Enhancing of Luminol-H₂O₂ Chemiluminescence System by Bimetallic Metal–Organic Frameworks with Mixed Ligands

Abstract

Chemiluminescence (CL) is regarded as a better method for the detection of reactive oxygen species (ROS). However, the weak CL intensity prevents its further application. Many nanomaterials have been developed to enhance CL intensity, and mixed-ligand MOFs incorporating additional metals or organic ligands exhibit high efficiency in catalyzing. In this work, one kind of bimetallic mixed-ligand metal–organic framework (Ni-Co m-MOF) was synthesized using solvothermal methods. The material was morphologically characterized and demonstrated to be a dense and spherical flower-like structure. The addition of Ni-Co m-MOFs significantly enhanced the CL intensity of the luminol-H₂O₂ system by nearly 2,000-fold. The enhancement was found through further research as hydrogen peroxide was catalyzed to create hydroxyl radicals, etc., which reacted more easily with luminol. Herein, significant enhancement of the CL system by Ni-Co m-MOFs was identified, which provides ideas for improving the sensitivity and signal-to-noise ratio development of CL detectors.

1. Introduction

Reactive oxygen species (ROS), generated by normal physiological activities or external stimuli, play critical roles in all life functions, from bioenergy to metabolism, and serve as a vital biomarker for various diseases and important analytes [1,2]. Beyond human health relevance, ROS are also essential for catalysis, industry, food safety, and agriculture [3,4]. Examples of ROS include, but are not limited to, hydrogen peroxide (H₂O₂), superoxide anion radical (^•O₂⁻), hydroxyl radical (^•OH), and singlet oxygen (¹O₂). Due to the short lifetime and high reactivity of ROS, the detection has been a challenge [5,6,7,8]. With the advantages of high sensitivity, a wide linear range, simple and inexpensive instrumentation, and reduced background noise, chemiluminescence (CL) provides a very useful method for detecting and quantifying ROS, particularly beneficial for bioimaging applications [9]. With the modification of CL probes by Schaap, Shabat, and Pu et al., CL is now well suited for disease detection and in vivo imaging [10,11,12,13]. However, the current efficiency of CL remains relatively low, so it is crucial to develop new CL systems to enhance intensity [14,15]. Within recent years, enhancement strategies employing nanomaterials have been widely used to enhance CL, resulting in improved CL performance, an increase in high sensitivity, and the reduction in detection limits [16]. Consequently, these allow CL to provide more effective detection of ROS and facilitate biological analysis.

Nanomaterials such as metal–organic frameworks (MOFs), carbon quantum dots, gold nanoparticles, etc., are commonly used for enhanced CL [15]. Among them, MOFs have been shown to have higher activity and stability, higher specific surface area, and excellent biocompatibility. These led to the rapid development of MOF-based catalysts as CL enhancers [17,18]. However, certain pure MOF materials exhibit limited catalytic activity in practical applications. As a result, they require modification through doping with transition metals or organic ligands to enhance their catalytic effectiveness [19]. It has been found that mixed-ligand MOFs incorporating additional metals or organic ligands exhibit high efficiency in catalyzing CL luminescence. In particular, bimetallic MOFs comprising two distinct metals have been demonstrated to generate a synergistic catalytic effect, significantly enhancing CL emission [20,21,22]. The classical and most popular CL substrates are luminol and its derivatives, which can be oxidized by ROS to generate the excited-state 3-amino-phenyl ester of phthalic acid (3-APA*), resulting in luminescence [23]. Employing the luminol-H₂O₂ system to explore the effect of MOF on CL enhancement would be a favorable choice.

