Peptide Nanosheet-Inspired Biomimetic Synthesis of CuS Nanoparticles on Ti3C2 Nanosheets for Electrochemical Biosensing of Hydrogen Peroxide

Abstract: Hydrogen peroxide (H2O2 ) is one of the intermediates or fifinal products of biological metabolism and participates in many important biological processes of life activities. The detection of H2O2 is of great signifificance in clinical disease monitoring, environmental protection, and bioanalysis. In this study, Ti3C2 -based nanohybrids are prepared by the biological modifification and self-assembled peptide nanosheets (PNSs)-based biomimetic synthesis of copper sulfifide nanoparticles (CuS NPs), which show potential application in the fabrication of low-cost and high-performance electrochemical H2O2 biosensors. The synthesized CuS-PNSs/Ti3C2 nanohybrids exhibit excellent electrochemical performance towards H2O2 , in which CuS NPs can catalyze the decomposition of H2O2 and realize the transformation from a chemical signal to an electrical signal to achieve the purpose of H2O2 detection. The prepared CuS-PNSs/Ti3C2 -based electrochemical biosensor platform exhibits a wide detection range (5 µM–15 mM) and a low detection limit (0.226 µM). In addition, it reveals good selectivity and stability and can realize the monitoring of H2O2 in a complex environment. The successful biomimetic synthesis of CuS-PNSs/Ti3C2 hybrid nanomaterials provides a green and friendly strategy for the design and synthesis of functional nanomaterials and also provides a new inspiration for the construction of highly effective electrochemical biosensors for practical detection of H2O2 in various environments. Keywords: biomimetic synthesis; Ti3C2 nanosheets; CuS nanoparticles; electrochemical biosensor; H2O2

1. Introduction As a very important biomolecule that is related to biological reaction processes, hydrogen peroxide (H2O2) is of great importance in food science, industrial production, clinical medicine, and biological life activities. In particular, H2O2 is a very important substance involved in the metabolism of living organisms, but excessive accumulation of H2O2 can cause irreparable damage to cells and living organisms [1,2]. Therefore, the level of H2O2 has become an important parameter to determine whether the cells are normal or not, and the detection of H2O2 has become a key factor in the early diagnosis of some diseases. Among various methods for the detection of H2O2, electrochemical detection has the characteristics of being a simple operation and having a low detection limit, high sensitivity, and good selectivity; therefore, it has great potential for the detection of H2O2 with high sensing performance [3–6]. In order to fabricate high-performance electrochemical H2O2 biosensors, it is necessary to fifind active materials with good electrochemical properties. The easy surface modifification, large specifific surface area, and unique layered structure of two-dimensional (2D) materials have tremendous advantages for the design and fabrication of electrochemical sensors and biosensors [7–11]. As one of the emerging 2D materials, MXene is mainly composed of layered transition metal carbides or nitrides. MXene has shown powerful capabilities in catalysis, biosensing, capacitors, and electrochemistry by virtue of its large specifific surface area, potential surface modififiability, good biocompatibility, excellent electrical conductivity, and high electron transfer effects [12–14]. Compared with graphene, which opens the door for 2D materials, Ti3C2 MXene materials with faster electron mobility are endowed with enhanced electrical conductivity, and studies have been conducted using Ti3C2 nanohybrids for various electrochemical sensors. For example, Ti3C2 has been modifified with platinum nanoparticles (Pt NPs) for electrochemical detection of H2O2. The Ti3C2/Pt NPs-modifified glassy carbon electrode (GCE) exhibited a stronger redox capability and also revealed a lower detection limit and better stability for the detection of H2O2 [15]. Therefore, it is important to take advantage of the electrochemical properties of Ti3C2 in combination with other materials to increase the abundance of Ti3C2 surface groups, in order to further improve the biocompatibility and electrochemical properties of Ti3C2 nanosheets [16]. Peptide is a kind of widely used biomolecule with a tailored self-assembly function, which consists of amino acids that are arranged in a certain sequence. The amino and carboxyl groups at the head and tail (or on the side chains) of peptides are able to complex and coordinate with metals or metal oxides, thus enhancing the biocompatibility of metals. It has been demonstrated that metal ions play a very important role in the self-assembly process of some peptides [17,18]. For instance, Vello et al. studied the reciprocal recognition of amyloid-β peptide (Aβ) by Zn(II) and Cu(II) ions