Bimetallic Pt/Pd encapsulated mesoporous-hollow CeO2 nanospheres for signal amplification toward electrochemical peptide-based biosensing for matrix metalloproteinase 2

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Highlights

  • A sensitive MMP-2 biosensor was constructed based on specific cleavage of peptide.

  • Pt/Pd/mhCeO2NS was used as nanocarriers and electrocatalysts for the biosensor.

  • The transduction of peptide cleavage events was combined with the electrocatalysts.

  • The catalytic capacity of Pt/Pd/mhCeO2NS was optimal to the signal amplification.

  • The electrochemical analytical performance was significantly improved and enhanced.

Abstract

A facile electrochemical biosensor for matrix metalloproteinase 2 (MMP-2) was developed based on the target induced cleavage of a special designed peptide by using bimetallic Pt and Pd nanoparticles encapsulated mesoporous-hollow ceria nanospheres (Pt/Pd/mhCeO2NS) as nanocarriers and electrocatalysts. Briefly, employing l-lysine as bridge and linker, Pt/Pd/mhCeO2NS simply synthesized was used as a loading platform to immobilize electroactive thionine (Thi) and streptavidin (SA), resulting in the final formation of SA/Thi/Pt/Pd/mhCeO2NS nanoprobes. A specific biotin-labeled peptide (biotin-GPLGVRGKGGC, P1) acted as a molecular recognition element was firstly anchored on the Au nanoparticles modified electrode surface. In the presence of MMP-2, the P1 was specifically cleaved into two fragments at a certain site between G and V, while SA/Thi/Pt/Pd/mhCeO2NS nanoprobes were bonded onto the resulting electrode surface through the inherent interaction between streptavidin and biotin derived from uncleaved P1. In the proposed protocol, the electrochemical signal amplification was achieved by the effectively catalysis of Pt/Pd/mhCeO2NS to the decomposition of H2O2. This could result in the significant enhancement of the electrochemical response for determining MMP-2 in the range of 0.1 pg mL−1–10 ng mL−1 with a detection limit of 0.078 pg mL−1. The present work demonstrated that the combination of the direct transduction of peptide cleavage events with the efficient Pt/Pd/mhCeO2NS catalysis method, providing a promising effective strategy for MMP-2 detection.

Graphical abstract

Based on specific target-induced cleavage of peptide, a sensitive electrochemical biosensor for MMP-2 was constructed by using Pt/Pd/mhCeO2NS as nanocarriers and electrocatalysts.

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Introduction

Development of simple, rapid, accurate, and sensitive method for the assay of disease-related proteins, especially cancer biomarkers, is of great importance for many aspects of modern clinical and biomedical fields [1]. Currently, immunoassay protocols based on antibody-antigen interaction is a common approach in the quantitative detection of target biomarkers [2], [3], [4], [5]. Nevertheless, there are some fundamental shortcomings to these approaches such as the requirement of costly antibodies, long incubation time and easily denaturing of antibodies with temperature change [6], [7]. Thus, it is necessary to develop new methods for protein assays. Peptides, with simple and defined structure, which was obtained using phage display technique, exhibit tremendous potential to act as the substitute for bioassay due to their advantages, including versatility, reliability, cost effectiveness, resistance to harsh environments [8], [9]. Several biosensors based on target induced cleavage of peptide have been employed to detect some proteases such as matrix metalloproteinases (MMPs) [10], prostate-specific antigen [11], collagenase [12], cathepsin B [13], caspase [14] and thrombin [15].

MMPs, as a class of zinc-dependent endopeptidases, have received extensive attention because their over-expression was observed in many human cancers [16], [17]. Among them, MMP-2 has been recognized as a biomarker of breast cancer, protease cancer and ovarian cancer [18], [19], [20], [21]. Since a specific peptide with the amino acid sequence PLGVR has been identified as a substrate to measure MMP-2, several peptide-based assay platforms have been explored for the determination of MMP-2, such as fluorescence [22], surface plasmon resonance [23], photoluminescence assays [24]. However, electrochemical methods, which possess such distinct merits as high sensitivity, cheapness, and small size, have been rarely used for detection of MMP-2 in clinical samples [25]. Thus, it is meaningful to search for more sensitive and effective electrochemical platform for the detection of MMP-2 based on specific peptide cleavage induced by target.

