Facile Synthesis of Cu2O@TiO2-PtCu Nanocomposites as a Signal

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Biological and Medical Applications of Materials and Interfaces

Facile synthesis of Cu2O@TiO2-PtCu nanocomposites as signal amplification strategy for the insulin detection Faying Li, Jinhui Feng, Zengqiang Gao, Li Shi, Dan Wu, Bin Du, and Qin Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01779 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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ACS Applied Materials & Interfaces

Facile synthesis of Cu2O@TiO2-PtCu nanocomposites as signal amplification strategy for the insulin detection

Faying Li†, ‡∇, Jinhui Feng†∇, Zengqiang Gao§, Li Shi†, ‡, Dan Wu†, Bin Du†, Qin Wei†,*

†: Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China ‡: Institut National de la Recherche Scientifique, Centre for Energy, Materials and Telecommunications, 1650 Boulevard Lionel-Boulet, Varennes, Québec J3X 1S2, Canada §: School of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo 255049, PR China

*: Corresponding author: Email address: [email protected]; Fax: + 86 531 82767367; Tel: + 86 531 82767872; (Qin Wei) 1

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Abstract A novel ultrasensitive sandwich-type electrochemical immunosensor was proposed for the quantitative detection of insulin, a representative biomarker for diabetes. To this end, molybdenum disulfide nanosheets loaded gold nanoparticles (MoS2/Au NPs) were used as substrates to modify bare glassy carbon electrodes (GCE). The MoS2/Au NPs not only present superior biocompatible and large specific surface area to enhance the loading capacity of primary antibody (Ab1), but also present good electrical conductivity to accelerate electron transfer rate. Moreover, the amino functionalized cuprous oxide @ titanium dioxide octahedral composites (Cu2O@TiO2-NH2) were prepared to load dendritic platinum-copper nanoparticles (PtCu NPs) to realize signal amplification strategy. The resultant nanocomposites (cuprous oxide @ titanium dioxide octahedral loaded dendritic platinum-copper nanoparticles) demonstrate uniform octahedral morphology and size, which effectively increases the catalytically active sites and specific surface area to load the secondary antibody (Ab2), even increases conductivity. Most importantly, the resultant nanocomposites possess superior electrocatalytic activity for hydrogen peroxide (H2O2) reduction, which present the signal amplification strategy. Under the optimal conditions, the proposed immunosensor exhibited a linear relationship between logarithm of insulin antigen concentration and amperometric response within a broad range from 0.1 pg/mL to 100 ng/mL, and a limit detection of 0.024 pg/mL. Meanwhile, the immunosensor was employed to detect insulin in human serum with satisfactory results. Furthermore, it also presents good reproducibility, selectivity and stability, which exhibits broad application prospects in biometric analysis. Keywords: sandwich-type immunosensor; insulin antigen; Cu2O@TiO2 octahedral composite; alloyed PtCu dendrimers; electrocatalytic activity 2


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1. Introduction Insulin, a polypeptide hormone secreted by islet cells, plays a vital physiological role in human metabolism.1-2 It not only promote the glucose in the blood circulation into the liver, muscle and other tissue cells to regulate the metabolism of carbohydrates, fats and protein,3 but also facilitate the oxidation and decomposition of glucose to release energy to sustains life activities. In general, the dysfunction of insulin secretion can lead to diabetes mellitus, and also hyperinsulinemia face increased risk factors for serious diseases such as obesity, myocardial infarction, kidney failure and neurodegenerative disease.2 As reported, several severe illness will happen when the insulin concentration is below 0.86 ng/mL in human blood under fasting conditions.4 Therefore, accurate and sensitive detection of insulin in human serum is essential for early clinical diagnosis and basic research of diabetes. In recent years, electrochemical immunosensors based on the specific interaction between antigen (Ag) and antibody (Ab) have attracted extensive attention due to their advantages such as simple instrument, high sensitivity, low background, and easy minimization, and become a popular technology for tumor marker detection.5 Especially, the sandwich-type electrochemical immunosensors have high sensitivity and selectivity because of dual specific conjugate between antibody and antigen.6 It applies a sandwich structure to capture and detect analytes by immobilized primary antibody (Ab1) and labeled secondary antibody (Ab2), respectively, providing a direct relationship between antigen concentration and electrocatalytic signal intensity.6 However, since the specific conjugate of antigens and antibodies form a hydrophobic insulating layer on the modified interface, hindering electron transfer, the interaction between antibody and antigen is insufficient to directly achieve a highly sensitive electrochemical detection signal.7 3



