)33+ to form the excited state of Rubpy32+∗, which can emit an ECL signal when it returns to the ground state. The specific process is as follows:(2)Rubpy32+−e−⟶Rubpy33++1.33V,(3)BNQDs−NH−e−⟶BNQDs−NH+•+productchemical oxidation,(4)BNQDs−NH+•−H+⟶BNQDs−N•deprotonation,(5)BNQDs−N•+Rubpy33+⟶Rubpy32+∗+product,(6)Rubpy32+∗⟶Rubpy32++hv.Figure 8
Reaction mechanism of BNQDs as the coreactant of Ru(bpy)32+ to enhance anode ECL signal intensity.


## 3.3. Optimization of Ru(bpy)32+/BNQDs ECL System

As shown in Figure9(a), after purging with N2 for 20 min, the ECL signal intensity of the Ru(bpy)32+/BNQDs system is higher than that under atmospheric environment and O2 conditions. In the presence of dissolved oxygen, the reducing intermediates BNQDs–N+• and Ru(bpy)32+ will interact with O2−•, respectively, resulting in insufficient excited state substance produced by the reaction of BNQDs–N• and Ru(bpy)33+, and eventually, the ECL signal will decrease. Therefore, all experiments thereafter were performed after 20 min of continuous N2 purging. Furthermore, the concentration of the coreactant has a significant effect on ECL signal. As shown in Figure 9(b), in the range of 0.5–30 mg/mL, the intensity of ECL varies with the concentration of BNQDs. In the range of 0.5–15 mg/mL, ECL intensity is linearly related to the concentration of BNQDs, R2 = 0.998. With further increase in the concentration of BNQDs (above 15 mg/mL), the ECL signal no longer changes. At this time, the content of Ru(bpy)32+ in the Ru(bpy)32+/BNQDs system is not enough to form more excited state of Rubpy32+∗. Therefore, 15 mg/mL was selected as the concentration of the coreactant in the luminescence system.Figure 9
Effects of gas conditions: (a) BNQDs concentration, (b) and pH, (c) on ECL signal intensity of Ru(bpy)32+/BNQDs system, and the stability of Ru(bpy)32+/BNQDs–ECL drug sensor (d).
(a)(b)(c)(d)The effect of pH on the ECL signal intensity of Ru(bpy)32+/BNQDs system indicates that the deprotonation progress of BNQDs also participates in ECL luminescence process. As shown in Figure 9(c), the ECL signal gradually increases with increasing alkalinity of the solution when the pH value is in between 5.5 and 8.0. Alkaline conditions contribute to the deprotonation process of BNQDs–NH+•, and the generated Rubpy32+∗ will also increase. However, when the pH value of the solution is higher than 8.0, the excess OH− in the system will react with Ru(bpy)33+ after the deprotonation process, which will lead to the consumption of Ru(bpy)33+, and finally, cause the decline of ECL signal. Therefore, 8.0 was considered to be the optimum pH value of Ru(bpy)32+/BNQDs–ECL system. Under optimal conditions, the stability of Ru(bpy)32+/BNQDs–ECL system was tested. The result is shown in Figure 9(d), there is no significant change in ECL intensity of this system under 40 cycles, indicating that this Ru(bpy)32+/BNQDs–ECL system has high stability and good potential for future applications.


