Plant microRNAs (miRNAs) differ from animal miRNAs in that they are 2′-O-methylated at the 3′ end nucleotide which limits the ability of plant miRNAs to serve as primers for polymerization reactions. Herein, we have rationally designed a three-way junction structure-based isothermal exponential amplification reaction (3WJ-EXPAR) which is well-suited for sensitive detection of 3′-terminal 2′-O-methylated miRNAs.

Plant microRNAs (miRNAs) differ from animal miRNAs in that they are 2′-O-methylated at the 3′ end nucleotide which limits the ability of plant miRNAs to serve as primers for polymerization reactions. Herein, we have rationally designed a three-way junction structure-based isothermal exponential amplification reaction (3WJ-EXPAR) which is well-suited for sensitive detection of 3′-terminal 2′-O-methylated miRNAs.

A novel one-step microRNA assay is developed based on a target-triggered loop-mediated isothermal amplification (TT-LAMP) mechanism, which enables the accurate detection of as low as 100 aM (1 zmol) microRNA with simple one-step operation by using only one-type of DNA polymerase.

A novel one-step microRNA assay is developed based on a target-triggered loop-mediated isothermal amplification (TT-LAMP) mechanism, which enables the accurate detection of as low as 100 aM (1 zmol) microRNA with simple one-step operation by using only one-type of DNA polymerase.

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.

A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

Telomerase is a crucial biomarker for cancers. Its reliable and sensitive detection, particularly in a single cell, has great significance for the early diagnosis of cancers, studies on tumor progression and anticancer therapy, which remain a challenge, due to nonspecific amplification. Herein, we developed a novel stem-loop primer-mediated exponential amplification (SPEA) strategy, which can specifically and efficiently amplify the telomerase-elongated telomere repeat unit with near zero nonspecific signal. The SPEA-based assay can accurately detect telomerase activity in the crude lysate of a single cell and is suited for detecting the cellular heterogeneity arising from cell-to-cell variations.

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs’ actions in a single cell, which is of great importance for the study of miRNAs’ biofunction as well as for the related biomedical studies.

Detection of a single nucleic acid molecule is of great significance for both fundamental biochemistry studies and clinical diagnostics. By using microRNA (miRNA) as a model target, herein, we have developed a single-microbead-based sensing (SMBS) platform, which simply enables the detection of miRNA at the single-molecule level. In this strategy, an isothermal exponential amplification reaction (EXPAR) is rationally designed towards specific miRNAs and all products of the EXPAR are integrated onto a single microbead for signal amplification and fluorescence enrichment. This pushes the detection of miRNAs down to 1 aM in a 5 μL sample, corresponding to 3 copies of the miRNA molecule. This new strategy also affords high selectivity and it is capable of distinguishing among homologous miRNA family members even with a single-base difference. Due to its ultrahigh sensitivity and selectivity, the proposed SMBS platform has been successfully applied to the detection of miRNA extracted from a single cell.

A novel RNA FRET probe that can produce target-dependent signal amplification with the catalysis of RNase H has been developed for detection of rolling circle amplification (RCA) products with greatly improved sensitivity.

A versatile flow cytometric bead assay (FCBA) has been developed for the ultrasensitive detection of T4 PNK activity by integrating the advantages of the phosphorylation-induced hybridization chain reaction (HCR) for fluorescence signal amplification and flow cytometry for the robust, sensitive and rapid signal readout of the microbeads (MBs).

We have developed a new colorimetric strategy that allows visual detection of Cu2+ under the irradiation of a UV lamp with high specificity based on the phenomenon that copper ions may lead to a fluorescence band shift of CdTe quantum dots (QDs) while other concomitant ions will not.

Accurate and rapid detection of protein kinase activities is of great significance because protein kinases play important regulatory roles in many vital biological processes. Herein, we wish to report a facile colorimetric protein kinase assay based on the phosphorylation-tuned crosslinking of gold nanoparticles (GNPs) by using protein kinase A (PKA) as a proof-of-concept target. In this new strategy, a biotinylated peptide (biotin-LRRASLG) is used as the PKA-specific substrate. When mixed with streptavidin-functionalized GNPs (STV-GNPs), the positively charged biotin-peptide will combine with different GNPs both through the specific STV–biotin binding and through electrostatic interactions, which will lead to the crosslinking and coagulation of GNPs. In contrast, under the catalysis of PKA, the biotin-peptide will be phosphorylated at the serine residue and its net charge will be obviously altered, which may significantly weaken the electrostatic interaction between the phosphopeptide and GNPs and thus effectively prevent the STV-GNPs from crosslinking and settlement. Therefore, by viewing the color changes of the GNPs, the PKA activity can be easily detected by the naked eye.