In recent years, varieties of functionalized labels were designed for signal amplification, such as functional mesoporous materials,8-9 metal oxides,10-12 noble metal nanoparticles.13-14 Particularly, labels with auxiliary catalytic activity towards hydrogen peroxide (H2O2) reduction are effective strategies for improving sensitivity. In general, smaller catalysts have a larger surface ratio and are advantageous for better catalytic performance.15-16 Cuprous oxide (Cu2O) nanocrystals, a transition metal oxide, have attracted much attention to the design of immunosensors due to their good environmental compatibility, large surface area and redox activity.17 In addition, Cu2O nanocrystals have good electrocatalytic properties for H2O2 reduction.18 Unfortunately, the individual Cu2O nanocrystals can not maintain sufficient stability. Meanwhile, nano-architectured titanium dioxide (TiO2) has also attracted considerable attention because of its good chemical and electrical stability. So far, TiO2 nanocrystals with large accessible surface area, good biocompatibility and conductivity, have also been widely used in various biosensors.19-21 In these biosensors, TiO2 was applied as substrate to promote the direct electron transfer and enhance the catalytic activity. Simultaneously, TiO2 has better peroxidase-like activity for H2O2 reduction.22-23 In addition, the TiO2-related derivate can carry more active regions or probes to couple biomolecules. In general, the core-shell nanostructures in which the metal composites are embedded in the oxide matrix can protect the former from agglomeration and are not exposed to the reactants and surrounding medium.24 Moreover, the combination of two or more signal variations may produce a synergistic effect compared to the single signal amplification strategy, which can effectively enhance the detection sensitivity.25 Most importantly, nanocomposites with a core-shell structure have multiple features that optimize interface contact. By combining Cu2O nanocrystals with TiO2 nanoparticles (NPs), the specific surface area, electron transfer capability, catalytic 4


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property and dispersibility of Cu2O@TiO2 NPs can improve dramatically. Subsequently, in order to combine the Cu2O@TiO2 NPs with antibodies as well as enhance the catalytic activity, the introduction of precious metal NPs has become an interesting strategy because of its good chemical stability, good biocompatibility and auxiliary catalytic properties. Among these tested noble metal NPs, platinum (Pt) NPs have attracted much attention because of their good biocompatibility and electrocatalytic property for H2O2 reduction.14,

26

Recently, Pt-based bimetallic

nanocrystals have received more attention, especially due to their possible bifunctional mechanism effect, reducing platinum consumption and improving the integral electrocatalytic performance.27 Among various Pt-based alloyed nanocrystals, PtCu bimetallic nanocrystals are considered as promising candidate for electrocatalytic reactions due to their relatively low cost and excellent electro catalytic activity.27-29 The PtCu bimetallic nanocrystals not only enhance the immobilization of antibodies by constructing stable Pt-N26 bond and Cu-N bond30-31 between PtCu NPs and -NH2 from antibodies, but also effectively improve the catalytic activity of the reactant.32-33 In this work, the alloyed PtCu dendrimers with high specific surface area and index surface that provide more catalytic active sites on the concave surface for catalytic reactions. In the proposed immunosensor, the cuprous oxide @ titanium dioxide octahedral loaded platinum copper dendrimer nanoparticles (Cu2O@TiO2-PtCu NPs) were used as signal amplification labels in combination with Ab2. The synergistic effect of Cu2O@TiO2PtCu NPs can improve electron transfer efficiency at electrode interface and improve reduction efficiency of the nanocomposite to H2O2. For the sandwich-type electrochemical immunosensor, effectively improving the immobilize of the primary antibody (Ab1) and the rapid electron transfer at the electrode interface are crucial to achieve high sensitivity. As a transition metal dichalcogenide, 5



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molybdenum disulfide (MoS2) has excellent thermal stability, good biocompatibility and high electro-catalytic activity, and is widely used in biosensors

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and

electrocatalysts.35-37 Hybridization of MoS2 nanosheets with precious metals is an ideal strategy further to optimize the performances of MoS2 for electronic devices.38 Moreover, gold NPs (Au NPs) have good biocompatibility, excellent stability and large surface area, which can increase the immobilization of biomolecules.