## 3.4. Application of ECL Sensor for Acetaminophen

AP is a commonly used antipyretic analgesic that can be rapidly metabolized in the body. It is worth noting that more than safe doses of AP metabolites can accumulate in the body, which will lead to some toxic side effects and may even be life-threatening. Considering its significant impact on the environment and human health, a large number of researchers are working to study high-sensitivity detection methods for AP. The Ru(bpy)32+/BNQDs–ECL sensor has good stability and simple operation, and we believe that it is expected to have simple and effective applications in the field of analysis. The quantitative detection principle of AP by this sensor can be attributed to the energy transfer between the excited state of Rubpy32+∗ and AP radicals. The CV curves in Figure 10 show that after adding 10 mM AP to the Ru(bpy)32+/BNQDs luminescent system, AP shows a distinct oxidation peak at +0.4 V compared with the Ru(bpy)32+/BNQDs luminescent system. It means that the phenolic hydroxyl group of AP is oxidized to benzoquinone radical at this time, and the energy is transferred from the excited state of Rubpy32+∗ to the benzoquinone radical, which finally quenches the ECL signal.Figure 10
CV behavior of AP in Ru(bpy)32+/BNQDs system.We also investigated the relationship between AP concentration and Ru(bpy)32+/BNQDs–ECL sensor signal intensity. Twenty microliter of AP at different concentrations were added to the Ru(bpy)32+/BNQDs–ECL system, and the obtained ECL curves are shown in Figure 11(a). As the AP concentration increased from 500 nM to 10 mM, the ECL signal intensity of the Ru(bpy)32+/BNQDs–ECL sensor became weaker and eventually disappeared. According to the relationship between the natural logarithmic concentration (lnC) of AP and the quenching value ΔI (I0−I) of ECL signal, the content of AP in the system can be quantitatively detected. Here, I0 represents the ECL intensity without AP and I represents the ECL intensity with AP. In Figure 11(b), ΔI was linearly related to the lnC of AP when AP concentrations ranged from 1 to 1,000 μM. The regression equation is Y = 34.79 + 70.82 lnC (R2 = 0.991). S/N = 3. LOD is as low as 4.8 × 10−9 mol/L.Figure 11
ECL intensity with different concentrations of AP from 500 nM to 10 mM (a) and the linear relationship between lnC of AP in the range of 1–1,000μM and ΔI (I0–I) (b).
(a)(b)In order to evaluate the selectivity of Ru(bpy)32+/BNQDs–ECL sensor, 1 mM β-cyclodextrin, glucose, AP, sulfamethoxazole (SMZ), bisphenol A (BPA), catechol (CL), cations (Na+, Mg2+, Zn2+), and anions (SO42−, SO32−, HCO3−) were tested, respectively. Results as shown in Figure 12, it can be found that the addition of ions has no significant effect on ECL signal, while the compounds with phenolic hydroxyl or aniline structure are easily oxidized to form free radicals, resulting in energy transfer quenching effect. The quenching effect of these compounds on ECL signals has also been found in other luminescence systems [33–35]. Therefore, these interfering substances should not be ignored when they are contained in solution. In addition, no obvious response of the Ru(bpy)32+/BNQDs–ECL sensor to other substances such as L-cysteine, glucose, and β-cyclodextrin was observed.Figure 12
Selectivity of Ru(bpy)32+/BNQDs–ECL sensor to different substances. A: β-cyclodextrin, B: glucose, C: AP, D: SMZ, E: BPA, F: CL, G: Na+, H: Mg2+, I: Zn2+, J: SO42−, K: SO32−, L: HCO3−, 1 mM, respectively.The constructed AP–Ru(bpy)32+/BNQDs–ECL sensor was compared with the results of other AP detection methods and phenolic drug ECL sensors. As shown in Table 1, the LOD of the AP–ECL sensor is well comparable to other phenolic ECL sensors, and these studies provide a basis for further development of ECL drug sensors. HPLC is a powerful tool for the separation of multiple analytes and has become an important technique in the field of liquid phase separations. But its operation process is cumbersome and the sensitivity is relatively low. Enzyme-linked immunosorbent assay (ELISA) has high sensitivity and accuracy, but requires expensive equipment and complicated sample preparation