Abstract
Exosomes are cell-derived nanovesicles that have recently gained popularity as potential biomarkers in liquid biopsies due to the large amounts of molecular cargo they carry, such as nucleic acids and proteins. However, most existing exosome-based analytical sensing methods struggle to achieve high sensitivity and high selectivity simultaneously. In this work, we present an electrochemical micro-aptasensor for the highly sensitive detection of exosomes by integrating a micropatterned electrochemical aptasensor and a hybridization chain reaction (HCR) signal amplification method. Specifically, exosomes are enriched on CD63 aptamer-functionalized electrodes and then recognized by HCR products with avidin-horseradish peroxidase (HRP) attached using EpCAM aptamers as bridges. Subsequently, the current signal that is generated through the enzyme reaction between the HRP enzyme and 3,3’,5,5’-tetramethylbenzidine (TMB)/H2O2 directly correlates to the amount of bound HRP on the HCR products and thus to the number of target exosomes. By introducing anti-EpCAM aptamers, micro-aptasensors can detect cancerous exosomes with high specificity. Due to the micropatterned electrodes and HCR dual-amplification strategy, the micro-aptasensors achieve a linear detection response for a wide range of exosome concentrations from 2.5×103 to 1×107 exosomes/mL, with a detection limit of 5×102 exosomes/mL. Moreover, our method successfully detects lung cancer exosomes in serum samples of early-stage and late-stage lung cancer patients, showcasing the great potential for early cancer diagnosis.
Highlights
Exosomes, nanosized vesicles with diameters of30–150 nm, are released by cells during the fusion of multivesicular endosomes with plasma membranes[1,2]
Biotins labeled with H1 and H2 were used to bind with the anti-EpCAM aptamers and further produce long chains with multiple biotins
Due to the specific recognition between avidin-horseradish peroxidase (HRP) and biotin, the current signal generated through the enzyme reaction is directly related to the amount of HRP on the hybridization chain reaction (HCR) products and the exosome concentration
Summary
Exosomes, nanosized vesicles with diameters of30–150 nm, are released by cells during the fusion of multivesicular endosomes with plasma membranes[1,2]. Exosomes transfer RNA and proteins to recipient cells, thereby facilitating cell-to-cell communication. Recent studies[3,4,5] have demonstrated that the expression profiles of exosomal nucleic acids and proteins are altered in. Zhang et al Microsystems & Nanoengineering (2021)7:63 concentration of cancer-specific exosomes could be used to determine the corresponding stage of cancer[15,16,17]. Various electrochemical biosensors have been developed for exosome detection using antibodies as recognition molecules[34,35,36,37]. One attractive alternative to antibodies is aptamers (i.e., short ssDNA and RNA molecules). Aptamers bind to desired targets with excellent specificity and exhibit merits such as high stability, easy synthesis, and low cost[10,38,39,40]
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