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In this work,

the MoS2/Au NPs were employed as substrates to improve the immobilization of Ab1 and accelerate the electron transfer at electrode interface. 2. Experimental Section 2.1 Assembly of the electrochemical immunosensor Scheme. 1A shown the preparation procedure of Cu2O@TiO2-PtCu/Ab2 label (Supporting Information). Scheme. 1B shown the fabricated schematic of the proposed immunosensor. In general, the GCE were polished with alumina powder to form a mirror-like before use. The eligible GCE were modified with 6.0 µL of MoS2/Au NPs aqueous solution (2.0 mg/mL) and dried. Then, Ab1 (6.0 µL, 10 μg/mL) was incubated onto the modified electrode by stable Au-N bond. After incubation for 1 h and washing, the modified electrode was incubated with 3.0 μL 1% BSA solution to eliminate nonspecific binding sites. Then, 6 μL of a series of different concentrations of insulin Ag solution were incubated on the surface of the modified electrodes. After that, 6.0 μL Cu2O@TiO2-PtCu/Ab2 solution (2.5 mg/mL) was incubated for another 1 h onto the modified electrodes surface. After dried, the immunosensor was rinsed thoroughly with PBS to remove any unbound Ab2 label and stored at 4°C. 2.2 Experimental Measurements A conventional three-electrode system was employed in entire electrochemical tests. Amperometric i-t curve was performed in PBS (pH = 6.64) with a voltage at -0.4 V 6


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(Figure S1). After the background current remained stable, 10 μL of H2O2 (5 mol/L) was injected into 10 mL of PBS under stirring.

Scheme.1 (A) The preparation process of Cu2O@TiO2-PtCu/Ab2; (B) The fabricated process of the proposed sandwich-type immunosensor. 3. Results and discussion 3.1. Characterization of the nanocomposites SEM, TEM and XRD characterization were helpful to verify the succeed preparation by observing their transformation of micro-morphologies. First, the micro-morphology and composition of the synthesized Cu2O nanocrystals were verified by SEM and EDX characterization. As shown in Figure 1A, the synthesized Cu2O NPs present average size approximately 300 nm were quasi-monodisperse octahedrons. Simultaneously, the octahedral Cu2O nanocrystals were relative tidy and smooth. The legible TEM image also confirmed the morphology (Figure 1D). To further explore the transformation of 7



crystallographic structures in the preparation process, XRD spectroscopy was utilized to characterize the synthetic process. As shown in Figure 1G, no obvious peak of CuO or Cu were detected, and the diffraction peaks of Cu2O belong to the cuprite (blue baseline, Powder Diffraction File (PDF) Card No. 78-2076). In addition, obvious O and Cu elements were detected in EDX spectrum (Figure S2A), which also confirmed the successful synthesis of octahedral Cu2O nanocrystals. After further synthesis, the surface of the octahedral nanocomposite became rough. Figure 1B shown the morphology of the synthesized Cu2O@TiO2 core-shell NPs. As shown in Figure 1B, the surface of octahedral Cu2O nanocrystals were surrounded by a dense layer of TiO2. The core-shell nanostructure can be clearly distinguished by TEM (Figure 1E). As shown in Figure 1E (a), the Cu2O@TiO2 core-shell NPs with average size about 330 nm were quasi-monodisperse octahedrons. Figure 1E (b) was partial enlarged view, demonstrating the marginalization of small particles of Cu2O@TiO2 surface. It was clearly viewed the surface of octahedral Cu2O nanocrystals were surrounded by a dense TiO2 layer composed of hundreds of ultra-small TiO2 NPs self-assembled. Then, XRD pattern was utilized to verify the transformation of crystallographic structures from Cu2O to Cu2O@TiO2. Particularly, the diffraction peaks of Cu2O (blue baseline) corresponds to XRD patterns of Cu2O@TiO2 core@shell NPs, clearly indicating that the core of Cu2O was well preserved during the synthesis. In addition, anatase TiO2 (sky blue baseline, PDF Card No. 71-1169) can index other diffraction peaks completely. Simultaneously, obvious O, Cu and Ti elements were detected in EDX spectra (Figure S2B), which further indicated that Cu2O@TiO2 NPs were synthesized successfully. Subsequently, the as-prepared PtCu nanocrystal was verified by TEM spectra in Figure 1F (a). It showed the magnified image of prepared alloyed PtCu nanocrystal with uniform size about 15 nm. In addition, the elemental composition and 8