•A simple but versatile FRET assay for T4 PNK is developed by using a coupled enzyme reaction.•T4 PNK-actuated λ exonuclease cleavage reaction will be reflected by the FRET ratio changes.•High sensitivity is achieved due to the conjugated polymer-amplified fluorescence signal.•The use of label-free hairpin DNA greatly simplifies the assay design and reduces the cost.A simple but robust strategy for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity is developed by means of a DNA phosphorylation-accelerated λ exonuclease cleavage reaction coupled with cationic conjugated polymer (CCP)-mediated fluorescence resonance energy transfer (FRET). Firstly, a label-free hairpin DNA with a 5′-hydroxyl end is designed as the substrate of T4 PNK. SYBR Green I (SGI), a double-stranded DNA (dsDNA)-specific fluorescent dye, can fluoresce only when intercalated to the stem region of the hairpin DNA. When mixed with CCP, the SGI-binding hairpin DNA will be brought in close proximity with the CCP due to strong electrostatic interaction, leading to efficient FRET from CCP to SGI. However, in the presence of T4 PNK, the hairpin DNA would be phosphorylated at its 5′-terminus and thus can be immediately recognized as the initial cleavage site of λ exonuclease. The phosphorylation-actuated λ exonuclease reaction will cleave the stem of the hairpin to yield a single-stranded DNA, which is unable to combine with SGI and as a result, the FRET signal would decrease gradually in correlation to the T4 PNK activity. Therefore, by recording the change of FRET ratio, T4 PNK activity can be facilely determined in a mix-and-read manner. Due to the light harvesting and fluorescence amplification properties of CCP, high sensitivity is achieved for this homogeneous assay. This new strategy provides a simple detection procedure, easy readout and cost-effective manner for T4 PNK analysis, which shows great potential in the study of polynucleotide kinase-related biological processes.

A simple but robust fluorescence turn-on assay is developed for highly sensitive detection of PTP1B activity by using calcein as the signaling element.

A versatile graphene oxide (GO)-based fluorescent assay is developed for the detection of protein tyrosine phosphatase activity by coupling with a chymotrypsin-assisted selective peptide cleavage reaction.

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample.

Protein kinases play important regulatory roles in intracellular signal transduction pathways. The aberrant activities of protein kinases are closely associated with the development of various diseases, which necessitates the development of practical and sensitive assays for monitoring protein kinase activities as well as for screening of potential kinase-targeted drugs. We demonstrate here a robust luminescence resonance energy transfer (LRET)-based protein kinase assay by using NaYF4:Yb,Er, one of the most efficient upconversion nanophosphors (UCNPs), as an autofluorescence-free LRET donor and a tetramethylrhodamine (TAMRA)-labeled substrate peptide as the acceptor. Fascinatingly, besides acting as the LRET donor, NaYF4:Yb,Er UCNPs also serve as the phosphopeptide-recognizing matrix because the intrinsic rare earth ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by protein kinases over the unphosphorylated ones. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read format without any requirement of surface modification, substrate immobilization, separation, or washing steps, showing great potential in protein kinases-related clinical diagnosis and drug discovery. To the best of our knowledge, this is the first report by use of rare earth-doped UCNPs as both the phospho-recognizing and signal reporting elements for protein kinase analysis.

An upconversion nanophosphor (UCNP)-based highly sensitive assay for the detection of protein kinase activity is developed with the assistance of Zr4+-functionalized magnetic beads.

In this work, a simple fluorescence turn-on assay is developed for a sensitive detection of microRNA (miRNA) through an enzyme-free signal amplification strategy. This design is based on the miRNA-catalyzed hairpin assembly (CHA). Two metastable hairpin DNA probes, one of which is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence, are rationally designed to preclude unexpected hybridization events between themselves. Therefore, in the absence of miRNAs, the two types of hairpins could stably coexist in the solution with low background fluorescence because of the stringent design. When target miRNAs are added, they work as catalysts to trigger the self-assembly pathway of the two probes and initiate the cycling of CHA circuits, which results in greatly enhanced fluorescence signal. With the help of an efficient signal amplification of CHA and the low-background design of hairpin probes, at concentrations as low as 1 pM miRNA can be detected using this simple and low-cost protocol. High specificity and a wide dynamic range from 1 pM to 2 nM are also obtained. Therefore, this method may have great potential for miRNA-related biological studies.