To improve the sensitivity of the electrochemical method, various nanomaterials with versatile properties have been extensively employed for signal amplification [26]. Particularly, notched mesoporous-hollow ceria nanospheres (mhCeO2NS), with uniform pore distribution, high surface area, and rigid framework, are desirable to be served as supporting materials to immobilize biomolecules [27], even though CeO2 nanoparticles suffer from low electron conductivity [28]. However, this could be greatly improved by forming nanocomposites of CeO2 with bimetallic Pt/PdNPs, which are of unique electronic conductivity and catalytic property [29]. According to previous reports [30], from the chemistry stand point, lanthanide ions such as cerium (CeO2) are hard Lewis acids. And they have high affinity with hard ligands such as phosphate and carboxyl groups. As a result, we attempted in this work to decorate Pt/PdNPs onto the surface of mhCeO2NS by using l-lysine with single bondNH2 and single bondCOOH as a bridge and linker [30], resulting in the formation of Pt/Pd/mhCeO2NS due to the strong interaction between single bondNH2 and metal NPs [31], [32]. Then, Pt/Pd/mhCeO2NS were served as nanocarriers to anchor streptavidin (SA) and electroactive Thi to obtain the proposed nanoprobes SA/Thi/Pt/Pd/mhCeO2NS. Meanwhile, a specific peptide labeled with biotin (biotin-GPLGVRGKGGC, P1) was firstly oriented onto the electrode surface electrodeposited with Au. Then the introduction of target MMP-2 resulted in the specific recognition and cleavage of P1 at a certain site between G and V. Through the high affinity of SA to biotin in remained peptides not cleaved by MMP-2, SA/Thi/Pt/Pd/mhCeO2NS could be bound to the resultant electrode surface. So the electrochemical response could be detected due to the presence of Thi. Based on our observations, the developed peptide-based detecting platform for MMP-2 exhibited improved analytical performance, which was originated from the desired conductivity and catalytic activity of Pt/Pd/mhCeO2NS as nanocarriers and signal enhancer, indicating the potential application for other proteases detection.

Section snippets

Reagents and material

Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-7 (MMP-7) were bought from Sino Biological Inc., (Beijing, China). Ammonium cerium (IV) nitrate ((NH4)2Ce(NO3)6) and l-lysine were from KeLong Bio. Co., Ltd. (Chengdu, China). Glutaraldehyde (GA) was obtained from Beijing Chemical Reagent Co., (Beijing, China). Palladium potassium chloride (K2PdCl4), chloroplatinic acid (H2PtCl6), gold chloride (HAuCl4), streptavidin (SA), bovine serum albumin (BSA), hemoglobin (Hb), l-cysteine (l

Characterizations of prepared nanoparticles

The morphology and size of CeO2 precursor, mhCeO2NS, and Pt/Pd/mhCeO2NS were investigated by SEM. As shown in Fig. 1A , CeO2 precursors have uniform shapes with a diameter of ∼80 nm. After calcination at 300 °C for 2 h, the obtained mhCeO2NS have rough surface and gaps between every nanospheres could be clearly observed. Moreover, the center of every mhCeO2NS was dark and the edge was bright, which were attributed to that the morphology of the prepared mhCeO2NS was a hollow structure at nanoscale (

Conclusion

In this work, a sensitive electrochemical peptide-based biosensor for MMP-2 detection was successfully fabricated based on the target induced peptide cleavage and the employment of mesoporous-hollow CeO2 nanospheres encapsulated with bimetallic Pt/PdNPs (Pt/Pd/mhCeO2NS) as nanocarriers and signal enhancer. Owing to the great catalytic capacity of Pt/Pd/mhCeO2NS, the amplified electrochemical response was successfully achieved, and the analytical performance of the proposed peptide-based

Acknowledgements

This work was financial supported by NNSF of China (21275119) and Fundamental Research Funds for the Central Universities (XDJK2013A008).

Wenju Xu is an associate professor of chemistry in Southwest University, China. She received her Ph.D. degree in analytical chemistry from Southwest University, China in 2009. Her main areas of interest are chemical sensors and electrochemical biosensors.

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    Wenju Xu is an associate professor of chemistry in Southwest University, China. She received her Ph.D. degree in analytical chemistry from Southwest University, China in 2009. Her main areas of interest are chemical sensors and electrochemical biosensors.

    Pei Jing is MS candidate in the College of Chemistry and Chemical Engineering, Southwest University, China. Her main areas of interest are electrochemical biosensors.

    Huayu Yi is MS candidate in the College of Chemistry and Chemical Engineering, Southwest University, China. Her main areas of interest are electrochemical biosensors.

    Shuyan Xue is MS candidate in the College of Chemistry and Chemical Engineering of Southwest University, China. Her main areas of interest are electrochemical biosensors.

    Ruo Yuan is a professor of chemistry in Southwest University, China. He received a Ph.D. degree in analytical chemistry from Hunan University, China in 1994. The main research interests of professor Yuan are chemical sensors and biosensors.

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    These authors contributed equally to this work.

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