Numerous long single-stranded DNAs produced by dual amplification reactions for electrochemical detection of exosomal microRNAs.

Exosomal microRNAs (miRNAs) have been explored as an extremely promising biomarker of liquid biopsy for the diagnosis, treatment and prognosis of diseases such as cancer, in which sensitive and selective detection is significant. Herein, we describe the construction and testing of an electrochemical biosensor for the sensitive detection of exosomal miRNAs. It is based on synthetizing numerous long single-stranded DNAs (ssDNAs), which are produced by dual amplification reactions of target-triggered cyclic strand displacement reaction (TCSDR) and primer exchange DNA amplification reaction (PEDAR). In the first signal amplification step, target miRNAs are captured by the hairpin DNA strands (capture probes, Cp) that are immobilized on electrode. After strand unfolding with target capture, primer probes (Pp) enable to hybridize with Cp. And then target miRNAs were displaced for starting the TCSDR process that enable the introduction of numerous primers in Pp. In the second signal amplification step, the primers associated with PEDAR produce copious amounts of elongated ssDNAs. These ssDNAs absorb abundant quantities of methylene blue (MB) that enables the highly sensitive and label-free detection of exosomal miRNAs. This dual amplification process is characterized by a low limit of detection (LOD) of 3.04 aM. In addition, the electrochemical biosensor exhibits good selectivity for miR-21 detection, and shows benefits of simple operation, low cost, portability. Overall, the electrochemical biosensor provides a promising platform for the early diagnosis and screening of tumor biomarkers and the development of devices for point-of-care testing (POCT).

Label-free fluorescent and electrochemical biosensors based on defective G-quadruplexes.

Abnormal levels of guanine closely associated with plenty of diseases are usually used as a biomarker for clinical diagnosis. In order to detect guanine and its derivatives accurately, in this paper, a defective G-quadruplex (DGQ) containing a G-vacancy at one of its G-quartet layers, and two kinds of G-quadruplex specific indicators including thioflavine T (ThT) and hemin were used for constructing a fluorescent and an electrochemical biosensor, respectively. In brief, a G-rich DNA probe is designed to form either hairpin or DGQ structure. In the absence of guanine, G-rich probes prefer to maintain hairpin structure and nearly have no interaction with ThT or hemin, leading to almost negligible signals. Upon addition of guanine, the G-rich probe fold into DGQ structure and then the G-vacancy in it is filled up immediately by guanine via Hoogsteen hydrogen bonds, resulting canonical G-quadruplex formation. Accordingly, ThT or hemin can selectively combine with G-quadruplex, giving rise to distinct fluorescent or current signal changes for label-free detection of guanine. Benefiting from the perfect discriminative ability of guanine towards DGQ and ThT/hemin against standard G-quadruplex, the fluorescent and electrochemical biosensors present better sensitivity and selectivity for guanine detection with the limit of detection (LOD) as low as 18.26 and 0.36 nM, respectively. Successful attempts were also made in applying the proposed electrochemical biosensor to detect guanine in drugs and urine, obtaining satisfactory recovery rates of 99~104% and 96~106%, respectively.

A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction.

Sensitive and selective detection of microRNAs (miRNAs) in cancer cells derived exosomes have attracted rapidly growing interest owing to their potential in diagnostic and prognostic applications. Here, we design a ratiometric electrochemical biosensor based on bipedal DNA walkers for the attomolar detection of exosomal miR-21. In the presence of miR-21, DNA walkers are activated to walk continuously along DNA tracks, resulting in conformational changes as well as considerable increases of the signal ratio produced by target-respond and target-independent reporters. With the signal cascade amplification of DNA walkers, the biosensor exhibits ultrahigh sensitivity with the limit of detection (LOD) down to 67 aM. Furthermore, owing to the background-correcting function of target-independent reporters termed as reference reporters, the biosensor is robust and stable enough to be applied in the detection of exosomal miR-21 extracted from breast cancer cell lines and serums. In addition, because locked nucleic acid (LNA) modified toehold mediate strand displacement reaction (TMSDR) has extraordinary discriminative ability, the biosensor displays excellent selectivity even against the single-base-mismatched target. It is worth mentioning that our sensor is regenerative and stable for at least 5 cycles without diminution in sensitivity. In brief, the high sensitivity, selectivity and reproducibility, together with cheap, make the proposed biosensor a promising approach for exosomal miRNAs detection, in conjunction with early point-of-care testing (POCT) of cancer.