Electrode immobilization-free and sensitive electrochemical sensing of thrombin via magnetic nanoparticle-decorated DNA polymers

Highlights

• An electrode immobilization-free biosensor for thrombin is developed.

• The MNPs/DNA polymer complexes lead to significantly amplified signal output.

• The sensor enables electrochemical detection of thrombin at the low picomolar level.

• The monitoring of thrombin in diluted human serums is demonstrated.

Abstract

The development of sensitive and simple sensing strategies for trace proteins has received increasing attentions in the diagnosis and treatment of diseases. We describe here an electrode immobilization-free and homogeneous electrochemical method for sensitively detecting thrombin in human serums. The strategy relies on target-promoted proximity binding hybridization chain reaction (HCR) formation of magnetic nanoparticle (MNP)-decorated DNA polymers. Simultaneous binding of thrombin by two distinct aptamers increases their local concentrations and enables the proximity binding-induced strand displacement reaction, which leads to subsequent initiation of HCR between the methylene blue (MB)- and biotin-labeled hairpins into long DNA polymers. The streptavidin-modified MNPs further bind the biotin moieties to form the MNPs/DNA polymers. Subsequent accumulation of the MNPs/DNA polymers on the AuNP-deposited electrode can thus yield substantially enhanced current, due to the oxidation of the many MB labels, for highly sensitive detection of thrombin ranging from 5 pM∼50 nM with a detection limit of 1.1 pM in a simple electrode immobilization-free way. Selective interrogation of low levels of thrombin in diluted human serums was also verified, revealing its potential for convenient and ultrasensitive monitoring of a variety of protein biomarkers for disease diagnosis at the early stages.

Introduction

Sensitive and specific detection of protein biomarkers shows great significance for the early diagnosis of diseases [1]. Enzyme-linked immunosorbent assay (ELISA) has been the standard common technique used in protein detection [2,3]. However, it involves in time-consuming operation processes with intensive washing steps and dedicated instrumentations. Additionally, the assay commonly has limited sensitivity and is inflexible to various targets [4,5]. For specific and sensitive determination of protein biomarkers, the emergence of aptamer-based detection methods has attracted much attention. Aptamers, which are selected by systematic evolution of ligands by exponential enrichment (SELEX) from random nucleic acid libraries, are short and single stranded sequences of RNA or DNA [6,7]. This selection process enables aptamer the ability of high specificity and affinity for binding versatile target molecules including proteins, peptides, cells, small molecules, and ions, which makes aptamers superior to antibody probes [8,9]. Owing to their nucleic acid nature, aptamers possess many advantages against antibodies, such as low cost, easy synthesis and modification, resistance to denaturation, and high stability [10,11]. Based on these merits, aptamer recognition molecules have been used for protein biomarker detections by using the proximity binding-induced strand displacement reaction (PBI-SDR) strategy [12]. The PBI-SDR is based on the high affinity of the recognition probes to the target protein, where the probes are brought to close proximity upon binding to the target molecules. This increases their local effective concentrations and facilitates their binding to accelerate the SDR [[13], [14], [15]]. In the meantime, many aptamer-based amplification strategies such as hybridization chain reaction (HCR) [16,17], polymerase chain reaction (PCR) [18,19], catalyzed hairpin assembly (CHA) [20] and rolling circle amplification (RCA) [21,22], were previously proposed to achieve ultra-sensitive biomarker detection. Compared with the methods of RCA, PCR, and LCR that are based on protein enzymes, the HCR possesses the advantages of enzyme-free, mild reaction condition and autonomous protocol features [23]. HCR is a useful non-enzymatic reaction means for the realization of autonomous amplification isothermally. There are also many electrochemical methods to detect thrombin by HCR. However, they required probe immobilization on the electrodes for detection and suffered from high background noises [[24], [25], [26]]. Therefore, it is useful to design electrochemical detection methods that can simplify the operation steps and enhance the sensitivity for their potential practical applications.

Many approaches, such as capillary electrophoresis, chromatography and magnetic separation, have been extensively established for enriching and separating complex samples to reduce background signals. Among them, magnetic nanoparticles (MNPs) have shown promises as excellent tools for separation in biological sample preparation and detection assays because of their high capacity for molecule loading, easy manipulation and reaction flexibility [[27], [28], [29]]. The detection method based on MNPs can offer rapid detection of target molecules and elimination of background signals, which is suitable for complex samples [30]. For example, a universal DNA detection method by integrating flow cytometry analysis with enzyme-free HCR signal amplification for direct measurement of fluorescence emissions on MNPs has been reported [31], while the Wang team developed a multiple-amplified and non-enzymatic electrochemiluminescence method for ultra-sensitive detection of HIV-DNA in complex samples using the enrichment and separation function of MNPs [32]. A DNA-templated formation of the polymer of quantum dot/MNP has also been synthesized to solve the problem of low capture efficiency for sensitive circulating tumor cell detection [33]. Most reported studies on magnetic separations were restricted to a 1:1 or n:1 association style, that is, a single magnetic nanoparticle can capture one or more target analytes, limiting the efficiency of magnetic separation. Therefore, it is important to establish electrochemical detection methods with high magnetic separation efficiency via DNA self-assembly, which not only can efficiently separate the target analytes, but also can obtain amplified electrochemical signals for specific and sensitive analysis of protein biomarkers.

Herein, we designed a homogeneous electrochemical detection method through proximity binding-triggered HCR signal amplification and further combined with MNPs for the efficient separation and detection of thrombin in complex serums. Two aptamer probes simultaneously recognize the same target thrombin to trigger PBI-SDR, which initiates hybridization of the complementary regions of aptamers and the release of the blocker DNAs. The resulting binding-induced hybridization duplexes can initiate the cross-hybridization of two hairpin structures respectively labeled with methylene blue (MB) and biotin to form long double-stranded DNA polymers. MNPs modified with streptavidin can then bind the biotin moieties of the DNA polymers, allowing the MNPs to bind the DNA polymer in an n:1 binding format to greatly improve the efficiency of magnetic separation. The magnetically separated precipitates can be concentrated onto the surface of the electrode to obtain an amplified current signal of the electroactive substance of MB for sensitively detecting thrombin. The homogeneous electrochemical detection method employed in this system is simpler and more convenient than traditional methods by eliminating complex electrode modification and incubation steps and by reducing the steric hindrance effect of the electrode surface for the recognition of the target molecules. In addition, compared with the traditional homogeneous bio-analysis based on exonuclease, MNPs-based method has simpler operation processes and can greatly reduce the influence of background noise, making it a promising method for sensitively detecting protein biomarkers.