Electrochemical sensing platform and electrochemical method for simultaneously detecting staphylococcus aureus and listeria monocytogenes

Abstract

The invention relates to an electrochemical sensing platform for simultaneously detecting staphylococcus aureus and listeria monocytogenes. The electrochemical sensing platform is prepared by the following steps: step 1, preparing a Ti < 3 > C < 2 > T < x > nanobelt Ti < 3 > C < 2 > T < x > NR; step 2, preparing a Ti < 3 > C < 2 > T < x > NR / Cu nano composite material by adopting a self-reduction method; step 3, constructing S-Apt / MB (at) UiO and L-Apt / Fc (at) UiO; step 4, preparing an electrode: dispensing the Ti3C2TxNR / Cu solution on the surface of a silk-screen printing carbon electrode SPCE to prepare a Ti3C2TxNR / Cu / SPCE modified electrode; the Ti3C2TxNR solution is dispensed on the surface of a silk-screen printing carbon electrode SPCE, and a Ti3C2TxNR / SPCE control electrode is prepared. The method can realize simultaneous quantitative detection of the two pathogenic bacteria, and has a wide application prospect in the field of food safety monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of common pathogenic bacteria. Background Art

[0002] Food safety, as a major global issue, has attracted much attention due to its direct impact on public health and socioeconomics. Foodborne diseases, as one of the major threats, not only endanger human health but also cause significant economic losses. It is worth noting that contaminated food often contains multiple pathogens, which accelerates food spoilage and increases health risks. Common pathogens include Staphylococcus aureus (S. aureus), Listeria monocytogenes (LM), Shigella flexneri (S. flexneri), Vibrio parahaemolyticus (V. para), Escherichia coli (E. coli), and Salmonella enteritidis (S. enteriti).

[0003] Electrochemical sensing technology has been widely used for the detection of various pathogens due to its advantages such as ease of operation, low cost, rapid response, and high sensitivity. However, studies on the simultaneous detection of two high-risk pathogens, Staphylococcus aureus and Listeria monocytogenes, remain rare. These two pathogens produce potent toxins that can cause serious diseases such as cancer, endocarditis, and abscesses. Therefore, the development of reliable, simultaneous qualitative and quantitative detection methods for these two pathogens is urgently needed to improve food safety monitoring.

[0004] The key to developing high-performance electrochemical sensors lies in designing electrode materials with signal amplification capabilities. Among the many emerging candidate materials, two-dimensional Ti3C2Tx MXene has attracted considerable attention for applications in hydrogen evolution reactions, photocatalysis, energy storage systems, and sensing due to its intrinsic properties, including ultrahigh surface area, excellent conductivity, and natural hydrophilicity. Particularly noteworthy is the unique non-bonded titanium state of Ti3C2Tx MXene, which provides additional electron transfer pathways, enabling the reduction of specific metal ions without the need for external reducing agents. This intrinsic reducing ability enables the construction of signal-amplifying nanocomposites by in situ modification of metal nanoparticles. Furthermore, recent research demonstrates that combining nanoporous materials with aptamers (aptamers) can enable the construction of intelligent stimuli-responsive systems based on biomolecular gating mechanisms. While porous frameworks such as ZIF-8, UiO-66, and mesoporous silica have been successfully used to construct biogated nanomaterials for target detection, the application of these materials in electrochemical pathogen sensing remains a major research gap. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that there is no method for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes in the prior art. The present invention proposes an electrochemical sensing platform and electrochemical method for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, which can simultaneously and quantitatively detect these two pathogens.

[0006] In order to solve the above technical problems, the present invention proposes the following technical solution: an electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, which is manufactured by the following steps:

[0007] Step 1: Preparation of Ti3C2T x Nanobelt Ti3C2T x NR;

[0008] Step 2: Preparation of Ti3C2T by self-reduction method x NR / Cu nanocomposites;

[0009] Step 3: Construction of S-Apt / MB@UiO and L-Apt / Fc@UiO;

[0010] Step 4: Electrode preparation: Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE reference electrode.

[0011] The above technical solution is further limited in that step 1 further includes step 1a and step 1b:

[0012] Step 1a: Etching treatment: Disperse Ti3AlC2 powder in hydrofluoric acid and stir continuously. After the reaction, collect Ti3C2T by centrifugation. x Nanosheet Ti3C2T x NS and washed repeatedly with deionized water until the supernatant was neutral;

[0013] Step 1b: Tearing treatment: The obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in KOH solution and reacted under N2 atmosphere at 25℃ to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR.

[0014] The above technical solution is further limited in that step 2 further includes step 2a and step 2b:

[0015] Step 2a: Add CuCl2 solution dropwise to Ti3C2T under stirring. x In NR solution;

[0016] Step 2b: Obtain Ti3C2T by centrifugation x The NR / Cu nanocomposite material was dried and set aside.

[0017] The above technical solution is further limited in that step 3 further includes step 3a and step 3b;

[0018] Step 3a: Synthesis of UiO-66: ZrCl4, NH2-BDC and benzoic acid were dissolved in DMF and ultrasonicated, and then transferred to a reactor for reaction to obtain UiO-66 crystals.

[0019] Step 3b: Probe encapsulation: UiO-66 was dispersed in MB solution and shaken, followed by the addition of the aptamer S-Apt and shaking. After centrifugation and washing, S-Apt / MB@UiO was obtained, which was stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken, followed by the addition of the aptamer L-Apt and shaking. After centrifugation and washing, L-Apt / Fc@UiO was obtained, which was stored in pH 7.0 buffer.

