Elsevier

Biosensors and Bioelectronics

Volume 134, 1 June 2019, Pages 49-56
Biosensors and Bioelectronics

Electrochemical dual-aptamer-based biosensor for nonenzymatic detection of cardiac troponin I by nanohybrid electrocatalysts labeling combined with DNA nanotetrahedron structure

https://doi.org/10.1016/j.bios.2019.03.049Get rights and content

Highlights

  • An enzyme-free electrochemical aptasensor was proposed for analysis of cTnI.

  • Three-dimensional nanotetrahedron-dual-aptamer probe can capture cTnI specifically.

  • Magnetic metal organic framework Fe3O4@UiO-66/Cu@Au was introduced as nanocarrier.

  • Cluster-based nanoprobe can amplify the signal significantly based on LBL assembly.

  • The proposed approach demonstrated detection limits as low as 16 pg/mL for cTnI.

Abstract

The accurate detection of biomarkers for acute myocardial infarction (AMI) plays an important role in clinical diagnosis and management process. In this work, we developed an electrochemical biosensor by using magnetic metal organic framework (MMOF) nanocatalysts and DNA nanotetrahedron (NTH) based dual-aptamer probes for nonenzymatic detection of cardiac troponin I (cTnI), a gold standard biomarker for the early diagnosis of AMI. Firstly, the NTH-assisted dual-aptamer (Tro4 and Tro6) capture probes were immobilized on the screen-printed gold electrode (SPGE) for the highly enhanced capture the target cTnI with steady support and optimized interface density. Then, the MMOF Fe3O4@UiO-66 nanozymes were decorated by bimetallic Cu@Au nanoparticles and two kinds of aptamer. This fabricated nonenzymatic nanoprobe1 (NP1) can be applied for recognizing the cTnI specifically and amplifying the current signal by catalyzing the oxidation of hydroquinone (HQ) to benzoquinone (BQ) with H2O2. The target proteins were captured to fabricate a supersandwich-like structure on a SPGE interface. Furthermore, the nanoprobe2 (NP2) of Cu@Au nanozymes labeled with dual-complementary DNA (cDNA) to the dual-aptamer, were anchored on the NP1 through DNA hybridization, leading to the formation of cluster-based nanoprobes for further enhancing detection sensitivity. Finally, this enzyme-free electrochemical aptasensor exhibited great analytical performance with a dynamic range of 0.05–100 ng/mL, a low detection limit of 16 pg/mL, high selectivity and good repeatability. The fabricated aptasensor has great potential development in the field of clinic disease diagnostics for AMI.

Introduction

Cardiovascular disease has turned into a severe public health problem worldwide, especially acute myocardial infarction (AMI) is a leading clinical disease and would cause serious damage in the myocardium (Smith and Gerszten, 2017). Following the myocardial damage, the individual proteins of troponin complex are released into the bloodstream within 90 min to 3 h upon symptom onset of AMI (Nandhikonda and Heagy, 2011). Early diagnosis of AMI play a vital role in clinical research for persons at high cardiovascular risk. Cardiac biomarkers can improve identification of high-risk patients for AMI events. A variety of cardiac biomarkers, including creatine kinase-MB (CK-MB), cardiac troponin T (cTnT), cardiac troponin I (cTnI) and myoglobin (MYO), can be employed to diagnose an AMI (Abdorahim et al., 2016; Nezami et al., 2018; Rezaei et al., 2016). Among various biomarkers, cTnI is regarded as the “gold standard” marker for AMI (Kemp et al., 2004; Takeda et al., 2003). Patients with a clear positive test for AMI have serum levels of cTnI as high as 5–50 ng/mL. Therefore, numerous studies have been performed to obtain reliable and sensitivity diagnostic tools for cTnI detection (Han et al., 2016).