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dispersion of the alloyed PtCu nanohybrid was confirmed by elemental mapping in Figure S3. As shown in Figure S3, the as-prepared PtCu nanocrystal present analogous sphere nanodendrites. The elemental mapping presented that Pt and Cu elements were uniform distribution. When the as-prepared alloyed PtCu dendrimers linked to the surface of Cu2O@TiO2-NH2 NPs, the surface morphology of resultant nanocomposites were greatly altered (Figure 1C). The transformation of morphology can be analyzed by SEM (Figure 1C) and TEM (Figure 1F-b), respectively. It was clear to demonstrate that some uniform quasi-monodisperse NPs attached on the surface of Cu2O@TiO2. Subsequently, XRD pattern of Cu2O@TiO2-PtCu verified the transformation of crystallographic structures after combination the Cu2O@TiO2 and PtCu dendrimers. According to the XRD pattern (Figure 1G), the diffraction peak of Cu2O and Cu was small, but the diffraction peak of Pt and TiO2 are obvious, which may be attribute to their low Cu2O contents and relative high Pt crystalline, respectively. Similarly, the element composition of the Cu2O@TiO2-PtCu was also verified by elemental mapping, presenting in Figure S4. From the element mapping in Figure S4, the Ti, O and most Cu elements spread out into a uniform rhombus when Pt and partial Cu dispersed in the epitaxy of the rhombus. These representations were fully substantiated the formation of Cu2O@TiO2-PtCu. N2 adsorption-desorption was applied to investigate the specific surface area of Cu2O@TiO2-PtCu. As shown in Figure 1H, an obvious hysteresis loop was observed in the nitrogen (N2) adsorption-desorption isotherm, the calculated specific surface area of the resultant Cu2O@TiO2-PtCu was about 24.2 m2/g. The upward tail of the N2 adsorption-desorption isotherm was caused by the microporous structure formed by the stacking of nanoparticles. Therefore, the relatively large specific surface area can effective increase the catalytically active site and increase the load of the Ab2. In addition, four elements (Cu, Ti, O and Pt) were detected by EDX 9



pattern in Figure 1I also confirmed the component of Cu2O@TiO2-PtCu. All of these above characterization verified that the succeed preparation of Cu2O@TiO2-PtCu labels.

Figure 1 SEM images of (A) Cu2O, (B) Cu2O@TiO2 and (C) Cu2O@TiO2-PtCu; TEM images of (D) Cu2O, (E-a) Cu2O@TiO2, (E-b) partial enlarged view of Cu2O@TiO2, (F-a) PtCu and (F-b) Cu2O@TiO2-PtCu; (G) XRD pattern of Cu2O, Cu2O@TiO2 and Cu2O@TiO2-PtCu; (H) BET and (I) EDX spectra of Cu2O@TiO2-PtCu. SEM, Raman, XRD and EDX characterization were used to investigate the microstructure of the prepared MoS2 and MoS2/Au nanohybrids. As shown in SEM image (Figure 2A), it can be clearly observed that the prepared MoS2 exhibits a nanoflower morphology. The average diameter of synthesized MoS2 nanoflowers was about 130-150 nm, which assembled by plentiful petal-like nanosheets. Moreover, the 10