Co, Fe, and Ni ions exhibit excellent performance in the catalysis of hydrogen peroxide decomposition [24]. They significantly boost the activity of ROS and have been used in several advanced oxidation reactions with satisfactory results [25]. Hence, we synthesized a hybrid ligand material known as Ni-Co m-MOFs, which incorporates bimetallic and ligand components based on previous research [26]. The obtained materials were prepared, and then we characterized their structure, composition, elemental valence, and distribution. Then, we evaluated their application in the luminol-H₂O₂ CL system. Subsequently, a variety of parameters were further optimized, including the concentrations of luminol and H₂O₂, the additions of Ni-Co m-MOFs dispersions, the pH value of H₂O₂, and the sequence of the additions of luminol and H₂O₂. The optimization resulted in the best catalytic conditions, and the CL intensity of luminol was significantly increased by a factor of two thousand. Furthermore, the mechanism of luminescence in the Ni-Co m-MOF-luminol-H₂O₂ system has been deduced based on a series of experiments. Compared with the traditional materials of MOFs, the presence of co-doped bimetallic ions contributes to their stabilization within the MOF framework structure, which greatly improves the intensity of CL. Research has indicated the excellent application of these MOFs for the detection of ROS and is expected to be further developed for the detection of other active substances with improved detection sensitivity and signal-to-noise ratios.

2. Results and Discussion

A hybrid MOF material with bimetallic and ligand characteristics was synthesized using a solvothermal method, incorporating Ni and Co metals as linkers and H₃BTC and H₂BDC as organic ligands. The morphology of Ni-Co m-MOFs was examined using SEM, as shown in Figure 1a. The images reveal a compact flower-like structure of Ni-Co m-MOFs. This structure is attributed to the synergistic interaction between nickel and cobalt bimetallic ions and the incorporation of dual ligands, which greatly enhance the catalytic efficiency of Ni-Co m-MOFs [26]. The material exhibits a significantly large surface area, enabling high exposure of metal active sites, thereby promoting enhanced CL. The results of the TEM are shown in Figure S1. It can be seen that the lattice stripe width of Ni-Co m-MOFs is 0.36 nm. Selected area electron diffraction (SAED) (Figure S1d) displays distinct diffraction spots and proved to be well crystallized. The elements are uniformly distributed on the surface of the Ni-Co m-MOFs, as can be seen in Figure S1f by the mapping of EDS. The Co content is relatively small compared to the other elements, but it is still distributed relatively evenly over the surface. To further understand the crystal structure of the material, the XRD spectrum of the material was obtained, and the results are shown in Figure 1b. The XRD diffraction peaks of the Ni-Co m-MOFs synthesized in this experiment match well with the simulated Ni-Co m-MOFs, providing evidence for the successful synthesis of Ni-Co m-MOFs and indicating their good crystallinity [27]. XPS analysis (Figure 1c) reveals the main types of elements present in Ni-Co m-MOFs, while EDS analysis (Figure 1d) further verifies the elemental composition of carbon, oxygen, nitrogen, nickel, and cobalt in Ni-Co m-MOFs. Nitrogen adsorption–desorption experimental treatments were measured for Ni-Co m-MOFs, as shown in Figure S2; the results showed that the synthesized Ni-Co m-MOFs have a large specific surface area, which can reach 35.18 m²/g. Their pore sizes are mostly spread from 2 to 10 nm. The Pore Volume of Ni-Co m-MOFs is 0.04 cc/g.

The CL intensity of the luminol-H₂O₂ system with and without the addition of MOFs is depicted in Figure 2a. As can be seen from the figure, the intensity of CL increases significantly after the addition of MOFs. Compared to the system without the addition of Ni-Co m-MOFs, the enhancement is about two thousandfold. Figure 2b reveals that the CL emission peak of the Ni-Co m-MOF-luminol-H₂O₂ system is observed at 450 nm. Notably, this emission peak aligns with the maximum emission peak at 450 nm for the luminol-H₂O₂ system on the instrument used. The difference from the widely reported result of 425 nm was interpreted as an instrumental error. Additionally, the Ni-Co m-MOFs PL spectra display a narrower emission range of 415–425 nm (Figure 2c), which does not overlap with the emission range of the Ni-Co m-MOF-luminol-H₂O₂ system. This observation suggests that chemically initiated electron exchange luminescence resonance transfer (CRET) did not occur during the emission of the Ni-Co m-MOF-luminol-H₂O₂ system. There was almost no change in the absorption peaks of the UV–Vis spectra for the luminol-H₂O₂ system after the addition of Ni-Co m-MOFs (Figure 2d), which has further confirmed that the Ni-Co m-MOF materials mainly act as catalysts in the emission process of the luminol-H₂O₂ system.