[0020] The above technical solution is further limited in that it is made by the following steps:

[0021] Step 1: Preparation of Ti3C2T x Nanobelt Ti3C2T x NR, including step 1a and step 1b;

[0022] Step 1a: Etching treatment: 1.0 g of Ti3AlC2 powder was dispersed in 30.0 mL of hydrofluoric acid and stirred for about 24 hours. After the reaction, the Ti3C2T x Nanosheet Ti3C2T x NS and washed repeatedly with deionized water until the supernatant was neutral;

[0023] Step 1b: Tearing treatment: The obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in a 6.0 M KOH solution and reacted for 108 h under N2 atmosphere and 25 °C to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR;

[0024] Step 2: Preparation of Ti3C2T by self-reduction method xNR / Cu nanocomposite material, comprising step 2a and step 2b;

[0025] Step 2a: Under vigorous stirring, 1.5 mL of 0.1 M CuCl2 solution was added dropwise to 50.0 mL of 1.0 mg / mL Ti3C2T x In NR solution;

[0026] Step 2b: After 20 minutes of reaction, Ti3C2T was obtained by centrifugation. x The NR / Cu nanocomposite material was dried and then used;

[0027] Step 3: Construction of S-Apt / MB@UiO and L-Apt / Fc@UiO, including steps 3a and 3b;

[0028] Step 3a: Synthesis of UiO-66: 75.0 mg of ZrCl4, 50.0 mg of NH2-BDC, and 1.5 g of benzoic acid were dissolved in DMF and ultrasonicated. The mixture was transferred to a reactor and subjected to hydrothermal reaction at 120°C for 12 hours to obtain UiO-66 crystals.

[0029] Step 3b: Probe encapsulation: UiO-66 was dispersed in MB solution and shaken overnight, followed by the addition of the aptamer S-Apt and shaking for 1 hour. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken overnight, followed by the addition of the aptamer L-Apt and shaking for 1 hour. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer.

[0030] Step 4: Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE reference electrode.

[0031] In order to solve the above technical problems, the present invention proposes the following technical solution: an electrochemical method for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, comprising the following steps:

[0032] Step 1: Preparation of Ti3C2T by simple self-reduction method x NR / Cu nanocomposites;

[0033] Step 2: The two electrochemical probes, MB and Fc, were encapsulated in the UiO-66 metal-organic framework, respectively, and the functionalized S. aureus aptamer S-Apt and LM aptamer L-Apt were used as biological gating elements, denoted as S-Apt / MB@UiO and L-Apt / Fc@UiO;

[0034] Step 3: Electrode preparation: Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE control electrode;

[0035] Step 4: Pathogen detection: Different concentrations of target pathogens S. aureus and LM were incubated with a mixed solution of S-Apt / MB@UiO and L-Apt / Fc@UiO in a 1:1 ratio, diluted with Tris-HCl buffer, and then added dropwise to the Ti3C2T x On the surface of the NR / Cu / SPCE electrode, differential pulse voltammetry was used for signal detection.

[0036] The above technical solution is further limited in that step 1 further includes the following steps:

[0037] Preparation of Ti3C2T x Nanobelt Ti3C2T x NR, including: Etching treatment: Disperse Ti3AlC2 powder in hydrofluoric acid, stir continuously, collect Ti3C2T by centrifugation after reaction x Nanosheet Ti3C2T x NS, and repeatedly washed with deionized water until the supernatant was neutral; tearing treatment: the obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in KOH solution and reacted under N2 atmosphere at 25℃ to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR;

[0038] Under stirring conditions, CuCl2 solution was added dropwise to Ti3C2T x In NR solution;

[0039] Ti3C2T was obtained by centrifugal separation x The NR / Cu nanocomposite material was dried and set aside.

[0040] The above technical solution is further limited in that step 2 further includes the following steps:

[0041] Synthesis of UiO-66: ZrCl4, NH2-BDC and benzoic acid were dissolved in DMF, ultrasonicated and transferred to a reactor for reaction to obtain UiO-66 crystals.

[0042] Probe encapsulation: UiO-66 was dispersed in MB solution and shaken, followed by the addition of the aptamer S-Apt and shaking. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken, followed by the addition of the aptamer L-Apt and shaking. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer.

[0043] The above technical solution is further limited in that the electrochemical method for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes comprises the following steps:

[0044] Step 1: Preparation of Ti3C2T by self-reduction method x The NR / Cu nanocomposite material further comprises the following steps:

[0045] Preparation of Ti3C2T x Nanobelt Ti3C2T x NR, including: Etching treatment: 1.0g Ti3AlC2 powder was dispersed in 30.0mL hydrofluoric acid and stirred for about 24 hours. After the reaction, Ti3C2T was collected by centrifugation. x Nanosheet Ti3C2T x NS, and repeatedly washed with deionized water until the supernatant was neutral; tearing treatment: the obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in a 6.0 M KOH solution and reacted for 108 h under N2 atmosphere and 25 °C to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR;

[0046] Under vigorous stirring, 1.5 mL of 0.1 M CuCl2 solution was added dropwise to 50.0 mL of 1.0 mg / mL Ti3C2T x In NR solution;

[0047] After 20 minutes of reaction, Ti3C2T x The NR / Cu nanocomposite material was dried and then used;

[0048] Step 2: Constructing S-Apt / MB@UiO and L-Apt / Fc@UiO, further including the following steps:

[0049] Synthesis of UiO-66: 75.0 mg of ZrCl4, 50.0 mg of NH2-BDC, and 1.5 g of benzoic acid were dissolved in DMF and ultrasonicated. The mixture was transferred to a reactor and hydrothermally reacted at 120°C for 12 hours to obtain UiO-66 crystals.