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EDX spectra was applied to characterize the elemental composition. The EDX spectrum of Mo and S elements further indicated the successful synthesize of pure MoS2 nanoflowers (Figure 2D). Compared with MoS2 nanoflowers, the morphology of prepared MoS2/Au NPs was greatly altered. As shown in SEM of MoS2/Au NPs (Figure 2B), the Au NPs uniformly distributed on the surface and inside of MoS2, which improved electron transfer efficiency. Subsequently, XRD patterns used to characterize their nanocrystal of the prepared MoS2 nanoflowers and MoS2/AuNP nanohybrids in Figure 2C. As shown the black curve in Figure 2C, the prepared MoS2 nanoflowers exhibit the broad diffraction peaks of MoS2 nanosheets, which reveal the typical crystal domains with a hexagonal structure. Moreover, four obvious diffraction peaks were detected in red curve, which could be indexed with Au (PDF Card No. 65-8601). However, the diffraction peaks of MoS2 was weak in MoS2/Au NPs, which may be attribute to their lower MoS2 contents and relative high crystallinity of Au contents, respectively. Subsequently, as shown in Figure 2D, the Raman spectrum of MoS2 nanoflowers reveals characteristic peaks of MoS2 at 377.8 and 402.7 cm−1, which agree well with the previously reported data38. Moreover, the intensities of the two bands in the spectrum of MoS2/Au NPs nanohybrids were enhanced dramatically, indicating the presence of a chemical interaction or bonding between MoS2 and Au NPs40. Simultaneously, obvious Mo, S and Au elements were detected which also reveal the succeed preparation of MoS2/Au NPs nanohybrids (Figure 2E).

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Figure 2. SEM images of (A) MoS2 and (B) MoS2/Au NPs; (C) XRD pattern and (D) Raman spectra of MoS2 and MoS2/Au NPs; EDX image of (E) MoS2 and (F) MoS2/Au NPs. 3.2. Mechanism Exploration In the proposed sandwich-type immunosensors, the sensitivity depends mainly on the superior electrocatalytic performance of the label for H2O2 reduction. The signal amplification mechanism was investigated by comparative experiments. About the electrocatalytic comparison, the as-synthesized MoS2 nanoflowers, MoS2/Au nanohybrid, Cu2O octahedral, Cu2O@TiO2 nanocrystals, PtCu dendrimers and Cu2O@TiO2-PtCu nanocomposites with the same concentration (6 µL, 2.0 mg/mL) were modified on bare GCE, respectively, and tested by amperometric i-t curve. As shown in Figure 3A, the bare GCE (curve a) has no catalytic effect on H2O2 reduction. However, the current response increased gradually when GCE were modified with MoS2 nanoflowers (curve b) and MoS2/Au nanohybrid (curve c), respectively, indicating that MoS2 nanoflowers and MoS2/Au nanohybrid can promote electron transfer and catalyze H2O2. Especially, the MoS2/Au nanohybrids was superior to MoS2 12


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nanoflowers as substrate to immobilize Ab1. Subsequently, the current response increased significantly (curve d) when octahedral Cu2O modified GCE. Since the octahedral Cu2O has a large specific surface area, good electrochemical performance, and as known to all, it has superior electro-catalytic activity for H2O2 reduction41. Then, the current response also increased when Cu2O@TiO2 nanocrystals modified the bare GCE (curve e). Simultaneously, the PtCu dendrimers exhibit a superior electro catalytic property (curve f) towards the H2O2 reduction because of the dendritic structure provide more active site and increased the surface area. In general, a combination of two or more signal amplification strategies may have a synergistic effect compared to a single signal amplification strategy, thanks to the optimized the interface. As expected, with the Cu2O@TiO2-PtCu nanocomposite as the label, the current changes the most (curve g), it can be inferred that the changes in surface morphology and electronic state may lead to synergistic effects. According to literatures42-44, the electrocatalytic mechanism of H2O2 electroreduction was as follows: (1) H2O2 + e− → OHad + OH−; (2) OHad + e− → OH−; (3) 2OH− + 2H+ → 2H2O. Based on above equality, the formation of OHad is crucial step to control the reaction process in the H2O2 reduction45. Therefore, a key issue is that the surface of the resultant nanocomposites can provide active sites for the adsorption of OHad, which can be demonstrated by the electrochemical properties of the Cu2O@TiO2-PtCu in H2O2 solution (Figure S1). The significant current increase of Cu2O@TiO2-PtCu confirmed the positive catalytic performance of Cu2O@TiO2-PtCu for H2O2 reduction, which was helpful to realize electrochemical signal amplification and high sensitive immunosensor. 13