and found that Aβ fifibrils encapsulated with Cu(II) were able to generate reactive oxygen species, which can lead to cell death in the presence of H2O2 and reducing agents [19]. Additionally, the charge carried by the peptide changes with the pH of the solution system. The pH of the peptide in electroneutrality is called its isoelectric point, and this property enables it to adsorb metal ions through electrostatic interactions under the modulation of the external environment. For instance, in the work of Liu et al., a peptide with the isoelectric point of 9.94 was used to adsorb Ag+ in a neutral environment, and then Na2S was used for the in-situ reduction of Ag+ on the peptides to form Ag2S NPs for photothermal therapy of tumors [20]. Peptidebased nanomaterials with good morphology and functionality can be easily obtained by designing the peptide sequence and controlling the self-assembly conditions, which can be further used as a bridge between materials to construct hybrid nanomaterials with unique structures and desirable properties [21–24]. In the conjugation process of peptides with 2D materials, the cross-linking agents are usually required to achieve the binding. Glutaraldehyde (GA) is a widely used biological cross-linking agent whose aldehyde groups can react with -OH or -NH4 groups to achieve the cross-linking. In the work of Ou et al., the binding of GO with cellulose was achieved by GA-based linking, where both GO and cellulose surfaces are rich in the presence of -OH groups, and where GA binds to the -OH groups on the cellulose surface at one end and to the -OH group on the GO surface at the other end [25]. Besides reacting with the -OH group, GA is also capable of cross-linking with the -NH4 group. The surface of Ti3C2 that is synthesized by the etching method is rich in the -OH group, which can react with the aldehyde group of GA, while the aldehyde group at the other end of GA can react with the -OH group or the -NH4 group of peptide molecules to realize the combination. Therefore, GA-modifified Ti3C2 will widen the distance between Ti3C2 nanosheets and narrow the force between nanosheets, making Ti3C2 have better dispersion and stability. In this work, GA is used as a cross-linking agent to connect Ti3C2 with self-assembled peptide nanosheets (PNSs) to enrich the Ti3C2 surface groups. The peptide with the sequence KLVFFAK is selected to modify the Ti3C2 surface for this aim. The peptide sequence KLVFFAK is derived from a partial fragment of β-amyloid and is able to form nanosheets of moderate thickness and size through π–π interactions [26–28]. By modulating the selfassembly of KLVFFAK peptides, smaller size PNSs are prepared and modifified onto the Ti3C2 surface by GA-based cross-linking, which leads to an increase in the type and number of groups on the Ti3C2 surface. At the same time, the isoelectric point of the KLVFFAK peptide is 10.6, and the PNSs formed by the peptide self-assembly under neutral and acidic environments have a negative charge, which enhances the electronegativity of Ti3C2 after the modifification and facilitates the subsequent binding with metal ions. CuS nanoparticles (NPs) were selected to improve the catalytic performance of PNSs/Ti3C2 hybrid materials. As a common metal, copper ion has good electrical conductivity and catalytic performance. It has been widely used in electrochemical sensors, carbon enrichment products, and hydrogen evolution reactions [29,30]. Additionally, sulfifides are important semiconductor materials due to their metal-like properties for chemical sensing, and it should be noted that there is an additional advantage of cost-effectiveness of copper sulfifide due to its Earth abundant property [31,32]. As shown in Scheme 1, the synthesized Ti3C2 nanosheets are cross-linked with selfassembled PNSs by GA linking to form the PNSs/Ti3C2 nanohybrids with abundant negative charges on the surface. Then, the adsorption of Cu2+ is achieved by electrostatic interaction between PNSs and Cu2+, and the in situ biomimetic synthesis of CuS NPs on the prepared PNSs/Ti3C2 nanohybrids is carried out by adding Na2S as the reaction agent, which is a green and friendly biomimetic synthesis method for the CuS-PNSs/Ti3C2 nanohybrids. Further, the created CuS-PNSs/Ti3C2 nanohybrids are modifified onto the surface of GCE to construct the electrochemical H2O2 sensor platform. The fabricated biosensor platform exhibits good stability and high sensitivity, with a detection range of 5 µM–15 mM and a minimum detection limit of 0.226 µM. As an economic, highly catalytic, and electroactive material for detecting H2O2, the CuS-PNSs/Ti3C2-based electrochemical sensor platform has the advantages of excellent sensitivity, extremely high interference immunity, and long-term stability, showing potential applications for high-performance determination of H2O2 in biological and natural environments.

