Accurate and Nonpurified Identification of Extracellular Vesicles Using Dual-Binding Recognition Mode

Abstract

Circulating extracellular vesicles (EVs) are promising biomarkers for the early diagnosis and prognosis of cancer in a non-invasive manner. However, the rapid and accurate identification of EVs in complex biological samples is technically challenging, which is attributed to the requirement of extensive sample purification and unsatisfactory detection accuracy due to the disturbance of interfering proteins. Herein, a simultaneous binding of double-positive EV membrane protein-based recognition mode (DRM) is proposed. By the combination of DRMmediated toehold activation and G-quadruplex DNAZymecatalyzed etching of Au@Ag nanorods (Au@Ag NRs), we have developed an accurate, non-purified, low-cost, and visual strategy for EV identification. The synchronous binding of double-positive proteins on EV membranes is validated by confocal laser scanning microscopy analysis. This approach exhibits excellent specificity and sensitivity toward EVs ranging from 1.0 × 10 5 to 1.0 × 10 9 particles/mL with a detection limit of 6.31 × 10 4 particles/mL. Moreover, we have successfully realized non-purified EV quantification in complex biological media. In addition, target-initiated catalyzed hairpin assembly (CHA) is integrated with G-quadruplex DNAZyme-catalyzed color variation of Au@Ag NRs; thus, lowbackground EV detection can be achieved by the naked eye. Furthermore, our strategy is easy to adapt to high-throughput formats by using an automatic microplate reader, which could be expected to meet the requirements for high-throughput detection of clinical samples. With its capacities of rapidness, portability, affordability, high throughput, non-purification, and visual detection, this strategy could provide a practical tool for accurate identification of EVs and early diagnosis of cancer.

Aptamer-Initiated Catalytic Hairpin Assembly Fluorescence Assay for Universal, Sensitive Exosome Detection

“…However, the lowest expression of EpCAM was observed for EXO-PANC-1 compared to that for the two other exosomes (Figure A), which agrees with reported studies . AICHA was also assessed by detecting CD63 for exosomes derived from the three cancer cell lines; the obtained results in Figure B found that the expression of CD63 in EXO-PANC-1 and EXO-LOVO were higher than that of EXO-MCF-7, which are in accordance with previous literature. ,, The obtained results indicate that AICHA enables adaptation for profiling, offering a promising platform for exosome proteomic studies, cancer diagnosis and classification, as well as monitoring cancer recurrence.…”

Ultrasensitive electrochemical aptasensor based on palindromic sequence mediated bidirectional SDA and a DNAzyme walker for kanamycin detection

“…16 Consequently, aptamers are widely used in the field of biosensors for disease diagnosis, the detection of antibiotics, toxins and microorganisms, and so on. 17–20…”

“…16 Consequently, aptamers are widely used in the field of biosensors for disease diagnosis, the detection of antibiotics, toxins and microorganisms, and so on. [17][18][19][20] A G-quadruplex (G4), a special secondary structure of nucleic acids, is composed of four successive guanine (G) segments on a single strand nucleic acid. 21,22 Numerous studies have found that the G-quadruplex structure plays a significant role in gene stability, telomere synthesis, gene transcription and translation regulation, gene recombination and other life processes.…”

Dual-Aptamer-Assisted Ratiometric SERS Biosensor for Ultrasensitive and Precise Identification of Breast Cancer Exosomes

“…Yang et al reported a double-aptamer recognition system based on a hyperbranched DNA superstructure that promotes signal amplification and ratiometric dual-signal strategies . Sun et al developed an identification strategy of nonpurified exosomes by the combination of the double-positive exosome membrane protein recognition-mediated toehold activation and the G-quadruplex DNAzyme-catalyzed etching of Au@Ag nanorods . Li et al proposed an aptamer-based logic gate with numerous protein biomarkers on single exosomes as input and thermophoretic accumulation amplified signals as output, achieving 97% identification accuracy for breast cancer patients .…”

“…29 Sun et al developed an identification strategy of nonpurified exosomes by the combination of the double-positive exosome membrane protein recognition-mediated toehold activation and the G-quadruplex DNAzyme-catalyzed etching of Au@Ag nanorods. 30 Li et al proposed an aptamer-based logic gate with numerous protein biomarkers on single exosomes as input and thermophoretic accumulation amplified signals as output, achieving 97% identification accuracy for breast cancer patients. 12 These double-aptamer detection strategies enhance the accuracy of exosome detection significantly, but they usually require a tedious signal amplification process to improve the sensitivity.…”