To achieve maximum enhancement of CL in the luminol-H₂O₂ system facilitated by Ni-Co m-MOFs, we conducted comprehensive optimization of the experimental conditions. We maintained a constant voltage of 500 V on the ultramicro CL instrument while varying the H₂O₂ concentration from 1 × 10⁻⁴ M to 1.0 M. Through this, we determined that the optimum CL intensity was achieved when using H₂O₂ at a concentration of 1 × 10⁻³ M (Figure 3a). Furthermore, we explored the impact of luminol concentration by ranging it from 1 × 10⁻⁵ M to 1 × 10⁻⁴ M and identified that the CL intensity was attained at a luminol concentration of 1 × 10⁻⁴ M (Figure 3b). The quantity of Ni-Co m-MOFs varied between 0 and 200 μL, and the highest CL intensity was attained when adding 100 μL of Ni-Co m-MOFs (Figure 3c).

Following that, we investigated the order of reagent addition (Figure 3d). When luminol was injected into the mixture of Ni-Co m-MOFs and H₂O₂, the CL signal was observed to be weak. However, when H₂O₂ was injected into the mixture of Ni-Co m-MOFs and luminol, a more significant CL signal was observed. This suggests that Ni-Co m-MOFs acted as a catalyst, facilitating the decomposition of H₂O₂ and generating reactive intermediates that readily reacted with luminol. In the first injection sequence, the active intermediates were released but did not participate in the CL reaction promptly due to the subsequent injection of luminol, resulting in weaker luminescence intensity. Next, we conducted pH optimization of H₂O₂ to investigate its effect on the luminescence of the Ni-Co m-MOF-luminol-H₂O₂ system. However, the optimized CL intensity at that pH exceeded the ultramicro CL instrument range. Consequently, the voltage was adjusted from 500 V to 200 V to generate the optimized pH curves for the H₂O₂ solution (Figure 3e). As the pH of the H₂O₂ solution increases gradually (initial pH = 6), the enhancement of the CL intensity in the system becomes negligible. Starting at pH = 10, the Ni-Co m-MOF-luminol-H₂O₂ system exhibits a rapid increase in CL intensity, peaking at pH 12. Subsequently, the luminescence intensity of the system exhibits a continuous increase in pH, followed by a rapid decrease. In this regard, the pH of H₂O₂ = 12 provides the optimal CL intensity. Although the CL intensity of the luminol-H₂O₂ system without optimized H₂O₂ solution pH at 200 V was nearly undetectable, these two systems involve multiple variables: (1) the presence or absence of Ni-Co m-MOFs and (2) the pH of H₂O₂. Consequently, we ultimately utilized the Ni-Co m-MOF-luminol-H₂O₂ system without optimized H₂O₂ solution pH to compare the CL with the luminol-H₂O₂ system without optimized H₂O₂ solution pH. Notably, both systems were not optimized for the pH of the H₂O₂ solution. However, it is undeniable that a stronger CL signal was obtained when the pH of H₂O₂ was 12. Therefore, the optimal conditions for achieving the maximum CL signals in the Ni-Co m-MOF-luminol-H₂O₂ system are as follows: Injecting 1 mL of a 1 × 10⁻³ M H₂O₂ solution into a mixture of 100 μL containing 1 mg·mL⁻¹ Ni-Co m-MOFs and 1 mL of a 1 × 10⁻⁴ M luminol enhances the CL intensity by nearly two thousand folds.

The common mechanisms of nanomaterial-enhanced CL in the system generally include the catalytic effect of nanomaterials on the system and their involvement in CRET during the CL process. In this study, we investigated the CL mechanism of the Ni-Co m-MOF-luminol-H₂O₂ system by analyzing a previous paper’s experimental and characterization sections. The CL spectrum of the Ni-Co m-MOF-luminol-H₂O₂ system reveals a maximum emission peak at 450 nm, suggesting that the emission of 3-APA* is mainly attributed to the presence of luminol in the system. The primary cause of luminescence in the CL reaction is the occurrence of a redox reaction, leading to the production of a significant quantity of reactive oxygen species. To ascertain the specific types of reactive oxygen radicals generated, free radical quenching experiments and EPR spectroscopy were employed to identify the specific free radical species present in the Ni-Co m-MOF-luminol-H₂O₂ system.