[0050] Probe encapsulation: UiO-66 was dispersed in MB solution and shaken overnight, followed by the addition of the aptamer S-Apt and shaking for 1 hour. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken overnight, followed by the addition of the aptamer L-Apt and shaking for 1 hour. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer.

[0051] Step 3: Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE control electrode;

[0052] Step 4: Pathogen detection: 20 μL of target pathogens S. aureus and LM at different concentrations were incubated with 40 μL of a 1:1 mixed solution of S-Apt / MB@UiO and L-Apt / Fc@UiO for 30 minutes, diluted to 0.2 mL with Tris-HCl buffer, and then added dropwise to the Ti3C2T x On the surface of the NR / Cu / SPCE electrode, differential pulse voltammetry was used for signal detection.

[0053] Compared with the prior art, the present invention has the following beneficial effects: the present invention synthesizes Ti3C2T for the first time through a simple self-reduction method. x MXene nanobelts / copper nanoparticles (Ti3C2T x NR / Cu) was used for electrode modification to achieve signal amplification, and a UiO-66 metal-organic framework encapsulating different probes and gated by targeting aptamers (Apts) was constructed as a biological gating system. Based on this, an innovative electrochemical method was proposed for the simultaneous detection of two typical pathogens, Staphylococcus aureus (S. aureus) and Listeria monocytogenes (LM).x NR / Cu nanohybrid materials through synergistic Ti3C2T x MXene nanoribbons and copper nanoparticles (Ti3C2T x The advantages of NR / Cu) significantly enhance the probe current signal. For S. aureus detection, methylene blue (MB) and S. aureus specific aptamer (S-Apt) are used as probes and biological gating elements respectively; while for LM detection, ferrocene (Fc) and LM specific aptamer (L-Apt) are used as probes and biological gating elements respectively. When the target pathogens are present, the aptamer dissociates from UiO-66 and releases the encapsulated probe (MB or Fc), which is then transferred to the Ti3C2T x The current signal generated by the NR / Cu / SPCE-modified electrode enables simultaneous quantitative detection of two pathogens. The modular design of this sensor allows for the detection of other bacteria by simply replacing the aptamer, showing broad application prospects in food safety monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Ti3C2T is prepared based on the self-reduction method x Schematic diagram, SEM image and TEM image of NR / Cu nanohybrid material; Figure 1 (A) is based on the self-reduction method to prepare Ti3C2T x Schematic diagram of NR / Cu nanohybrid material; Figure 1 Middle (B) is Ti3C2T x SEM image of NS; Figure 1 (C) is Ti3C2T x SEM image of NR; Figure 1 Middle (D) is Ti3C2T x SEM image of NR / Cu; Figure 1 Middle (E) is Ti3C2T x TEM image of NS; Figure 1 (F) is Ti3C2T x TEM image of NR; Figure 1 (G) is Ti3C2T x TEM image of NR / Cu.

[0055] Figure 2 Ti3C2T x Energy dispersive spectrometry (EDS) spectrum of NR / Cu nanohybrid materials.

[0056] Figure 3 Ti3C2T x Differential pulse voltammetry (DPV) curves of NR / Cu / SPCE electrode.

[0057] Figure 4 Based on Ti3C2T x Schematic diagram of the principle of simultaneous electrochemical detection of Staphylococcus aureus (S. aureus) and Listeria monocytogenes (LM) using NR / Cu electrodes coupled with S-Apt / MB@UiO and L-Apt / Fc@UiO.

[0058] Figure 5 is a graph showing the impact of key parameters on detection performance; Figure 5 (A) is Ti3C2T x NR / Cu dosage 10 3 CFU mL -1 The effect of (a) S. aureus and (b) LM detection under different concentrations; Figure 5 (b) is the effect of probe concentration on 10 3 CFU mL -1 The effect of (a) S. aureus and (b) LM detection under different concentrations; Figure 5 (C) is the incubation time for 10 3 CFU mL -1 Effect of concentration on (a) S. aureus and (b) LM detection.

[0059] Figure 6 Ti3C2T x Plot of the differential pulse voltammetry (DPV) curves of the NR / Cu / SPCE electrode in response to (A) Staphylococcus aureus (S. aureus) and (B) Listeria monocytogenes (LM) and their linear relationships.

[0060] Figure 7 Ti3C2T x Graphs showing specificity analysis of the NR / Cu / SPCE electrode responses to (A) Staphylococcus aureus (S. aureus) and (B) Listeria monocytogenes (LM). DETAILED DESCRIPTION

[0061] The present invention provides an electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, which is manufactured by the following steps:

[0062] Step 1: Preparation of Ti3C2T x Nanobelts (Ti3C2T x NR), comprising step 1a and step 1b.

[0063] Step 1a: Etching treatment: 1.0 g of Ti3AlC2 powder was dispersed in 30.0 mL of hydrofluoric acid (HF) and stirred for about 24 hours. After the reaction, the Ti3C2Tx Nanosheets (Ti3C2T x NS) and washed repeatedly with deionized water until the supernatant was neutral (pH ≈ 7.0).