Scheme 1. Schematic presentation of the biomimetic synthesis of CuS-PNSs/Ti3C2 and the fabrication of electrochemical H2O2 biosensors: (A) preparation of Ti3C2 nanosheets; (B) preparation of CuSPNSs/Ti3C2 and fabrication of electrochemical H2O2 biosensors.

2. Materials and Methods 2.1. Materials and Reagents A peptide with the sequence of KLVFFAK was bought from SynPeptide Biotechnology Co., Ltd. (Nanjing, China). Copper chloride, sodium sulfifide, ethylene diamine tetracetic acid (EDTA), dimethyl sulfoxide (DMSO), Nafifion, lysine (Lys), L-ascorbic acid (AA), and uric acid (UA) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, PR China). Ethanol, hydrochloric acid, and sodium hydroxide (96%) were provided by Shanghai Test Laboratory Equipment Co., Ltd. (Shanghai, China). Lithium flfluoride (LiF), titanium aluminum carbide (Ti3AlC2), sodium bicarbonate, and triflfluoroacetic acid (TFA) were bought from Shanghai Yien Chemical Technology Co., Ltd. (Shanghai, China). 2.2. Synthesis of Ti3C2 Nanosheets In order to obtain Ti3C2 monolayered nanosheets, the methods of HF etching of Ti3AlC2 and DMSO intercalation of multilayer Ti3C2 nanosheets were used to synthesize the Ti3C2 monolayer nanosheets on the basis of a previously reported method [33,34]. In brief, 1 mg LiF was dissolved in 20 mL HCl (12 M) for 10 min with magnetic stirring. Next, 1 mg Ti3AlC2 was added and magnetically stirred at 35 ◦C in a water bath for 24 h. Then, the black Ti3C2 solution was fifirst washed with HCl (1 M) to remove the unreacted LiF and other impurities before being washed by deionized water at 8000 rpm rotating speed for 5 min until the fifinal pH of the solution reached 6.0–7.0. Monolayer Ti3C2 nanosheets were obtained by sonicating the obtained solution for 30 min, and the middle part of the supernatant was collected for morphological characterizations. Clay-like multilayer Ti3C2 on the bottom of the centrifuge tubes was added into DMSO to intercalate under magnetic stirring overnight. To remove DMSO, a dialysis operation was applied. The sodium ascorbate solution was used as the exchange solvent to exchange DMSO for 24 h. Finally, freeze-drying was used to obtain the monolayer Ti3C2 pounds for further use. 2.3. Tailoring the Self-Assembly of Peptides into PNSs PNSs are created by the self-assembly method by controlling the reaction condition, and the creation of PNSs in the whole study followed the following procedure: 20 mg of peptide (KLVFFAK) was dissolved in 10 mL 0.1% TFA and ethanol solution with a volume ratio of 1:9. The obtained peptide solution with a concentration of 2 mg mL−1 was then incubated at 47 ◦C in a water bath for 2 h to obtain PNSs. 2.4. Synthesis of CuS-PNSs/Ti3C2 Nanohybrids The synthesis of the CuS-PNSs/Ti3C2 nanohybrids followed the following procedure: 10 mg Ti3C2 pounds was dissolved in 5 mL sodium ascorbate solution. To further modify the surface of the Ti3C2 nanosheets, 0.1% GA was added into the Ti3C2 solution with a volume ratio of 1:10 to obtain the GA-modifified Ti3C2 under stirring for 12 h. After removing the uncombined GA through centrifuging, 2 mg mL−1 GA-modifified Ti3C2 solution was mixed with prepared PNSs in the same volume under stirring for 12 h to obtain the PNSs/Ti3C2 nanohybrids. After 12 h reaction, a centrifuge was applied to remove the PNSs that did not combine with Ti3C2 nanosheets. After that, CuCl2 (10 mM) solution was added into PNSs/Ti3C2 solution with the volume ratio of 1:5 under stirring for 12 h, and Na2S was used to react with Cu2+ with the same volume to obtain the CuS-PNSs/Ti3C2 nanohybrids. 