Laborious and costly detection of miRNAs has brought challenges to its practical applications, especially for home health care, rigorous military medicine, and the third world. In this work, we present a pH-responsive miRNA amplification method, which allows the detection of miRNA just using a pH test paper. The operation is easy and no other costly instrument is involved, making the method very friendly. In our strategy, a highly efficient isothermal amplification of miRNA is achieved using an improved netlike rolling circle amplification (NRCA) technique. Large amounts of H can be produced as a byproduct during the amplification to induce significant changes of pH, which can be monitored directly using a pH test paper or pH-sensitive indicators. The degree of color changes depends on the amount of miRNA, making it possible for quantitative analysis. As an example, the method is successfully applied to quantify a miRNA (miR-21) in cancer cells. The results agree well with that from the prevalent qRT-PCR analysis. It is the first time that a paper-based point-of-care testing (POCT) is developed for the detection of miRNAs, which might promote the popularization of miRNAs working as biomarkers for diagnostic purposes.
Abnormal expression of miRNAs always occurs in solid tumors. Thus, it is critical to sensitively and selectively detect such biomarkers for the diagnosis and prognosis of diseases. Here, we report a biosensing scheme for the determination of miRNA with triple signal amplification based on target-triggered cyclic duplex specific nuclease digestion and bridge DNA-gold nanoparticles. Electrochemical signals are recorded to present initial levels of miRNA. This method is ultrasensitive with a wide linear range of 10 to 10 M. The limit of detection is down to 6.8 aM. Moreover, the overexpression of miR-21 is confirmed in lung cancer patients by the proposed method, which is in good accordance with qRT-PCR results. In addition, the developed biosensor does not need a reverse transcription process or any thermal cycling processes. Its performance satisfies the requirement for convenient, rapid, sensitive, and specific early diagnosis of cancers. Therefore, it may have great potential utility in the near future.
Molecular
diagnostics devoted to discover and monitor new biomarkers
is gaining increasing attention in clinical diagnosis. In this work,
a programmable DNA-fueled electrochemical analysis strategy is designed
for the determination of an emerging biomarker in lung cancer, PD-L1-expressing
exosomes. Specifically, PD-L1-expressing exosomes are first enriched
onto magnetic beads functionalized with PD-L1 antibody and are able
to interact with cholesterol-modified hairpin templates. Then, programmable
DNA synthesis starts from the hairpin template-triggered primer exchange
reaction and generates a large number of extension products to activate
the trans-cleavage activity of CRISPR-Cas12a. After that, CRISPR-Cas12a-catalyzed
random cleavage boosts the degradation of methylene blue-labeled signaling
strands, so electro-active methylene blue molecules can be enriched
onto a cucurbit[7]uril-modified electrode for quantitative determination.
Our method demonstrates high sensitivity and specificity toward electrochemical
analysis of PD-L1-expressing exosomes in the range from 103 to 109 particles mL–1 with a low detection
limit of 708 particles mL–1. When applied to clinical
samples, our method reveals an elevated level of circulating PD-L1-expressing
exosomes in lung cancer patients, especially for those at the advanced
stages. Therefore, our method may provide new insight into liquid
biopsy for better implementation of immunotherapy in lung cancer in
the future.
Passivation of electrode surface and tedious reconstruction of biosensing architectures have long plagued researchers for the development of electrochemical biosensors. Here, we report a novel self-cleaning electrode by modifying the commonly used working electrode with superhydrophobic and conductive nanocomposite. Owing to the superhydrophobicity and the chemical stability, the electrode avoids passivation result from both adsorption of molecules and oxidation in air. The high conductivity and the high effective area also allow the achievement of enhanced electrochemical signals. On the basis of comprehensive studies on this novel electrode, we have applied it in the fabrication of refreshable electrochemical biosensors for both electro-active and electro-inactive targets. For both cases, detection of the targets can be well performed, and the self-cleaning electrode can be refreshed by simply washing and applied for successive measurements in a long period.
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