[0064] Step 1b: Tearing treatment: The obtained Ti3C2T x Nanosheets (Ti3C2T x NS) was placed in a 6.0 M KOH solution and reacted for 108 h under N2 atmosphere and 25 °C to obtain Ti3C2T by longitudinal tearing. x Nanobelts (Ti3C2T x NR).

[0065] Step 2: Preparation of Ti3C2T by self-reduction method x The NR / Cu nanocomposite material includes steps 2a and 2b.

[0066] Step 2a: Under vigorous stirring, 1.5 mL of 0.1 M CuCl2 solution was added dropwise to 50.0 mL of 1.0 mg / mL Ti3C2T x NR solution.

[0067] Step 2b: After 20 minutes of reaction, Ti3C2T was obtained by centrifugation. x The NR / Cu nanocomposite material was dried and set aside.

[0068] Step 3: Construction of S-Apt / MB@UiO and L-Apt / Fc@UiO, including steps 3a and 3b.

[0069] Step 3a: Synthesis of UiO-66: 75.0 mg of ZrCl4, 50.0 mg of NH2-BDC and 1.5 g of benzoic acid were dissolved in DMF (N,N-dimethylformamide) and ultrasonically treated. The mixture was transferred to a reactor and hydrothermally reacted at 120°C for 12 hours to obtain UiO-66 crystals.

[0070] Step 3b: Probe encapsulation: UiO-66 was dispersed in MB (methylene blue) solution and shaken overnight, followed by the addition of the aptamer S-Apt and shaking for 1 hour. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc (ferrocene) solution and shaken overnight, followed by the addition of the aptamer L-Apt and shaking for 1 hour. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer.

[0071] Step 4: Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T xNR / Cu solution was drop-coated on the surface of screen-printed carbon electrode (SPCE) to prepare Ti3C2T x NR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode (SPCE) to make Ti3C2T x NR / SPCE control electrode. Thus, an electrochemical sensing platform for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes was obtained.

[0072] The present invention also provides an electrochemical method for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, comprising the following steps:

[0073] Step 1: According to the steps 1 and 2 of the above-mentioned electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, Ti3C2T was prepared by a simple self-reduction method. x NR / Cu nanocomposites are used to make modified electrodes to enhance the response signal.

[0074] Step 2: According to step 3 of the above-mentioned electrochemical sensing platform for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, two electrochemical probes, methylene blue (MB) and ferrocene (Fc), were encapsulated in the UiO-66 metal-organic framework, respectively, and functionalized S. aureus aptamer (S-Apt) and LM aptamer (L-Apt) were used as biological gating elements, denoted as S-Apt / MB@UiO and L-Apt / Fc@UiO.

[0075] Step 3: Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode (SPCE) to prepare Ti3C2T x NR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode (SPCE) to make Ti3C2T x NR / SPCE reference electrode.

[0076] Step 4: Pathogen detection: 20 μL of target pathogens (S. aureus / LM) at different concentrations were incubated with 40 μL of a 1:1 mixed solution of S-Apt / MB@UiO and L-Apt / Fc@UiO for 30 minutes, diluted to 0.2 mL with Tris-HCl buffer, and then added dropwise to the Ti3C2T xOn the surface of NR / Cu / SPCE electrode, differential pulse voltammetry (DPV) was used for signal detection.

[0077] The principle of pathogen detection in step 4 is: when there are no pathogens, MB and Fc generate almost no electrochemical signals because the probe molecules are confined in the UiO-66 nanopores; when the target pathogens, Staphylococcus aureus (S. aureus) and Listeria monocytogenes (LM), are added, S-Apt and L-Apt will specifically recognize S. aureus and LM respectively, causing the aptamer-gated UiO-66 probe system to open, release the encapsulated probe and generate an electrochemical response. Finally, through Ti3C2T x The NR / Cu / SPCE modified electrode detected the characteristic signals of MB and Fc, respectively, and achieved the quantitative analysis of S. aureus and LM.

[0078] 1. Experimental

[0079] 1.1 Reagents and Materials

[0080] Ti3AlC2 (99.0%) was purchased from Beijing Forsman Technology Co., Ltd.; hydrofluoric acid (HF, ≥40%), CuCl2, zirconium chloride (ZrCl4), 2-aminoterephthalic acid (NH2-H2BDC), benzoic acid (BEN), trishydroxymethylaminomethane (Tris), ferrocene (Fc) and sodium hydroxide (NaOH) were purchased from Aladdin Biotechnology Co., Ltd.; Staphylococcus aureus (S. aureus), Listeria monocytogenes (LM), Shigella flexneri (S. flexneri), Vibrio parahaemolyticus (V. para), Escherichia coli (E. coli) and Salmonella enteritidis (S. enteriti) strains were obtained from the China Industrial Microbiological Culture Collection Center; S. aureus aptamer (S-Apt) and LM aptamer (L-Apt) were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; all solutions used in the experiments were prepared with ultrapure water.

[0081] 1.2 Ti3C2T x Preparation of NR / Cu

[0082] Refer to the literature and improve it to prepare Ti3C2T x Nanobelts (Ti3C2T x NR), the specific process is as follows:

[0083] (a) Etching treatment: 1.0 g of Ti3AlC2 powder was dispersed in 30.0 mL of hydrofluoric acid (HF) and stirred for about 24 h. After the reaction, the Ti3C2T x Nanosheets (Ti3C2T xNS) and washed repeatedly with deionized water until the supernatant was neutral (pH ≈ 7.0).