In this experimental phase, all the reagents and the appropriate injection sequence were utilized in optimal conditions. EPR testing was conducted on a Bruker E500 spectrometer for detecting ¹O₂ in the Ni-Co m-MOF-luminol-H₂O₂ system with TEMP dissolved in a phosphate buffer solution. From Figure 4a, a typical 1:1:1 triplet signal peak was observed. It was found that the addition of Ni-Co m-MOFs to the luminol-H₂O₂ system significantly increased the intensity of the ¹O₂ signal peak. These findings suggest that Ni-Co m-MOFs facilitate the generation of a substantial amount of ¹O₂ within the system and play a vital role in CL emission. The DMPO methanol solution was used to detect the presence of ^•O₂⁻ in the Ni-Co m-MOF-luminol-H₂O₂ system. From Figure 4b, it can be observed that the characteristic peak of the luminol-H₂O₂ system is not prominent. However, after the addition of Ni-Co m-MOFs, a distinct 1:1:1:1 quadruple characteristic peak appears in the system. This indicates that the addition of Ni-Co m-MOFs leads to the formation of newly generated ^•O₂⁻ in the system. Furthermore, Figure 4d shows that BQ (benzoquinone) has a significant inhibitory effect on ^•O₂⁻, further confirming the importance of ^•O₂⁻ in the chemiluminescence emission process of the Ni-Co m-MOF-luminol-H₂O₂ system. An aqueous solution of DMPO was utilized to determine the presence of ^•OH in the system. Figure 4c barely exhibits the characteristic peak of ^•OH in the luminol-H₂O₂ system. However, upon the addition of Ni-Co m-MOFs, the 1:2:2:1 quadruple EPR signal peak of DMPO-^•OH becomes prominently visible, indicating the significant generation of ^•OH in the system facilitated by the introduction of Ni-Co m-MOFs. The substantial inhibitory effects observed when introducing two free radical scavengers, isopropanol (Figure 4e) and p-benzoquinone (Figure 4f), further confirm the intensified generation of ^•OH in the Ni-Co m-MOF-luminol-H₂O₂ system and its impact on the chemiluminescence emission intensity. Moreover, ascorbic acid, functioning as an oxygen-containing free radical scavenger, effectively eliminates three reactive oxygen species, signifying the participation of ^•O₂⁻, ¹O₂, and ^•OH in the chemiluminescence process of the system. Compared with the luminol-H₂O₂ system, the intensity of the EPR signals of the three radicals was significantly enhanced in the Ni-Co m-MOF-luminol-H₂O₂ system, indicating that the presence of Ni-Co m-MOFs facilitated the production of a considerable quantity of reactive oxygen radicals within the system.