Figure 3. (A) Current responses of different nanocomposites to 5 mmol/L of H2O2: (a) bare GCE, (b) MoS2 nanoflowers, (c) MoS2/Au nanohybrid, (d) Cu2O octahedral, (e) Cu2O@TiO2 nanocrystals, (f) PtCu dendrimers and (g) Cu2O@TiO2-PtCu nanocomposites; (B) Nyquist diagram of A.C. impedance: bare (a) GCE, (b) MoS2/Au NPs/GCE, (c) Ab1/MoS2/Au NPs/GCE, (d) BSA/Ab1/ MoS2/Au NPs/GCE, (e) insulin Ag/BSA/Ab1/MoS2/Au

NPs/GCE

and

(f)

Cu2O@TiO2-PtCu/Ab2/Insulin

Ag/BSA/Ab1/MoS2/Au NPs/GCE. 3.3. Characterization of the immunosensor Figure 3B shown the Nyquist diagram of A.C. impedance spectroscopy in gradient modification process. The Nyquist diagram consists mainly of two portions. The linear portion at low frequency was associated with the electrochemical behavior limited by diffusion, when the semicircular portion at high frequency correspond to the electrochemical process of electron transfer.46-48 In general, the diameter of the semicircular portion is proportional to the electron transfer resistance (Ret). As shown in Figure 3B, the Ret of bare GCE was very small (curve a). However, the Ret decreased (curve b) when the MoS2/Au nanocomposites were dropped onto GCE surface because of better electron transfer performance of MoS2/Au NPs. Subsequently, the Ret gradually increase when Ab1 (curve c), BSA (curve d) and insulin Ag (curve e) were sequentially modified the GCE, due to the nonconductive property of biomolecules. 14


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After modification of Cu2O@TiO2-PtCu/Ab2 (f), the Ret further increased, indicating that the specific recognition of insulin Ag and Ab2 interaction was successful. Obviously, the proposed immunosensor was prepared successfully. The inset of Figure 3B shown a model of Randles equivalent circuit, .including four components: the apparent charge transfer resistance (Ret), the ohmic resistance of the electrolyte (Rs), the Warburg impedance (Zw) and the double layer capacitance (Cdl).49 Their values were simulated by ZSimpWin software and the results were shown in Table S1. 3.4. Optimization of experimental conditions To achieve the optimal sensitivity for insulin Ag quantitative detection of, it was vital to optimize the experimental conditions. All optimization experiments were based on the proposed immunosensor employed for insulin Ag detection (1.0 ng/mL). In general, pH value primarily affects the electro-catalytic process and biological protein activity of the label for H2O2 reduction. Figure 4A shown the amperometric response of the proposed immunosensor in PBS at different pH values for H2O2 reduction. As shown in Figure 4A, the optimal amperometric response was obtained at pH 6.64. Thus, PBS at pH =6.64 was optimized for testing in the entire measurement. The concentration of MoS2/Au NPs was an important factor affecting the immobilization of Ab1, charge transfer efficiency at the electrode interface and electrocatalytic process for H2O2 reduction. Figure 4B shown the electro-catalytic response when the proposed immunosensor was constructed with different concentrations of MoS2/Au NPs for 1.0 ng/mL of insulin Ag detection. As shown in Figure 4B, the 2.0 mg/mL of MoS2/Au NPs was selected as optimal concentration to modify GCE. In addition, the concentration of Cu2O@TiO2-PtCu/Ab2 also affected the process of 15



electro-catalytic H2O2. Figure 4C shown the amperometric response when different concentrations of Cu2O@TiO2-PtCu/Ab2 were used as labels for insulin Ag detection. As shown in Figure 4C, the concentration of 2.5 mg/mL exhibited the optimal amperometric response. However, the current response decreased when the concentration was further increased. It can infer that as the thickness of the Cu2O@TiO2-PtCu/Ab2 film increases, the electron transfer resistance at interface may increase. Thus, 2.5 mg/mL of Cu2O@TiO2-PtCu/Ab2 was selected as optimal concentration to fabricate immunosensor in the work.