[0084] (b) Tearing treatment: The obtained Ti3C2T x Nanosheets (Ti3C2T x NS) was placed in a 6.0 M KOH solution and reacted for 108 h under N2 atmosphere and 25 °C to obtain Ti3C2T by longitudinal tearing. x Nanobelts (Ti3C2T x NR).

[0085] Subsequently, Ti3C2T was prepared by self-reduction method. x NR / Cu nanocomposite materials, the specific process is as follows:

[0086] (a) Under vigorous stirring, 1.5 mL of 0.1 M CuCl2 solution was added dropwise to 50.0 mL of 1.0 mg / mL Ti3C2T x In NR solution;

[0087] (b) After 20 minutes of reaction, Ti3C2T x The NR / Cu nanocomposite material was dried and set aside.

[0088] 1.3 Construction of S-Apt / MB@UiO and L-Apt / Fc@UiO

[0089] Synthesis of UiO-66: 75.0 mg of ZrCl4, 50.0 mg of NH2-BDC and 1.5 g of benzoic acid were dissolved in DMF (N,N-dimethylformamide) and ultrasonically treated. The mixture was transferred to a reactor and hydrothermally reacted at 120°C for 12 hours to finally obtain UiO-66 crystals.

[0090] Probe encapsulation: UiO-66 was dispersed in MB (methylene blue) solution and shaken overnight, followed by the addition of the aptamer S-Apt and shaking for 1 hour. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc (ferrocene) solution and shaken overnight. After the addition of the aptamer L-Apt and shaking for 1 hour, L-Apt / Fc@UiO was obtained after centrifugation and washing, and stored in pH 7.0 buffer.

[0091] 1.4 Electrochemical Detection of Staphylococcus aureus and Listeria monocytogenes

[0092] Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode (SPCE) to prepare Ti3C2T xNR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode (SPCE) to make Ti3C2T x NR / SPCE reference electrode.

[0093] Pathogen detection: 20 μL of target pathogens (S. aureus / LM) at different concentrations were incubated with 40 μL of a 1:1 mixed solution of S-Apt / MB@UiO and L-Apt / Fc@UiO for 30 minutes, diluted to 0.2 mL with Tris-HCl buffer, and then added dropwise to the Ti3C2T x On the surface of NR / Cu / SPCE electrode, differential pulse voltammetry (DPV) was used for signal detection.

[0094] 2. Results and Discussion

[0095] 2.1 Material characterization

[0096] Figure 1 (A) is based on the self-reduction method to prepare Ti3C2T x Schematic diagram of NR / Cu nanohybrid material. Figure 1 Middle (B) is Ti3C2T x SEM image of NS. Figure 1 (C) is Ti3C2T x SEM image of NR. Figure 1 Middle (D) is Ti3C2T x SEM image of NR / Cu. Figure 1 Middle (E) is Ti3C2T x TEM image of NS. Figure 1 (F) is Ti3C2T x TEM image of NR. Figure 1 (G) is Ti3C2T x TEM image of NR / Cu. Figure 2 Ti3C2T x Energy dispersive spectrometry (EDS) spectrum of NR / Cu nanohybrid materials.

[0097] Figure 1 (A) shows Ti3C2T x The self-reduction preparation process of NR / Cu was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). x NS, Ti3C2T x NR and Ti3C2T x The microstructure and morphology of NR / Cu were characterized. Figure 1Middle (B) shows Ti3C2T x The SEM image of NS shows a two-dimensional loose wrinkled morphology. x NR (see Figure 1 (C) and Ti3C2T x NR / Cu(see Figure 1 The SEM image of (D) shows a similar nanobelt structure with no obvious difference, which may be due to the extremely small size of copper nanoparticles (CuNPs). TEM image shows that Ti3C2T x NSs aggregate with each other (see Figure 1 (E)), and the prepared Ti3C2T x NR is loosely arranged with a width of about 20 nm (see Figure 1 (F)), which increases the number of active sites and is conducive to loading more CuNPs. Figure 1 Middle (G) shows Ti3C2T x A large number of tiny CuNPs were formed on the surface of NR. Energy dispersive spectroscopy (EDS) confirmed the successful introduction of Cu, which accounted for 16.3% (see Figure 2 ). These results confirm the successful preparation of Ti3C2TxNR / Cu.

[0098] The crystal structures of these materials were characterized and analyzed by X-ray diffraction (XRD). x The diffraction peak of NS at 8.8° corresponds to the (002) crystal plane, indicating the presence of Ti3C2T x When Ti3C2T x NS is alkalized to form Ti3C2T x When NR is used, the diffraction peak of (002) crystal plane shifts from 8.8° to 7.0°, which is consistent with the expansion of interlayer spacing. x NR is covered by CuNPs, so Ti3C2T x The diffraction peak of NR / Cu at (002) plane is higher than that of Ti3C2T x NR is sharper, and the diffraction peak shifts from 7.0° to 6.1°, moving toward lower angles, corresponding to the increase in interlayer spacing.

[0099] 2.2 Method feasibility verification

[0100] To verify the Ti3C2T x The feasibility of the detection scheme constructed by NR / Cu electrode, S-Apt / MB@UiO and L-Apt / Fc@UiO was demonstrated, and the differential pulse voltammetry (DPV) curves of the sensing system towards Staphylococcus aureus (S. aureus) and Listeria monocytogenes (LM) were measured.

