A nature-inspired colorimetric and fluorescent dual-modal biosensor for exosomes detection.

As non-invasive biomarkers, exosomes are of great significance to diseases diagnosis. However, sensitive and accurate detection of exosomes still remains technical challenges. Herein, inspired by nature's "one-to-many" concept, we design a biosensor mimicking the cactus with numerous thorns to detect exosomes. The biosensor is composed of CD63 antibodies, resembling the roots of cactus, to capture exosomes, and the exosomes resemble the stems. Cholesterol-labeled DNA (DNA anchor) binding to streptavidin modified horseradish peroxidase (HRP) can insert into exosomes membrane, which seems the thorns. The readout signal is produced through HRP-catalyzed hydrogen peroxide (H2O2) mediated oxidation of 1,4-phenylenediamine (PPD) to form 2,5-diamino-NN'-bis-(p-aminophenyl)-1,4-benzoquinone di-imine (PPDox). The PPDox can quench fluorescence of fluorescein through inner filter effect (IFE), which provides fluorescent signal for exosomes detection. Based on this principle, the obtained exosomes solution is qualitatively and quantitatively analyzed by our biosensor, with the comparison to current standard methods by nanoparticle tracking analysis (NTA) and commercial enzyme-linked immunosorbent assay (ELISA) kit. The linear range is from 1.0 × 104 to 5.0 × 105 particles µL-1 with the limit of detection 3.40 × 103 particles µL-1 and 3.12 × 103 particles µL-1 for colorimetric and fluorescent assays, respectively. Meanwhile, our biosensor exhibits good selectivity, and can eliminate the interference from proteins. This dual-modal biosensor shows favorable performance towards analytical application in clinic samples, pushing one step further towards practical clinical use.

A ratiometric electrochemical DNA biosensor for detection of exosomal MicroRNA.

The detection of exosomal microRNAs (miRNAs) derived from cancer cells with sensitive and selective methods has stimulated increasing interest due to its potential utility in the application of tumor diagnosis. Here, we developed a ratiometric electrochemical DNA biosensor based on a locked nucleic acid (LNA)-modified "Y" shape-like structure for the detection of exosomal miRNA-21 (miR-21). When miR-21 is present, the LNA-assisted strand displacement reaction on the "Y" shape-like structure is activated, leading to a structure change and augmentation of the signal ratio, which reflects the different distances between the electrode surface and two electroactive molecules labeled on the "Y" shape-like structure. With this dual signal ratiometric method, the biosensor shows high accuracy and sensitivity with a limit of detection as low as 2.3 fM. Moreover, because of the logarithm of the signal ratio displays a linear relationship with the logarithm of the miR-21 concentration, the biosensor is stable enough to be used in the detection of miR-21 in MCF-7 cell-derived exosomes. In addition, the biosensor shows good selectivity even in the detection of even a single base-mismatched target due to the LNA-assisted strand displacement reaction. Notably, the sensor is both regenerative and robust. In brief, the high sensitivity and selectivity, combined with the low cost of the glassy carbon electrode, make this biosensor a promising tool for the development of point-of-care testing in cancer.

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.