Split-aptamer mediated regenerable temperature-sensitive electrochemical biosensor for the detection of tumour exosomes

Highlights

• A split-aptamer mediated regenerable temperature-sensitive (SMRT) biosensor was built for the first time.

• The sensitive and specific detection of SMMC-7721 exosomes was successful implementation.

• The SMRT biosensor could be regenerated simply and quickly by adjusting the temperature to 37 °C in 30 s.

Abstract

In this paper, a split-aptamer mediated regenerable temperature-sensitive (SMRT) electrochemical biosensor was constructed for the detection of exosomes. The split-aptamer used in this SMRT biosensor was composed of two fragments, one of which was immobilized on the surface of an electrode via sulfhydryl groups and named split-a and the other was labelled with methylene blue and named split-b. The two fragments could form sandwich structures at the electrode surface via target-induced self-assembly in the presence of target exosomes at 4 °C in PBS, and then realizing the detection of exosomes via voltammetry. In addition, due to the temperature sensitivity of the split-aptamer, the electrode could be regenerated through temperature-induced disassembly of the sandwich structures. Consequently, the SMRT biosensor realized sensitive and specific analysis of target exosomes with a limit of detection of 1.5 × 10⁶ particles/mL and could be quickly and easily regenerated by washing with PBS at 37 °C for 30 s without any additives. This is the first study on the construction of a reproducible electrochemical biosensor using a split-aptamer for the specific detection of tumour exosomes, and may provide an innovative strategy for the economical and efficient design of regenerable electrochemical biosensors.

Introduction

Exosomes, which are double-layered vesicles with sizes of 40–160 nm, are widespread in the supernatants of cell cultures and in multiple body fluids [1]. These vesicles possess a variety of biomolecules from parental cells, including nucleic acids, proteins and lipids [2], and play significant roles in various physiological and pathological processes of the body, including tumour proliferation, metastasis, and immune modulation [[3], [4], [5]]. Hence, exosomes have become new and valid biomarkers for tumour screening, diagnosis and treatment [6]. However, due to their small volume and low content in biofluids at the early phases of cancer, realizing highly sensitive and selective detection of exosomes remains challenging [7].

Recently, numerous methods that are based on employing the surface proteins of exosomes as targets have been applied for the analysis of exosomes, including fluorescence [8,9], colorimetry [10], surface plasmon resonance [11], surface-enhanced Raman spectroscopy [12], and electrochemical methods [13,14]. Among them, the electrochemical method has aroused the interest of researchers due to its various advantages, such as rapid response speed, hypersensitivity, simple device, and low cost [15]. However, electrochemical biosensors have poor reproducibility and poor stability, which hinders their wide application as diagnostic equipment [16]. This is due mainly to discrepancies in the modifications of electrodes [17]. To solve this problem, a sensor regeneration strategy was proposed, which enables a sensor to repeatedly perform multiple detections, thereby substantially reducing divergence from sensor to sensor [18]. To realize the regeneration of electrochemical biosensors, most current strategies require additional chemical reagents, such as guanidine chloride [19], glycine [20], and urea [21]. Moreover, these strategies not only affect the type and dispersion of ions on the biosensor surface but also damage the probes that have been modified onto the electrode [22]. Consequently, the design of a reliable, gentle, simple, and fast strategy for the regeneration of electrochemical biosensors is vital.

Aptamers, which are single-stranded oligonucleotides with 20–60 bases that can identify targets with high affinity and specificity, have attracted extensive attention in the development of simple and universal electrochemical sensors [[23], [24], [25], [26]]. Among them, split-aptamers have shown significant advantages in constructing electrochemical sensors [[27], [28], [29]]. A split-aptamer originates from one intact aptamer sequence that is artificially divided into two or more fragments [30]. They are more flexible and easier to design for sensors due to their short sequences [31,32]. In addition, the split-aptamer fragments can be induced to aggregate to form a recognition configuration in the presence of the target [33,34]. The recognition configuration is mostly a sandwich structure, which is very convenient for the construction of electrochemical sensors [[35], [36], [37]]. However, building reproducible electrochemical biosensors using aptamers or split-aptamers under mild conditions remains challenging.

Recently, our group and Wang's group explored a few successful split-aptamer based probes with temperature sensitivity for the detection of tumour cells or exosomes [8,38]. In this work, we report a split-aptamer mediated regenerable temperature-sensitive (SMRT) electrochemical biosensor for the sensitive and selective detection of tumour exosomes for the first time. As displayed in Scheme 1, the two fragments, namely, split-a and split-b, that originate from an integrated aptamer that can specifically recognize the N-glycoprotein on the surfaces of SMMC-7721 cells [8,39], are used as recognition elements in this study. After modification with a sulfhydryl group, split-a can be immobilized on a gold electrode surface by Au–S bonds. Redox moiety methylene blue is modified onto split-b. In the presence of exosomes at 4 °C, fragments split-a and split-b move close to each other to capture the target and form a recognition configuration that can realize the specific and sensitive detection of exosomes as the methylene blue approaches the electrode, thereby generating a redox signal. The recognition configuration that is formed by the split-aptamer and exosomes can be destroyed by rinsing the electrode in PBS at 37 °C due to the split-aptamer losing its recognition ability at higher temperatures. Therefore, the electrodes can be regenerated in as little as 30 s and then used directly for subsequent testing without any additional modifications.