procedures, which is not conducive to repeated analysis. In contrast to chromatographic techniques, electrochemical sensors can be used directly to provide real-time sample information. Many studies have improved the performance of analyte detection in this method by modifying electrode materials. Among various electrochemical techniques, CV and differential pulse voltammetry (DPV) have been mainly used for the high-sensitivity determination of paracetamol. Despite the simplicity of the electrochemical method, there is room for further research in terms of detection limit and sensitivity. Compared with other AP detection methods, the LOD of the AP–Ru(bpy)32+/BNQDs–ECL sensor is lower than that of other AP detection methods, and the advantages of simple operation, high sensitivity, and fast response make it a good application prospect in the field of drug analysis and detection.Table 1
Comparison of different detection methods for phenols.
SubstanceMethodSystemLinear concentration rangesLODRef.CatecholECLCDs/HKUST-15.0 × 10−9 − 2.5 × 10−5 mol/L3.8 × 10−9 mol/L[36]DopamineECLAu@SiO2–NPs/Fe–SAC/luminol1.0 × 10−12 − 1.0 × 10−9 mol/L1.0 × 10−10 mol/L[37]DopamineECLCNQDs/Ru(bpy)32+1.0 × 10−6 − 1.0 × 10−3 mol/L5.0 × 10−7 mol/L[38]HydroquinoneECLZnSe QDs/GCE/K2S2O81.0 × 10−9 − 1.0 × 10−6 mol/L3.4 × 10−10 mol/L[39]APFluorescence spectrometryThiol-capped core/shell quantum dot/millipore water3.0 × 10−9 − 1.0 × 10−7 mol/L1.6 × 10−9 mol/L[40]APHPLC–UVC18 column/deionized water/MeOH0.17 − 10.35 mg/L3.5 × 10−9 mol/L[25]APRP–HPLC–PDAC18 column/methanol/phosphate0.8 – 270µg/mL1.4 × 10−6 mol/L[26]APFIASPCE/NFG/tyrosinase-GTA/Nafion2.36 × 10−7 − 1.98 × 10−7 mol /L1.1 × 10−6 mol/L[27]APELISA3-O-3-BSA0.91 − 48.54 ng/mL2.3 × 10−9 mol/L[41]APDPVGraphitic-N-rich nitrogen-doped graphene2.0 × 10−6 − 5.0 × 10−5 mol /L3.8 × 10−7 mol/L[42]APDPVCeO2–SPE1.5 × 10−4 − 5.7 × 10−5 mol /L5.1 × 10−8 mol/L[43]APDPVCe0.75Bi0.25Ox NFs–SPE2.5 × 10−6 − 1.3 × 10−4 mol/L2.0 × 10−7 mol/L[44]APDPVP-RG0/GCE1.5 × 10−6 − 1.2 × 10−4 mol/L3.6 × 10−7 mol/L[45]APDPVGdTiO3 perovskite–graphene7.2 × 10−10 − 1.5 × 10−6 mol /L5.9 × 10−8 mol/L[46]APDPVSMCNTM–GPE2.0 × 10−5 − 3.5 × 10−4 mol /L4.1 × 10−8 mol/L[47]APECLPt–Ru(bpy)32+/BNQDs5.0 × 10−7 − 1.0 × 10−5 mol /L4.8 × 10−9 mol/LThis workUnder the same conditions, five AP–Ru(bpy)32+/BNQDs–ECL sensors were prepared to detect AP solution with the concentration of 1 × 10−6 mol/L. The ECL signal intensities are 78, 75, 86, 82, and 84, respectively; the relative standard deviation (RSD) is 4.47%, indicating that the AP–Ru(bpy)32+/BNQDs–ECL sensor has good reproducibility. To study the feasibility of Ru(bpy)32+/BNQDs–ECL sensor, tap water is selected in the experiment as the actual sample. As shown in Table 2, the standard addition recovery rate is between 92.3% and 108.0% and the RSD range is 3.74%–4.26%, indicating that the AP–Ru(bpy)32+/BNQDs–ECL sensor has a good application prospect.Table 2
Recoveries of AP spiked from tap water samples (n = 3).
SampleStandard value of AP (M)Found (M)Recovery (%)RSD (%,n = 5)Tap water1 × 10−69.23 × 10−7–9.87 × 10−792.3%–98.7%4.031 × 10−59.58 × 10−6–1.08 × 10−595.8%–108.0%3.745 × 10−55.08 × 10−5–5.32 × 10−5101.6%–106.4%4.26


## 4. Conclusion

In conclusion, BNQDs with excellent fluorescence properties were prepared by a simple ultrasonic-solvothermal method, and this low-cost and environmentally friendly preparation method of BNQDs can be extended to the synthesis of other nanomaterials. BNQDs have good fluorescence photostability, which provides a basis for their further development and wide application. Based on the surface amine groups and electrocatalysis, BNQDs can be considered as potential coreactants for Ru(bpy)32+ anode ECL. The Ru(bpy)32+/BNQDs–ECL system has high stability and sensitivity. Based on the quenching effect of AP on ECL signal, an AP–Ru(bpy)32+/BNQDs–ECL drug sensor was constructed. Compared with other AP detection methods, this ECL drug sensor has the advantages of simple operation and high sensitivity, which opens up new prospects for the wide application of BNQDs.



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*Source: 3179008-2023-02-13.xml*