Various developed methods, such as electrochemistry immunoassay (Akter et al., 2017; Dhawan et al., 2018; Yan et al., 2018; Zhang et al., 2018a), photoelectrochemical immunoassay (Fan et al., 2018; Tan et al., 2017), electrochemiluminescence immunoassay (Yang et al., 2018b; Zhang et al., 2017), fluorescence immunoassay (Liu et al., 2018a) and enzyme-linked immunosorbent assay (ELISA), were utilized for the determination of cTnI. Compared with other approaches, electrochemical immunoassay has attracted particular attention due to its properties of portable, sensitive, and rapid response. However, these antibody based methods have several limitations such as the difficulty for the chemical modification, high cost for the production, and low stability at high temperatures. Recently, electrochemical aptamer-based biosensor has been established for cTnI research as they offer many advantages to overcome the limitations of antibodies (Chekin et al., 2018; Negahdary et al., 2017; Qiao et al., 2018). Aptamers are single-stranded RNA or DNA oligonucleotides that can specifically and efficiently bind to a series of proteins and cells. For instance, Ban's group has demonstrated that Tro4 and Tro6 aptamer can be used for recognizing cTnI selectively (Jo et al., 2015, 2017). Compared with antibodies, aptamers have been widely performed in the biosensor fields with high affinity, good stability, small size, and flexible modification. Hence, dual-aptamer as recognition molecule on the electrode interface can increase the recognition efficiency and target accessibility for cTnI analysis.

However, single-stranded DNA aptamer is easy to nonspecifically absorb and aggregate on the electrode interface, impeding the effective binding of aptamers to target proteins. Framework nucleic acids especially DNA nanotetrahedron (NTH) structure with well-defined and highly rigid architectures can ensure the precise orientation and density of the aptamer on the sensing interface (Goodman et al., 2005). DNA NTH structure is assembled from four single-stranded nucleic acids and can be firmly attached on the electrode surface uniformly. The relative far vertical space between aptamer and electrode can provide a solution-native-like recognition microenvironment and suppress the entangling of aptamer recognition probes. Because of the superior properties of NTH-based electrode surface, it has been used for sensitive and selective detection of protein, exosome and cell (Lin et al., 2016; Sun et al., 2018a, 2018b; Yang et al., 2018a). Herein, NTH-based dual-aptamer capture probe on the electrode surface can greatly increase the target protein accessibility and recognition efficiency, and further improve detection sensitivity of aptasensor for cTnI detection.

Only with the DNA NTH structure, the detection sensitivity for cTnI may not meet the requirement of clinical sample assay. For electrochemical aptasensing, the detection sensitivity can be improved with specifically designed nanoprobes. The common signal amplification method is to immobilize natural enzymes onto nanomaterials for enhancing the detection sensitivity through enzymatic electrochemical processes (Sun et al., 2016; Zheng et al., 2014). However, the native conformations of enzymes can be easily disrupted by changes of pH and temperature, leading to the loss of catalytic activities. In addition, the preparation and storage of nature enzymes are expensive and time-consuming. Hence, it is urgently desirable to design robust nonenzymatic nanocatalysts with high electrocatalytic activities for electrochemical biosensor.

Recently, nanozymes, a term defined for nanomaterial with enzyme-mimicking properties, have emerged as a new kind of artificial enzymes due to its striking merits of low cost, robustness and ease of mass production (Liu et al., 2018b; Wei and Wang, 2013). In 2007, it was discovered that magnetic iron oxide (Fe3O4) nanoparticles possess an intrinsic enzyme-like activity similar to that of natural horseradish peroxidase (HRP) (Zheng et al., 2014). To date, a large amount of nanomaterials have been demonstrated to possess the HRP-mimicking properties, ranging from metals and metal oxides to metal organic frameworks (MOFs) (Wang et al., 2018; Zhang et al., 2018b). For instance, noble metal-related nanomaterials (Au, Pt and Pd) have extensive applications owing to the great catalytic ability and high ratio surface area. Especially, core-shell bimetallic nanoparticles have showed outstanding catalytic property over the corresponding monometallic nanomaterials (Sun et al., 2016). In addition, MOFs, an emerging class of porous crystalline material, have received increasing attention owing to unique properties of permanent porosity, tunable pore sizes and high interface area. Furthermore, MOFs coupled with other nanomaterials has become a hot pursuit (Song et al., 2017; Zhao et al., 2016). This is because the novel hybrid nanomaterials readily inherit the advantages of both parent materials, significantly boosting their applications. Therefore, by the combination of magnetic metal organic framework (MMOF) nanocomposites and core-shell bimetallic nanoparticles, the nanohybrid electrocatalysts can be performed as the nanocarriers to immobilize aptamer for amplifying electrochemical signals. Compared with Fe3O4 magnetic nanoparticles, the nanoelectrocatalysts exhibited outstanding catalytic performance since the MOFs and bimetallic nanoparticles can greatly improve the catalytic activity of Fe3O4 nanoparticles (Ma et al., 2018).