In addition, we conducted liquid UV tests on three systems: Ni-Co m-MOF-H₂O₂, Ni-Co m-MOF-luminol, and Ni-Co m-MOF-luminol-H₂O₂. The dispersion of Ni-Co m-MOFs exhibits almost no UV absorption peaks. In contrast, both Ni-Co m-MOF-luminol and Ni-Co m-MOF-luminol-H₂O₂ systems demonstrate two absorption peaks at 295 nm and 350 nm, respectively. These peaks align with the UV absorption peaks of luminol. Notably, the UV spectra of the Ni-Co m-MOF-luminol-H₂O₂ system show no emergence of new characteristic peaks, indicating no initial reaction between Ni-Co m-MOFs and the luminol solution. Combined with the fluorescence spectra of Ni-Co m-MOFs, it is observed that fluorescence within a narrower range (413–427 nm) is only detected at an excitation wavelength of 425 nm (Figure 1d). Significantly, this fluorescence falls within the emission range of the luminescence spectrum of the Ni-Co m-MOF-luminol-H₂O₂ system. This observation leads to the speculation that a portion of the CL process of Ni-Co m-MOFs undergoes CRET. Moreover, when combined with the liquid UV diagram, it indicates that the Ni-Co m-MOFs primarily contribute to the enhancement of CL emission in the luminol-H₂O₂ system through their catalytic effect. To conclude, the solvothermally synthesized bimetallic and ligand Ni-Co m-MOFs exhibited remarkable peroxidase activity in the luminol-H₂O₂ system. The incorporation of Ni-Co m-MOFs induced the disruption of the O−O bond in H₂O₂, leading to the generation of two reactive oxygen radicals, namely ^•O₂⁻ and ^•OH. In a subsequent energy transfer process, ^•O₂⁻ transferred energy to luminol, resulting in the production of ¹O₂. Ultimately, the catalytic effect of Ni-Co m-MOFs facilitated the generation of high-intensity CL. The presence of Ni-Co m-MOFs enhanced the production of radicals and promoted the electron transfer process within the luminol-H₂O₂ system. Subsequently, we employed Ni-Co m-MOFs, Co MOFs, and Ni MOFs to augment the CL of the luminol-H₂O₂ system. As shown in Figure 5, the CL enhancement achieved by Ni MOFs was relatively weak, with only about a five-fold increase. Co MOFs exhibited a moderately enhanced effect on the luminol-H₂O₂ system, although prior studies have already reported CL enhancement using similar materials. In contrast, Ni-Co m-MOFs demonstrated the most pronounced enhancement effect, exhibiting an enhancement of nearly two thousand-fold. Based on these results, we postulate that the exceptional enhancement achieved by Ni-Co m-MOFs in the luminol-H₂O₂ system primarily originates from the synergistic catalytic effect of the bimetallic coordination centers present in the MOFs.

Based on the studies conducted, we present a summary of the roles played by Ni-Co m-MOFs in the luminol-H₂O₂ system and propose the possible CL mechanism of the Ni-Co m-MOF-luminol-H₂O₂ system, illustrated by reaction Equations (1) to (8). Initially, H₂O₂ undergoes a reaction with OH⁻ in the solution, resulting in the formation of a small quantity of H₂O⁻ (Reaction Equation (1)). Furthermore, H₂O₂ decomposes, primarily catalyzed by Ni-Co m-MOFs, yielding a substantial amount of ^•OH (Reaction Equation (2)). Subsequently, HO₂⁻, generated during the first step of the reaction, reacts with a fraction of ^•OH, leading to the formation of ^•O₂⁻ (Reaction Equation (3)). Luminol then becomes involved in the reaction, as it reacts with ^•O₂⁻ to produce a significant amount of L⁻ and ¹O₂ (Reaction Equation (4)). Simultaneously, luminol also reacts with ^•OH, resulting in the formation of L⁻ and HO₂⁻ (Reaction Equation (5)). Lastly, L⁻, produced during the initial two steps of the reaction, reacts, respectively, with ¹O₂ and ^•OH, leading to the formation of 3-APA* (Reaction Equation (6) and Reaction Equation (7). Finally, 3-APA* returns from the excited state to the ground state, resulting in the release of energy in the form of light (Reaction Equation (8)). The mechanistic diagrams illustrated are shown in Figure 6.

H₂O₂ + OH⁻ → HO₂⁻ + H₂O

(1)

H₂O₂ + Ni-Co MOF → 2^•OH

(2)

HO₂⁻ + ^•OH → ^•O₂⁻ + H₂O

(3)

Luminol + OH⁻ → L⁻

(4)

L⁻ + ^•O₂⁻ → 3-APA*

(5)

L⁻ + ¹O₂ → 3-APA*

(6)

L⁻ + ^•OH → 3-APA*

(7)

3-APA* → 3-APA + hv

(8)

