A facile and label-free electrochemical aptasensor for tumour-derived extracellular vesicle detection based on the target-induced proximity hybridization of split aptamers.

Facile detection of tumour-derived extracellular vesicles (EVs) is crucial to cancer diagnosis. Herein, a facile and label-free electrochemical aptasensor was fabricated to detect tumour-derived EVs based on the target-induced proximity hybridization of split aptamers. In this assay, two designed oligonucleotide probes containing fragments of a protein tyrosine kinase-7 (PTK7) aptamer were used to recognize and capture EVs containing PTK7. In the presence of target EVs, the aptamer-target ternary complex could induce proximity hybridization and form a DNA duplex on the electrode. The DNA duplex could bind more electroactive Ru(NH3)63+ through electrostatic attraction, resulting in an increased cathodic current signal. By virtue of the excellent electrochemical signal reporter RuHex, the specificity of the aptamer and proximity ligation, a facile EV electrochemical aptasensor with a detection limit of 6.607 × 105 particles per mL was realized. Furthermore, this aptasensor showed good selectivity to distinguish different tumour-derived EVs and was applied to detect EVs in complex biological samples. The proposed electrochemical aptasensor can be further extended to the detection of other EVs, thus showing great potential in clinical diagnosis.

An electrochemiluminescent aptasensor for amplified detection of exosomes from breast tumor cells (MCF-7 cells) based on G-quadruplex/hemin DNAzymes.

Exosomes are non-invasive biomarkers for cancer diagnosis. Herein, we describe an electrochemiluminescent (ECL) aptasensor for the detection of exosomes from breast tumor cells. Mercaptopropionic acid (MPA)-modified Eu3+-doped CdS nanocrystals (MPA-CdS:Eu NCs) and H2O2 were used as ECL emitters and coreactant, respectively. The exosomes are recognized and captured by the CD63 aptamer, and then form a G-quadruplex/hemin DNAzyme, which efficiently catalyzes the decomposition of H2O2, resulting in the decreased ECL signal of MPA-CdS:Eu NCs. The exosomes from breast tumor cells (MCF-7 cells) can be detected in the range of 3.4 × 105 to 1.7 × 108 particles per mL. The limit of detection (LOD) was estimated to be 7.41 × 104 particles per mL at a signal-to-noise ratio of 3. The aptasensor has been successfully used to detect exosomes in the serum.

Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognition-Induced Multi-DNA Release and Cyclic Enzymatic Amplification.

Sensitive and specific detection of tumor exosomes is of great significance for early cancer diagnosis. In this paper, we report an aptamer strategy for exosome detection based on aptamer recognition-induced multi-DNA release and cyclic enzymatic amplification. First, we use aptamer-magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells, leading to the release of three kinds of messenger DNAs (mDNAs). After magnetic separation, the released mDNAs hybridized with the probe DNAs immobilized on a gold electrode. Electroactive Ru(NH3)63+ was used as the signal reporter because of its electrostatic attraction to DNA. Subsequent Exo III cyclic digestion caused the electrochemical signal to "turn off". Because the electrochemical signal reflects the concentration of Ru(NH3)63+ and the concentration of Ru(NH3)63+ is correlated with the mDNA concentration, which is correlated with the exosome concentration, the tumor exosomes can be detected by examining the decrease in the peak current of Ru(NH3)63+. In this paper, the signal was amplified by the numerous mDNAs released from the magnetic bead and the Exo III-assisted mDNA recycling. Under the optimal conditions, a detection limit down to 70 particles/µL was achieved, which is lower than the LODs of most currently available methods. Furthermore, this assay can be used to detect tumor exosomes in complex biological samples, demonstrating potential application in real sample diagnosis.

Electrochemical aptasensor for exosomal proteins profiling based on DNA nanotetrahedron coupled with enzymatic signal amplification.

Exosomes are extracellular nanovesicles for transferring and delivering membrane and cytosolic molecules between cells. Detection and profiling of exosomal proteins can provide direct information on disease progression, which is important to the early diagnosis and monitoring of diseases. Herein, a well-designed electrochemical aptasensor was fabricated for the profiling of cancerous exosomal proteins based on DNA nanotetrahedron (NTH) coupled with Au nanoparticles (NPs) and enzymatic signal amplification. In this assay, the aptamer modified DNA NTHs were used as the recognition and capture unit, Au NPs-DNA conjugates coupled with horseradish peroxidase were used to realize signal amplification. This aptasensor achieves a detection limit down to 1.66 × 104 particles/mL for HepG2 liver cancer exosomes. In addition, the analysis of plasma-derived exosomes in HepG2 liver cancer bearing mice at different cancer stages was also achieved. More importantly, the aptasensor can be used to profile four kinds of exosomal proteins by using the corresponding aptamer. The proposed electrochemical aptasensor may be served as a potential platform for exosome detection and exosomal proteins profiling.

Sensitive electrochemical biosensor for MicroRNAs based on duplex-specific nuclease-assisted target recycling followed with gold nanoparticles and enzymatic signal amplification.

The detection of sequence-specific microRNAs (miRNAs) is an important factor to the diseases diagnosis. Herein, a triple signal amplification electrochemical biosensor for highly sensitive detection of miRNA-21 was developed based on a duplex-specific nuclease (DSN)-assisted target recycling combined with gold nanoparticles (NPs), horseradish peroxidase (HRP) enzymatic signal amplification. The electrochemical biosensor generated significantly amplified amperometric current changes (Δi) for the detection of miRNA-21 down to 43.3 aM, and Δi was proportional to the logarithm of the concentration of miRNA-21 within the range of 0.1 fM to 100 pM. Meanwhile, the inherent selectivity of the term hairpin capture probe endowed the biosensor with high differentiation of similar miRNAs. The good feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in A549 lysates, which indicates its promising potential in biomedical research and clinical analysis.