2.5. Electrochemical Detection of H2O2 GCEs (4.0 mm in diameter) were fifirst polished with 0.3 mm and 0.05 mm alumina powder and then ultrasonically cleaned in ultrapure water and ethanol solution. The materials for the modifification of GCEs were prepared by mixing 50 µL Nafifion solution with 1 mL of PNSs, PNFs/Ti3C2, and CuS-PNSs/Ti3C2 solutions, respectively. After that, 10 mL of the modifification solution was added dropwise onto the surface of GCE to obtain the Ti3C2/GCE, PNSs/Ti3C2/GCE, and CuS-PNSs/Ti3C2/GCE for subsequent electrochemical tests.

2.6. Characterization Techniques All atomic force microscope (AFM) samples were prepared by dropping 10 mL of sample solution onto freshly cleaved mica substrates and air-dried for characterization. AFM measurements were performed in air using the ‍‍FM-Nanoview 6800 AFM (FSM-Precision, Suzhou FSM Precision Instrument Co., Ltd., Jiangshu, China) ‍‍using the tapping mode. Tap300Al-G (300 kHz, 40 N m−1 ) silicon probes were used for capturing AFM images. The tapping mode images were recorded and analyzed with Gwyddion software (Version 2.57). A transmission electron microscope (TEM, Tecnai G2 F20, FEI Co; Tokyo, Japan) was used to observe the structure and morphology of various nanocomposites. A scanning electron microscope (SEM, Regulus 8100, Hitachi, Japan) was used to observe the microstructure of the peptide-based nanocomposites. X-ray spectroscopy (XPS) characterization of the samples was performed on a PHI 5000 VersaProbe III spectrometer (UlVAC-PHI Company, Tokyo, Japan). All the electrochemical experiments were carried out at room temperature using an electrochemical workstation (CHI660E, Shanghai Chenhua, China) using a traditional three-electrode system, in which the working electrode was the modifified GCE, the auxiliary electrode was platinum wire, and the reference electrode was a saturated calomel electrode.

3. Results 3.1. Characterizations of Ti3C2 and PNSs The AFM technique was fifirst used to characterize the prepared Ti3C2 monolayered nanosheets and self-assembled PNSs. It can be seen from Figure 1a that the Ti3C2 nanosheets were successfully prepared by HF etching for 24 h in a 37 ◦C water bath. In the obtained AFM image, the height of the Ti3C2 nanosheets is measured to be about 1.5 nm, which reveals good height distribution and agrees well with the height data previously reported for the Ti3C2 monolayer. In the corresponding TEM image (Figure 1b), the Ti3C2 nanosheets can also be seen clearly, and the size of the Ti3C2 nanosheets is between 200 nm and 1 µm, which is similar to the size that is shown in the presented AFM image. Therefore, we suggest that both AFM and TEM images prove that the Ti3C2 monolayered nanosheets with good distribution and uniform size were synthesized successfully.