3. Materials and Methods

3.1. Reagents and Materials

Cobalt (II) chloride hexahydrate (CoCl₂·6H₂O), 1,3,5-Homobenzenetricarboxylic acid (H₃BTC), benzene dicarboxylic acid (H₂BDC) were bought from Beijing Enochian Reagent Co. 1,4-benzoquinone (C₆H₄O₂) and thiourea (CH₄N₂S) were bought from Aladdin Reagent Co., Ltd. (Shanghai, China). N-N-Dimethylformamide (C₃H₇NO, DMF), Nickel (II) nitrate hexahydrate (Ni(NO₃)₂·6H₂O), isopropanol (C₃H₈O), and ethanol (C₂H₅OH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). H₂O₂ and hydrochloric acid (HCl) were purchased from Beijing Chemical Reagent Co., Ltd. (Beijing, China). Caustic soda (NaOH), 5,5-Dimethyl-1-pyrrole-n-oxide oxide (C₆H₁₁NO, DMPO), and 2,2,6,6,6-tetramethylpiperidine (C₉H₁₉N, TEMP) were purchased from Sigma-Aldrich Reagents Ltd. (Saint Louis, MO, USA). All reagents were used as specified without any additional purification unless specifically instructed for this experiment. All reagents used were of analytical purity, and deionized water was utilized to prepare all aqueous solutions.

3.2. Apparatus

Batch CL experiments were carried out with a BPCL ultraweak CL analyzer (BPCL-GP21Q-TGC, Guangzhou Microlight Technology Co., Ltd., Guangzhou, China). The CL profiles were integrated at intervals of 0.01 s. The voltage of the photomultiplier tube (PMT) was set at 1000 V. The photoluminescent (PL) spectra were obtained on an Agilent Cary Eclipse spectrophotometer (F-7000, Hitachi, Tokyo, Japan). The slit width was set as 5 nm, and the voltage of PMT was controlled to be 700 V. Electron paramagnetic resonance (EPR) spectra were conducted on a Bruker (Billerica, MA, USA) E500 spectrometer. The field position was set at 3400 G, and the center field was 3500 G. The sweep width was 200 G, and the number of points was 1024. The attenuation of the microwave was 15 dB. Scanning electron microscope (SEM) images and energy dispersive spectroscopy (EDS) spectrograms were obtained by using a field emission SEM (Zeiss, Oberkochen, Germany). Transmission electron microscopy (TEM) images were recorded by a JEM-F200 transmission electron microscope (JEOL, Tokyo, Japan) with a voltage of 120 kV. X-ray photoelectron spectrum (XPS) was measured by an X-ray photoelectron spectrometer (AXIS SUPRA, SHIMADZU, Tokyo, Japan) equipped with a monochromated Al-K X-ray source (1486.6 eV) at a pass energy of 40 eV.

3.3. Synthesis of Ni-Co m-MOFs

Mixed-ligand MOFs, known as Ni-Co m-MOFs, incorporating nickel and cobalt ions along with double gold and double ligands, were synthesized using a straightforward solvothermal method. A total of 1.164 g of Ni(NO₃)₂·6H₂O, 0.096 g of CoCl₂·6H₂O (the molar ratio is 10:1), 0.740 g of H₃BTC, and 0.584 g of H₂BDC (the molar ratio is 1:1) were dissolved in a pre-mixed solution comprising 20 mL of DMF and 20 mL of water. The resulting mixture was sonicated at room temperature until it became clear. Subsequently, the solution was transferred to a 100 mL PTFE-lined reactor and placed in an oven at 120 °C for 24 h. After the reactor naturally cooled to room temperature, the sample underwent diafiltration, being washed three times with DMF and then three times with anhydrous ethanol. The washed solid was subsequently dried in an oven at 80 °C for 12 h, resulting in a light-green solid powder.