In this work, an enzyme-free electrochemical biosensor was proposed for cTnI detection based on NTH-based dual-aptamer as capture probe and hybrid nanoelectrocatalysts as signal amplification probe. Firstly, the self-assembled NTH-based Tro4 and Tro6 aptamer probes were conjugated on a screen-printed gold electrode (SPGE) interface through gold−thiol interactions for the greatly enhanced recognition of cTnI. Then, the nonenzymatic nanoprobe1 (NP1) was fabricated through the two kinds of aptamer immobilized on the surfaces of bimetallic Cu@Au nanoparticles modified MMOF Fe3O4@UiO-66 (Fe3O4@UiO-66/Cu@Au). The target cTnI were captured to fabricate an NTH-dual-aptamer/cTnI/NP1 supersandwich-like structure on a SPGE interface. Furthermore, the nanoprobe2 (NP2) of Cu@Au nanoparticles labeled with two types of complementary DNA (cDNA) to the dual-aptamer, were immobilized on the NP1 via the DNA hybridization, leading to the formation of cluster-based nanoprobes. The cluster-based nanoprobes can be applied to catalyze the oxidation of hydroquinone (HQ) for amplifying the electrochemical signal and improving the detection sensitivity significantly. As a result, the aptasensor exhibited outstanding sensitivity and selectivity for cTnI, showing great potential applications in the clinic diagnosis.

Section snippets

Materials and apparatus

All oligonucleotides were synthesized and purified by Invitrogen Biotech. Co., Ltd. (Shanghai China). The sequences were showed in Table S1. Recombinant cardiac troponin I (cTnI), recombinant myoglobin (MYO), bovine serum albumin (BSA) and ELISA kit for cTnI were purchased from Cloud-clone Co., Ltd. (Wuhan, China). Other detailed chemicals and apparatus are provided in the Supplementary material.

Synthesis of hybrid nanoprobes

According to the reported methods, Fe3O4@UiO-66/Cu@Au nanomaterials were prepared. Detailed steps

Principle of the electrochemical aptasensor

The design principle of the nonenzymatic electrochemical aptasensor is exhibited in Scheme 1. In this paper, a designed nanohybrid electrocatalyst were fabricated through a layer-by-layer (LBL) assembly process. UiO-66, one class of Zr(IV)-based MOFs, is constructed with zirconium ions and terephthalic acid and has attracted great attention due to the chemical and solvent stability. Furthermore, UiO-66 was selected for fabricating the shell over the Fe3O4 nanoparticles to form novel MMOF Fe3O4

Conclusions

In summary, a novel nonenzymatic electrochemical aptasensor was designed for highly sensitive and selective detection of cTnI using NTH assisted dual-aptamer capture probes and cluster-based signal nanoprobes. With the assistance of DNA NTH structure, dual-aptamer technology, Fe3O4@UiO-66/Cu@Au hybrid nanozymes and LBL assembly method, supersandwich-type architectures were established on a SPGE interface. The enzyme-free aptasensor exhibits a low detection limit of 16 pg/mL, wide linear range,

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81803521 and 21675177), the Natural Science Foundation of Guangdong Province (No. 2018A030310142), the Fundamental Research Funds for the Central Universities (No. 18zxxt69), the Medical Scientific Research Foundation of Guangdong Province (No. A2017033), and a Start-up Grant from Guangdong Pharmaceutical University (No. 51377003). The authors acknowledge the help from Dr. Xiangan Lin in Sun Yat-Sen Memorial

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