[0101] like Figure 3 As shown, when there is no target pathogen, the molecular probes (MB and Fc) are confined in the nanochannels of UiO-66, and Ti3C2T x The current signals of MB and Fc on the NR / Cu / SPCE electrode are extremely low; however, when S. aureus and LM are added, two obvious current peaks appear at -0.12V and 0.29V, corresponding to the oxidation peaks of MB and Fc, respectively. This is because after the target pathogens bind to the corresponding aptamers, the biological gating systems S-Apt / MB@UiO and L-Apt / Fc@UiO are turned on, thereby releasing the encapsulated MB and Fc probes, respectively. It is worth noting that Ti3C2T x The current signal intensity of NR / Cu / SPCE is significantly higher than that of Ti3C2T x NR / SPCE, indicating that the introduction of CuNPs can significantly improve the sensing performance of the modified electrode.

[0102] Figure 3 Ti3C2T x Differential pulse voltammetry (DPV) curves of the NR / Cu / SPCE electrode. Curve a is the response curve in the absence of target pathogens, curve c is the response curve in the presence of S. aureus and LM, and curve b is the response curve of Ti3C2T x DPV responses of the NR / SPCE electrode in the presence of S. aureus and LM.

[0103] Based on Ti3C2T x The sensing strategy of NR / Cu / SPCE electrode coupled with bio-gated probes can achieve specific and simultaneous detection of S. aureus and LM. Figure 4 Based on Ti3C2T x Schematic diagram of the principle of simultaneous electrochemical detection of Staphylococcus aureus (S. aureus) and Listeria monocytogenes (LM) using NR / Cu electrodes coupled with S-Apt / MB@UiO and L-Apt / Fc@UiO.

[0104] 2.3 Detection performance optimization and evaluation

[0105] Before establishing the linear relationship between the probe signal and the target concentration, in order to optimize the performance of the sensing system, the Ti3C2T x Key parameters such as NR / Cu dosage, probe concentration, and incubation time were systematically evaluated (see Figure 5 ). The experiment shows that: with the addition of Ti3C2T x As the amount of NR / Cu increased, the response signals of MB and Fc gradually increased, and when the amount reached 15 μL, the signal reached the maximum value (see Figure 5(A)), so 15μL was selected as the optimal modification amount. At the same time, the concentrations of MB and Fc have a significant impact on the detection results because they directly determine the encapsulation amount of the probe in UiO-66, which in turn affects the analytical signal intensity. By evaluating different concentrations of probes (see Figure 5 (b) found that the DPV signal intensity increased with the increase of probe concentration. When the MB concentration reached 1.2 mg mL -1 (curve a), Fc concentration reaches 1.8 mg mL -1 (curve b) the signal tends to be stable. In addition, the incubation time of the target pathogens and the aptamer has an important influence on the detection signal. Figure 5 As shown in (C), when the incubation time was extended to 30 minutes, the probe signals corresponding to S. aureus and LM both reached peak values, so 30 minutes was selected as the optimal incubation time.

[0106] Figure 5 is a graph showing the impact of key parameters on detection performance. Figure 5 (A) is Ti3C2T x NR / Cu dosage 10 3 CFU mL -1 Effect of concentration on (a) S. aureus and (b) LM detection. Figure 5 (b) is the effect of probe concentration on 10 3 CFU mL -1 Effect of concentration on (a) S. aureus and (b) LM detection. Figure 5 (C) is the incubation time for 10 3 CFU mL -1 Effect of concentration on (a) S. aureus and (b) LM detection. Figure 5 Curve a in (b) detects MB usage in S. aureus. Figure 5 Curve b in (b) detects the amount of Fc used in LM.

[0107] Under the optimal parameter conditions, differential pulse voltammetry (DPV) was used to detect the concentration of pathogenic bacteria through the probe signal. Figure 6 As shown in (A), with the increase of Staphylococcus aureus (S. aureus) concentration, Ti3C2T x The current signal intensity (Is) of methylene blue (MB) on the NR / Cu / SPCE electrode increases gradually, which is due to the release of MB triggered by the binding of S. aureus to S-Apt. 7 CFU mL -1 It shows good linearity within the range, and the linear equation is ΔI s =0.6228log C-0.5231(R 2=0.9934). Similarly, Figure 6 Middle (B) shows the DPV curve and corresponding linear relationship of Listeria monocytogenes (LM) response. It can be seen that the ferrocene (Fc) current signal increases with the increase of LM concentration, and the linear range is 10-10 8 CFU mL -1 , the corresponding equation is ΔI L =0.8272log C-0.7181(R 2 =0.9942). According to the signal-to-noise ratio (S / N=3), the detection limits (LOD) of S. aureus and LM were 2 CFU mL -1 and 3 CFU mL -1 .

[0108] Figure 6 Ti3C2T x Differential pulse voltammetry (DPV) curves of the NR / Cu / SPCE electrode in response to (A) Staphylococcus aureus (S. aureus) and (B) Listeria monocytogenes (LM) and their linear relationships.

[0109] To evaluate the selectivity / specificity of the designed sensor, the responses of pathogens other than the target pathogens (S. aureus and LM) including E. coli, V. para, S. flexner and S. enteriti were tested ( Figure 7 The results showed that only S. aureus (10 5 CFU mL -1 ) and LM(10 5 CFU mL -1 ) can independently produce significant ΔI S and ΔI L The signal changes; however, other pathogens only caused a negligible ΔI response even when their concentration increased 10-fold. Further electrochemical response studies confirmed that the ΔI fluctuations caused by non-target pathogens were minimal, fully demonstrating the high selectivity and specificity of the sensor.