Figure 4. The optimization of experimental conditions: (A) pH value, concentration of (B) MoS2/Au NPs and (C) Cu2O@TiO2-PtCu/Ab2. Error bar = RSD (n = 5). 3.5 Analysis and detection The prepared immunosensors were used to detect different insulin Ag concentrations under the optimal conditions by amperometric i-t curve, using MoS2/Au as substrate and Cu2O@TiO2-PtCu/Ab2 as labels. Due to the conjugate binding between Ab2 and 16


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label, the different insulin Ag concentration on electrode surface decide the amount of the catalytic active site because of the specific binding between antigen and Ab2, which also quantitatively correlative with the change of amperometric response. As shown in Figure 5A, the amperometric responses increased as the insulin concentration increased from 0.1 pg/mL to 100 ng/mL. Especially, the electro-catalytic response was ignorable when no any insulin Ag (curve a), indicating the background current and other nonconductive bioactive substances were negligible.49 As shown in Figure 5B, the amperometric responses is linear with the logarithm of insulin Ag concentration as the insulin concentration increased from 0.1 pg/mL (curve b) to 100 ng/mL (curve n). Accordingly, the linear regression equation for the calibration curve was I = 29.14 log c + 144.08, with a correlation coefficient of 0.9985. The detection limit was calculated about 0.024 pg/mL at a signal to noise ratio (S/N) of 3 (detailed calculations shown in Supporting Information). Moreover, the proposed immunosensors exhibit a lower detection limit and wider linear range compared to previous reports for insulin detection (Table S2).

Figure 5. (A) Amperometric response of immunosensors for different concentrations of insulin Ag detection: (a-i) 0, 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500 pg/mL; (j-n) 1.0, 5.0, 10, 50 and 100 ng/mL; (B) Calibration curves of the immunosensor to different concentrations of insulin Ag. Error bar = RSD (n = 5). 17



3.6 Application of the immunosensor in human serum To verify the reliability and accuracy of the proposed immunosensor in actual sample analysis, a recovery experiments was designed by adding a series of standard insulin solution to human serum samples. The average recovery was 98.9% ~ 100.3%, and the RSD was 2.54% ~ 4.28% (Table S3), suggesting the proposed immunosensor can effective quantify detection insulin Ag in human serum. 4. Conclusions In this work, MoS2/Au NPs was used as the substrate, and cotahedral Cu2O@TiO2PtCu was used as a label to develop an ultrasensitive sandwich electrochemical immunosensor for the quantitative detection of insulin Ag. Using MoS2/Au NPs as substrate to modify GCE can effectively increase the immobilization of Ab1 and significantly accelerate the electron transfer efficiency at the electrode interface. More significantly, utilizing Cu2O@TiO2-PtCu NPs as signal amplification labels that efficient strengthen the electro-catalytic performance for H2O2 reduction, and effective amplify the current signal due to the synergy between the components. Finally, the proposed immunosensor exhibited excellent sensitivity, reproducibility, selectivity and stability, provides a new prospective for realizing the sensitive detection of other biomolecules in clinical diagnosis.

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Associated Content Supporting Information The contents of Supporting Information are listed as follows: Apparatus and reagents, Preparation of MoS2/Au NPs composites, Preparation of octahedral Cu2O@TiO2 coreshell nanohybrid, Preparation of Cu2O@TiO2-PtCu nanocomposite, Preparation of Cu2O@TiO2-PtCu/Ab2, Cyclic Voltammograms of Cu2O@TiO2-PtCu, EDX spectra, TEM of alloyed PtCu nanocrystal, TEM of Cu2O@TiO2-PtCu nanocrystal, XPS analytical of MoS2/Au NPs and Cu2O@TiO2-PtCu, Electrochemical properties of MoS2/Au NPs, Limit of detection, Reproducibility, selectivity and stability, Equivalent circuit of A.C. impedance, Simulation parameters of the equivalent circuit components, Comparison of different methods for Insulin Ag detection and Determination of insulin Ag in human serum sample (PDF).

Author Contributions ∇: F. Li and J. Feng contributed equally. The authors declare no competing financial interest

Acknowledgments This study was supported by the National Key Scientific Instrument and Equipment Development Project of China (No.21627809), National Natural Science Foundation of China (Nos. 21575050, 21777056, 21505051).

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Facile Synthesis of Cu2O@TiO2-PtCu Nanocomposites as a Signal

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