3.4. Chemiluminescence Investigation of Ni-Co m-MOF-luminol-H₂O₂ System

Chemiluminescence Emission Experiment

To investigate the enhancing effect of Ni-Co m-MOFs on the CL strength of the luminol-H₂O₂ system, we prepared a Ni-Co m-MOF suspension at a concentration of 1 mg·mL⁻¹ using water as the solvent. During the assay conducted using the ultramicro CL instrument, the H₂O₂ solution was injected into a mixture comprised of a suspension of Ni-Co m-MOFs and a solution of luminal. In the control experiments, the suspension of Ni-Co m-MOFs was not added. The comparison of the chemiluminescent strengths between the two systems was utilized to evaluate the potential of Ni-Co m-MOFs to enhance the CL. The concentrations of luminol and H₂O₂, the quantity of Ni-Co m-MOFs dispersion added, and the pH of H₂O₂ were optimized in the Ni-Co m-MOF-luminol-H₂O₂ optimization to achieve the most favorable CL emission. Through the single variable method, the concentrations of luminol (1 × 10⁻⁵, 4 × 10⁻⁵, 6 × 10⁻⁵, 8 × 10⁻⁵, and 1 × 10⁻⁴ M) and hydrogen peroxide (1 × 10⁻⁴, 1 × 10⁻³, 1 × 10⁻², 1 × 10⁻¹, and 1 M) were adjusted independently. The optimal conditions for the CL experiment were determined by optimizing the pH of H₂O₂ under the best concentration conditions and varying the amount of Ni-Co m-MOFs dispersion (0, 50, 100, 150, and 200 μL).

The effect of reagent injection sequence on the chemiluminescence intensity

All reagents were used at the optimal concentrations obtained in the above experiments. To explore the optimal injection sequence for the Ni-Co m-MOF-luminol-H₂O₂ system, we investigated the impact of different injection sequences of luminol and H₂O₂ reagents on the CL intensity of the system. This experimental phase primarily focused on two injection sequences: one involved injecting a 1 × 10⁻⁴ M luminol solution into a mixture of 100 μL Ni-Co m-MOFs suspension and a 1 × 10⁻³ M H₂O₂ solution, whereas the other method entailed adding a 1 × 10⁻³ M H₂O₂ solution to a mixture of 100 μL Ni-Co m-MOFs suspension and a 1 × 10⁻⁴ M luminol solution.

Chemiluminescence spectra of the Ni-Co m-MOF-luminol-H₂O₂ system

Both the injection sequence and the solution concentration used in the Ni-Co m-MOF-luminol-H₂O₂ system were the optimal choices obtained from previous explorations. To acquire the CL spectra of the Ni-Co m-MOF-luminol-H₂O₂ system, a filter ranging from 350 to 625 nm was positioned between the photomultiplier tube and the reaction cell in the ultramicro CL instrument. The CL intensity was measured at intervals of 0.001 s until it reached a point where its decrease was almost negligible.

3.5. Investigation of the Chemiluminescence Mechanism of Ni-Co m-MOF-luminol-H₂O₂ System

The luminescence mechanism of the Ni-Co m-MOF-luminol-H₂O₂ system was deduced through EPR tests and free radical quenching experiments. For the detection of the three reactive oxygen species, namely ¹O₂, ^•O₂⁻, and ^•OH, methanol solutions of TEMP, DMPO, and aqueous solutions of DMPO dissolved in phosphate buffer were utilized, respectively. Four different free radical quenchers, namely p-benzoquinone, isopropanol, thiourea, and L-ascorbic acid, were added to the Ni-Co m-MOF-luminol-H₂O₂ system. Observations of the changes in the CL intensity were conducted to identify the reactive oxygen radicals present.

4. Conclusions

In conclusion, we successfully synthesized Ni-Co m-MOFs using a solvothermal method, resulting in structures with a dense, round, spherical, flower-like morphology. These synthesized Ni-Co m-MOFs exhibited a remarkable enhancement in the weak CL of the luminol-H₂O₂ system. After repeatedly optimizing all experimental conditions, we obtained the best chemical experimental conditions, leading to a more than two-thousand-fold enhancement in the CL signal of the luminol-H₂O₂ system. By elucidating the mechanism of the Ni-Co m-MOF-luminol-H₂O₂ system, we uncovered that Ni-Co m-MOFs effectively facilitated the decomposition of H₂O₂ and acted as catalysts for the generation of ¹O₂ on the surface of the material. Unfortunately, luminol’s emission wavelength is in the visible region, and the system needs to be in an alkaline environment to reach its strongest emission. This results in a system that is only suitable for in vitro detection, and the use of catalysts to enhance chemiluminescence for in vivo imaging should be further investigated. This research contributes to the advancement of the field by expanding the understanding of constructing novel CL systems based on mixed-ligand MOF materials. Furthermore, it provides valuable insights into the broader applications of the CL analytical method.