[0110] Figure 7 Ti3C2T x Specificity analysis of the NR / Cu / SPCE electrode responses to (A) Staphylococcus aureus (S. aureus) and (B) Listeria monocytogenes (LM).

[0111] To investigate the reproducibility, eight independent sensing platforms were used to detect 10 5 CFU mL -1Pathogenic bacteria. The results showed that the relative standard deviations (RSDs) of the ΔI signals generated by S. aureus and LM were 3.37% and 2.95%, respectively, demonstrating excellent reproducibility. Stability testing showed that the sensor maintained ≥91.06% of its initial current response after 56 days of storage, demonstrating good long-term stability.

[0112] Finally, the sensor's practical performance was verified in milk samples using a standard addition method. After spiking the samples with pathogenic bacteria solutions at varying concentrations, the probe response signal showed good correlation with the spiked concentrations of S. aureus and LM, with recoveries ranging from 91.3% to 97.6% and relative standard deviations (RSDs) ranging from 2.78% to 3.62%, confirming the sensor's reliable performance in real-world sample testing.

[0113] 3. Conclusion

[0114] In this study, Ti3C2T x By using NR / Cu nanohybrid materials to amplify the electrode response signal, and constructing S-Apt / MB@UiO and L-Apt / Fc@UiO biogating systems for specific pathogen recognition, a highly sensitive electrochemical sensing method for the simultaneous detection of Staphylococcus aureus (S. aureus) and Listeria monocytogenes (LM) was developed. After optimizing key parameters, the sensing method demonstrated excellent analytical performance: the detection limits for S. aureus and LM were as low as 2 CFU·mL, respectively. -1 and 3 CFU·mL -1 The method exhibits remarkable selectivity, specificity, reproducibility, and stability, and can be successfully applied to the detection of two pathogens in real samples with satisfactory results. Based on these comprehensive performance characteristics, this study innovatively provides a reliable technical path for the simultaneous detection of multiple pathogens, demonstrating significant application value in the field of food safety monitoring.

Claims

1. An electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, characterized in that: It is made by the following steps: Step 1: Preparation of Ti3C2T x Nanobelt Ti3C2T x NR; Step 2: Preparation of Ti3C2T by self-reduction method x NR / Cu nanocomposites; Step 3: Construction of S-Apt / MB@UiO and L-Apt / Fc@UiO; Step 4: Electrode preparation: Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE reference electrode.

2. The electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 1, characterized in that: Step 1 further includes step 1a and step 1b: Step 1a: Etching treatment: Disperse Ti3AlC2 powder in hydrofluoric acid and stir continuously. After the reaction, collect Ti3C2T by centrifugation. x Nanosheet Ti3C2T x NS and washed repeatedly with deionized water until the supernatant was neutral; Step 1b: Tearing treatment: The obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in KOH solution and reacted under N2 atmosphere at 25℃ to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR.

3. The electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 1, characterized in that: Step 2 further comprises steps 2a and 2b: Step 2a: Add CuCl2 solution dropwise to Ti3C2T under stirring. x In NR solution; Step 2b: Obtain Ti3C2T by centrifugation x The NR / Cu nanocomposite material was dried and set aside.

4. The electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 1, characterized in that: Step 3 further includes step 3a and step 3b; Step 3a: Synthesis of UiO-66: ZrCl4, NH2-BDC and benzoic acid were dissolved in DMF and ultrasonicated, and then transferred to a reactor for reaction to obtain UiO-66 crystals. Step 3b: Probe encapsulation: UiO-66 was dispersed in MB solution and shaken, followed by the addition of aptamer S-Apt and shaking. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken, and then the aptamer L-Apt was added and shaken. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer.

5. The electrochemical sensing platform for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 1, characterized in that: It is made by the following steps: Step 1: Preparation of Ti3C2T x Nanobelt Ti3C2T x NR, including step 1a and step 1b; Step 1a: Etching treatment: 1.0 g of Ti3AlC2 powder was dispersed in 30.0 mL of hydrofluoric acid and stirred for about 24 hours. After the reaction, the Ti3C2T x Nanosheet Ti3C2T x NS and washed repeatedly with deionized water until the supernatant was neutral; Step 1b: Tearing treatment: The obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in a 6.0 M KOH solution and reacted for 108 h under N2 atmosphere and 25 °C to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR; Step 2: Preparation of Ti3C2T by self-reduction method x NR / Cu nanocomposite material, comprising step 2a and step 2b; Step 2a: Under vigorous stirring, 1.5 mL of 0.1 M CuCl2 solution was added dropwise to 50.0 mL of 1.0 mg / mL Ti3C2T x In NR solution; Step 2b: After 20 minutes of reaction, Ti3C2T was obtained by centrifugation. x The NR / Cu nanocomposite material was dried and then used; Step 3: Construction of S-Apt / MB@UiO and L-Apt / Fc@UiO, including steps 3a and 3b; Step 3a: Synthesis of UiO-66: 75.0 mg of ZrCl4, 50.0 mg of NH2-BDC, and 1.5 g of benzoic acid were dissolved in DMF and ultrasonicated. The mixture was transferred to a reactor and subjected to hydrothermal reaction at 120°C for 12 hours to obtain UiO-66 crystals. Step 3b: Probe encapsulation: UiO-66 was dispersed in MB solution and shaken overnight. Then, the aptamer S-Apt was added and shaken for 1 hour. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken overnight, followed by the addition of aptamer L-Apt and shaking for 1 hour. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer. Step 4: Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE reference electrode.

