•A cleavage-mediated isothermal exponential amplification reaction (IEXPAR) is developed for mRNA assay.•Three specifically recognize regions on the target mRNA guarantees the approach with high specificity.•The specificity of RNase H avoids the contamination of genomic DNA.•The superior properties of IEXPAR warrant the proposed method with high sensitivity.Messenger RNA (mRNA) as a bridge links the organism’s genotypes to its phenotypes. Quantitative detection of mRNA is vital in basic studies of life science and medical diagnostics. In this work, a sensitive, specific, and reverse transcription-free approach for quantification of mRNA based on cleavage-mediated isothermal exponential amplification reaction (IEXPAR) is established. The target mRNA firstly hybridizes with the flexibly designed DNA probe to form a loop structure. RNase H specifically digests the hybridized mRNA and releases the loop part of the mRNA, which can serve as the trigger to initiate IEXPAR. In this design, the DNA probe and the IEXPAR templates can specifically recognize three regions on the target mRNA, so that the high specificity can be achieved. Moreover, the specificity of RNase H can avoid the contamination of genomic DNA. IEXPAR is a powerful nucleic acid amplification technique with rapid, simple and cost-effective features, making the proposed assay highly sensitive and fast. With this assay, as low as 100 zmol target mRNA can be quantitatively detected and wide linear range with six orders of magnitude can be obtained. The assay can also be successfully applied to determine p53 mRNA in 5 ng total RNA sample extracted from MCF-7 breast cancer cells.

•Ligation-depended polymerase colony is developed for digital microRNA assay.•As low as 60 copies miRNA molecules can be detected with high specificity.•This method is able to dissect cell-to-cell variations of miRNA expression at single-cell level.The ability to dissect cell-to-cell variations of microRNA (miRNA) expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases. Herein, we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony (polony). Firstly, two simply designed target-specific DNA probes were ligated by using individual miRNA as the template. Then the ligated DNA probe acted as polony template that was amplified by PCR process in the thin polyacrylamide hydrogel. Due to the covalent attachment of a PCR primer on polyacrylamide matrix and the retarding effect of the polyacrylamide hydrogel matrix itself, as the polony reaction proceeds, the PCR products diffused radially near individual template molecule to form a bacteria colony-like spots of DNA molecules. The spots can be counted after staining the polyacrylamide gel with SYBR Green I and imaging with a microarray scanner. Our polony-based method is sensitive enough to detect 60 copies of miRNA molecules. Meanwhile, the new strategy has the capability of distinguishing singe-base difference. Due to its high sensitivity and specificity, the proposed method has been successfully applied to analysis of the expression profiling of miRNA in single cell.

Alternative messenger RNA (mRNA) splicing is a basic mechanism of gene regulation. In general, reverse transcription and polymerase based primer extension limit the sensitivity and selectivity of the current detection of mRNA splice variants, respectively. Here, we show that, using the ligation of two properly designed probes at the exon junction combined with universal PCR amplification, as little as a single copy of a mRNA splice variant per cell can be accurately determined, and the dynamic range covers six orders of magnitude. Three mRNA splice variants were measured from total RNA samples derived from different cell lines. Moreover, by encoding the ligation probes with different lengths, multiplexed mRNA splice variants can be simultaneously detected in one-tube PCR amplification using electrophoretic separation.

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.

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs’ population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

A novel loop-mediated isothermal amplification (LAMP)-based methylation assay for simple, robust and cost-effective detection of site-specific DNA methylation has been developed. DNA targets are first treated with methylation-sensitive restriction endonuclease (HpaII), where the DNA targets will be cleaved at specific unmethylated-cytosine residues while leaving the methylated DNA intact. Subsequently, the methylated DNA targets can serve as templates to perform LAMP for the detection of DNA methylation with real-time fluorescence measurements by using a common fluorescent dye (SYBR Green I). Taking advantage of the simplicity and high specificity of HpaII digestion and the isothermal nature and high sensitivity of LAMP, the proposed assay can greatly simplify the detection of DNA methylation and achieve ultrahigh sensitivity and specificity. With this assay, as low as 10 aM methylated DNA can be detected and 0.1% methylated DNA can be determined in the presence of a large excess of unmethylated DNA.

In this work, we have developed a sensitive, simple, and enzyme-free assay for detection of microRNAs (miRNAs) by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains. In the presence of miRNA target, it can hybridize with one of the stem-loop DNA to open the stem and to produce a miRNA/DNA hybrid and a single strand (ss) DNA, the ssDNA will in turn hybridize with another stem-loop DNA and finally form a double strand (ds) DNA to release the miRNA. One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence. The formation of dsDNA can produced specific fluorescence signal for miRNA detection. The released miRNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor, which results in great fluorescence amplification. With the efficient signal amplification, as low as 1 pmol/L miRNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained. Moreover, by designing different stem-loop DNAs specific to different miRNA targets and labeling them with different fluorophores, multiplexed miRNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum (SFS) technique.

DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and the occurrence of diseases, including cancers. DNA methylation has been increasingly utilized as a biomarker for cancer detection and differential diagnosis. Generally, one type of cancer is associated with several CpG methylation sites and detection of multiplexed CpG methylation can greatly improve the accuracy of cancer diagnosis. In this paper, we have developed a novel ligase chain reaction (LCR)-based method for multiplexed detection of CpG methylation in genomic DNA at single-base resolution. By rationally designing the two pairs of DNA probes for LCR, the bisulfite-treated methylated DNA target can be exponentially amplified by thermal cycling of the ligation reaction, in which one-base mismatch can be discriminated against, and thus high sensitivity and specificity for the detection of DNA methylation can be achieved. The LCR-based method can accurately determine as low as 10 aM methylated DNA fragment and 10 ng methylated genomic DNA. 0.1% methylated DNA can be detected in the presence of a large excess of unmethylated DNA. Moreover, by simply encoding one of the DNA probes in the LCR with a different length of poly(A) for detection of methylation at different CpG sites, the CpG methylation at different sites can produce LCR products with different lengths, and thus, can be simultaneously detected with one-tube LCR amplification and separation by capillary electrophoresis.

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 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).

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.

We have developed a new ligase chain reaction-based colorimetric assay for detection of DNA methylation with ultrahigh sensitivity and selectivity. Using the proposed assay, as low as 0.01 fM methylated DNA can be detected by visualization of color changes of gold nanoparticles with the naked eye.

Optical transition of a polyethylene oxide electrospun micro/nanofibrous mesh from opaque to transparent has been studied by UV-Vis spectroscopy and scanning electron microscopy. This transition occurs only above approximately 88% relative humidity and could be used for humidity monitoring by the naked eye for food quality control in which low toxicity and irreversibility are highly required.

•A new real-time fluorescence LCR assay is developed for detection of SNP.•Single-base mutation can be specifically discriminated with high sensitivity.•This method can be directly applied to detect SNP in genomic DNA with one-step.•This approach is promising for detection of various genetic biomarkers.Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis.

A highly sensitive and specific assay is developed for quantification of messenger RNA with a real-time fluorescence ligase chain reaction by using a ribonucleotide-modified DNA probe.

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.

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.

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

Alternative messenger RNA (mRNA) splicing is a basic mechanism of gene regulation. In general, reverse transcription and polymerase based primer extension limit the sensitivity and selectivity of the current detection of mRNA splice variants, respectively. Here, we show that, using the ligation of two properly designed probes at the exon junction combined with universal PCR amplification, as little as a single copy of a mRNA splice variant per cell can be accurately determined, and the dynamic range covers six orders of magnitude. Three mRNA splice variants were measured from total RNA samples derived from different cell lines. Moreover, by encoding the ligation probes with different lengths, multiplexed mRNA splice variants can be simultaneously detected in one-tube PCR amplification using electrophoretic separation.

We have developed a new ligase chain reaction-based colorimetric assay for detection of DNA methylation with ultrahigh sensitivity and selectivity. Using the proposed assay, as low as 0.01 fM methylated DNA can be detected by visualization of color changes of gold nanoparticles with the naked eye.

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).

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 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 highly sensitive and specific assay is developed for quantification of messenger RNA with a real-time fluorescence ligase chain reaction by using a ribonucleotide-modified DNA probe.

DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and the occurrence of diseases, including cancers. DNA methylation has been increasingly utilized as a biomarker for cancer detection and differential diagnosis. Generally, one type of cancer is associated with several CpG methylation sites and detection of multiplexed CpG methylation can greatly improve the accuracy of cancer diagnosis. In this paper, we have developed a novel ligase chain reaction (LCR)-based method for multiplexed detection of CpG methylation in genomic DNA at single-base resolution. By rationally designing the two pairs of DNA probes for LCR, the bisulfite-treated methylated DNA target can be exponentially amplified by thermal cycling of the ligation reaction, in which one-base mismatch can be discriminated against, and thus high sensitivity and specificity for the detection of DNA methylation can be achieved. The LCR-based method can accurately determine as low as 10 aM methylated DNA fragment and 10 ng methylated genomic DNA. 0.1% methylated DNA can be detected in the presence of a large excess of unmethylated DNA. Moreover, by simply encoding one of the DNA probes in the LCR with a different length of poly(A) for detection of methylation at different CpG sites, the CpG methylation at different sites can produce LCR products with different lengths, and thus, can be simultaneously detected with one-tube LCR amplification and separation by capillary electrophoresis.

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.