Here, a non-destructive amplification strategy is developed to image miRNAs in living cells, utilizing the enzyme-free hybridization chain reaction (HCR) with graphene oxide (GO) as a carrier. This provides a new tool for highly sensitive and simultaneous imaging of multiple low-level biomarkers, improving the accuracy of early disease diagnosis.

In this paper, a padlock probe-based exponential rolling circle amplification (P-ERCA) assay is developed for highly specific and sensitive detection of microRNA (miRNA). The padlock probe is composed of a hybridization sequence to miRNA and a nicking site for nicking endonuclease. Using the miRNA as a template, specific ligation to the padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as triggers in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate the rolling circle amplification reaction, and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of the padlock probe. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to single-nucleotide difference discrimination between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics.

In this work, a novel dual photoelectrochemical/colorimetric cyto-analysis format was first introduced into a microfluidic paper-based analytical device (μ-PAD) for synchronous sensitive and visual detection of H2O2 released from tumor cells based on an in situ hydroxyl radicals ((•)OH) cleaving DNA approach. The resulted μ-PAD offered an excellent platform for high-performance biosensing applications, which was constructed by a layer-by-layer modification of concanavalin A, graphene quantum dots (GQDs) labeled flower-like Au@Pd alloy nanoparticles (NPs) probe, and tumor cells on the surface of the vertically aligned bamboo like ZnO, which grows on a pyknotic Pt NPs modified paper working electrode (ZnO/Pt-PWE). It was the effective matching of energy levels between GQDs and ZnO levels that lead to the enhancement of the photocurrent response compared with the bare ZnO/Pt-PWE. After releasing H2O2, the DNA strand was cleaved by (•)OH generated under the synergistic catalysis of GQDs and Au@Pd alloy NPs and thus, reduced the photocurrent, resulting in a high sensitivity to H2O2 in aqueous solutions with a detection limit of 0.05 nmol observed, much lower than that in the previously reported method. The disengaged probe can result in catalytic chromogenic reaction of substrates, resulting in real-time imaging of H2O2 biological processes. Therefore, this work provided a truly low-cost, simple, and disposable μ-PAD for precise and visual detection of cellular H2O2, which had potential utility to cellular biology and pathophysiology.

MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Sensitive and accurate detection of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in clinical diagnosis. Here, we developed a stable, sensitive, and specific miRNAs detection method on the basis of cooperative amplification combining with the graphene oxide (GO) fluorescence switch-based circular exponential amplification and the multimolecules labeling of SYBR Green I (SG). First, the target miRNA is adsorbed on the surface of GO, which can protect the miRNA from enzyme digest. Next, the miRNA hybridizes with a partial hairpin probe and then acts as a primer to initiate a strand displacement reaction to form a complete duplex. Finally, under the action of nicking enzyme, universal DNA fragments are released and used as triggers to initiate next reaction cycle, constituting a new circular exponential amplification. In the proposed strategy, a small amount of target miRNA can be converted to a large number of stable DNA triggers, leading to a remarkable amplification for the target. Moreover, compared with labeling with a 1:1 stoichiometric ratio, multimolecules binding of intercalating dye SG to double-stranded DNA (dsDNA) can induce significant enhancement of fluorescence signal and further improve the detection sensitivity. The extraordinary fluorescence quenching of GO used here guarantees the high signal-to-noise ratio. Due to the protection for target miRNA by GO, the cooperative amplification, and low fluorescence background, sensitive and accurate detection of miRNAs has been achieved. The strategy proposed here will offer a new approach for reliable quantification of miRNAs in medical research and early clinical diagnostics.

An ultrasensitive photoelectrochemical (PEC) biosensor for N-glycan expression based on the enhancement of nanogold-assembled mesoporous silica nanoparticles (GMSNs) was fabricated, which also combined with multibranched hybridization chain reaction (mHCR) and graphene quantum dots (GQDs). In this work, the localized surface plasmon resonance, mHCR and GQDs-induced signal amplification strategies were integrated exquisitely and applied sufficiently. In the fabrication, after porous ZnO spheres immobilized on the Au nanorod-modified paper working electrode were sensitized by CdTe QDs, the GMSNs were assembled on the CdTe QDs. Then the photocurrent efficiency was improved by the sensitization of the CdTe QDs and the localized surface plasmon resonance of GMSNs. Successively, the products of mHCR with multiple biotins for multiple horseradish peroxidase binding and multiple branched arms for capturing the target cells were attached on the as-prepared electrode. The chemiluminescent (CL) emission with the aid of horseradish peroxidase served as an inner light source to excite photoactive materials for simplifying the instrument. Furthermore, the aptamer could capture the cancer cells by its highly efficient cell recognition ability, which avoided the conventional routing cell counting procedures. Meanwhile, the GQDs served as the signal amplication strategy, which was exerted in the process of N-glycan evaluation because the competitive absorption of exciting light and consumption of HO served as the electron donor of the PEC system and the oxidant of the luminol-based CL system. This judiciously engineered biosensor offered a promising platform for the exploration of N-glycan-based physiological processes.

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