6. An electrochemical method for the simultaneous detection of Staphylococcus aureus and Listeria monocytogenes, characterized in that: The following steps are involved: Step 1: Preparation of Ti3C2T by simple self-reduction method x NR / Cu nanocomposites; Step 2: The two electrochemical probes, MB and Fc, were encapsulated in the UiO-66 metal-organic framework, respectively, and the functionalized S. aureus aptamer S-Apt and LM aptamer L-Apt were used as biological gating elements, denoted as S-Apt / MB@UiO and L-Apt / Fc@UiO; Step 3: Electrode preparation: Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE control electrode; Step 4: Pathogen detection: Different concentrations of target pathogens S. aureus and LM were incubated with a mixed solution of S-Apt / MB@UiO and L-Apt / Fc@UiO in a 1:1 ratio, diluted with Tris-HCl buffer, and then added dropwise to the Ti3C2T x NR / Cu / SPCE electrode surface, signal detection was performed using differential pulse voltammetry.

7. The electrochemical method for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 6, characterized in that: Step 1 further comprises the following steps: Preparation of Ti3C2T x Nanobelt Ti3C2T x NR, including: Etching treatment: Disperse Ti3AlC2 powder in hydrofluoric acid, stir continuously, collect Ti3C2T by centrifugation after reaction x Nanosheet Ti3C2T x NS, and washed repeatedly with deionized water until the supernatant was neutral; tearing treatment: the obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in KOH solution and reacted under N2 atmosphere at 25℃ to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR; Under stirring conditions, CuCl2 solution was added dropwise to Ti3C2T x In NR solution; Ti3C2T was obtained by centrifugal separation x The NR / Cu nanocomposite material was dried and set aside.

8. The electrochemical method for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 6, characterized in that: Step 2 further includes the following steps: Synthesis of UiO-66: ZrCl4, NH2-BDC and benzoic acid were dissolved in DMF, ultrasonicated and transferred to a reactor for reaction to obtain UiO-66 crystals. Probe encapsulation: UiO-66 was dispersed in MB solution and shaken, followed by the addition of aptamer S-Apt and shaking. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken, and then the aptamer L-Apt was added and shaken. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer.

9. The electrochemical method for simultaneous detection of Staphylococcus aureus and Listeria monocytogenes according to claim 6, characterized in that: The following steps are involved: Step 1: Preparation of Ti3C2T by self-reduction method x The NR / Cu nanocomposite material further comprises the following steps: Preparation of Ti3C2T x Nanobelt Ti3C2T x NR, including: Etching treatment: 1.0g Ti3AlC2 powder was dispersed in 30.0mL hydrofluoric acid and stirred for about 24 hours. After the reaction, Ti3C2T x Nanosheet Ti3C2T x NS, and washed repeatedly with deionized water until the supernatant was neutral; tearing treatment: the obtained Ti3C2T x Nanosheet Ti3C2T x NS was placed in a 6.0 M KOH solution and reacted for 108 h under N2 atmosphere and 25 °C to obtain Ti3C2T by longitudinal tearing. x Nanobelt Ti3C2T x NR; Under vigorous stirring, 1.5 mL of 0.1 M CuCl2 solution was added dropwise to 50.0 mL of 1.0 mg / mL Ti3C2T x In NR solution; After 20 minutes of reaction, Ti3C2T x The NR / Cu nanocomposite material was dried and then used; Step 2: Constructing S-Apt / MB@UiO and L-Apt / Fc@UiO, further including the following steps: Synthesis of UiO-66: 75.0 mg of ZrCl4, 50.0 mg of NH2-BDC, and 1.5 g of benzoic acid were dissolved in DMF and ultrasonicated. The mixture was transferred to a reactor and hydrothermally reacted at 120°C for 12 hours to obtain UiO-66 crystals. Probe encapsulation: UiO-66 was dispersed in MB solution and shaken overnight, followed by the addition of the aptamer S-Apt and shaking for 1 hour. After centrifugation and washing, S-Apt / MB@UiO was obtained and stored in pH 7.0 buffer. UiO-66 was dispersed in Fc solution and shaken overnight, followed by the addition of the aptamer L-Apt and shaking for 1 hour. After centrifugation and washing, L-Apt / Fc@UiO was obtained and stored in pH 7.0 buffer. Step 3: Electrode preparation: 10 μL of 0.5 mg / mL Ti3C2T x NR / Cu solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / Cu / SPCE modified electrode; 10 μL of 0.5 mg / mL Ti3C2T x NR solution was drop-coated on the surface of screen-printed carbon electrode SPCE to produce Ti3C2T x NR / SPCE control electrode; Step 4: Pathogen detection: 20 μL of target pathogens S. aureus and LM at different concentrations were incubated with 40 μL of a 1:1 mixed solution of S-Apt / MB@UiO and L-Apt / Fc@UiO for 30 min. The mixture was diluted to 0.2 mL with Tris-HCl buffer and then added dropwise to the surface of the Ti3C2T2NR / Cu / SPCE electrode. Signal detection was performed using differential pulse voltammetry.

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