A flow cytometry-assisted mix-and-read assay is developed for ultrasensitive detection of protein kinase activity by use of Zr4+-functionalized mesoporous SiO2 microspheres (ZrMMs). This strategy integrates the distinct advantages of ZrMMs for highly specific recognition as well as high capacity binding of kinase-induced fluorescent phosphopeptides and flow cytometry for powerful and separation-free bead analysis, leading to an ultrahigh sensitivity for kinase analysis in a extremely simple mix-and-read manner. Furthermore, this ultrasensitive design is well suitable for detection of cell kinase activities in complex biological samples and for screening of potential protein kinase inhibitors, which is of great significance for the development of targeted therapy, clinical diagnosis, and studies of cellular signal transduction pathways.

A versatile graphene oxide (GO)-based fluorescent assay is developed for the detection of protein tyrosine phosphatase activity by coupling with a chymotrypsin-assisted selective peptide cleavage reaction.

A simple but robust fluorescence turn-on assay is developed for highly sensitive detection of PTP1B activity by using calcein as the signaling element.

Detection of a single nucleic acid molecule is of great significance for both fundamental biochemistry studies and clinical diagnostics. By using microRNA (miRNA) as a model target, herein, we have developed a single-microbead-based sensing (SMBS) platform, which simply enables the detection of miRNA at the single-molecule level. In this strategy, an isothermal exponential amplification reaction (EXPAR) is rationally designed towards specific miRNAs and all products of the EXPAR are integrated onto a single microbead for signal amplification and fluorescence enrichment. This pushes the detection of miRNAs down to 1 aM in a 5 μL sample, corresponding to 3 copies of the miRNA molecule. This new strategy also affords high selectivity and it is capable of distinguishing among homologous miRNA family members even with a single-base difference. Due to its ultrahigh sensitivity and selectivity, the proposed SMBS platform has been successfully applied to the detection of miRNA extracted from a single cell.

A novel RNA FRET probe that can produce target-dependent signal amplification with the catalysis of RNase H has been developed for detection of rolling circle amplification (RCA) products with greatly improved sensitivity.

A versatile flow cytometric bead assay (FCBA) has been developed for the ultrasensitive detection of T4 PNK activity by integrating the advantages of the phosphorylation-induced hybridization chain reaction (HCR) for fluorescence signal amplification and flow cytometry for the robust, sensitive and rapid signal readout of the microbeads (MBs).

In this work, a simple fluorescence turn-on assay is developed for a sensitive detection of microRNA (miRNA) through an enzyme-free signal amplification strategy. This design is based on the miRNA-catalyzed hairpin assembly (CHA). Two metastable hairpin DNA probes, one of which is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence, are rationally designed to preclude unexpected hybridization events between themselves. Therefore, in the absence of miRNAs, the two types of hairpins could stably coexist in the solution with low background fluorescence because of the stringent design. When target miRNAs are added, they work as catalysts to trigger the self-assembly pathway of the two probes and initiate the cycling of CHA circuits, which results in greatly enhanced fluorescence signal. With the help of an efficient signal amplification of CHA and the low-background design of hairpin probes, at concentrations as low as 1 pM miRNA can be detected using this simple and low-cost protocol. High specificity and a wide dynamic range from 1 pM to 2 nM are also obtained. Therefore, this method may have great potential for miRNA-related biological studies.

A simple, homogeneous and generic method for detecting protein tyrosine (Tyr) kinase activity is developed based on a tyrosinase-assisted fluorescence turn-on strategy. The tyrosinase-mediated oxidation of the Tyr residue in a fluorescently-labeled peptide may lead to efficient fluorescence quenching, while the tyrosine kinase-catalyzed phosphorylation of the peptide can prevent the Tyr oxidation and thus maintain strong fluorescence.

A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

Plant microRNAs (miRNAs) differ from animal miRNAs in that they are 2′-O-methylated at the 3′ end nucleotide which limits the ability of plant miRNAs to serve as primers for polymerization reactions. Herein, we have rationally designed a three-way junction structure-based isothermal exponential amplification reaction (3WJ-EXPAR) which is well-suited for sensitive detection of 3′-terminal 2′-O-methylated miRNAs.

Plant microRNAs (miRNAs) differ from animal miRNAs in that they are 2′-O-methylated at the 3′ end nucleotide which limits the ability of plant miRNAs to serve as primers for polymerization reactions. Herein, we have rationally designed a three-way junction structure-based isothermal exponential amplification reaction (3WJ-EXPAR) which is well-suited for sensitive detection of 3′-terminal 2′-O-methylated miRNAs.