Using the ribonucleotide-modified DNA probes, multiple microRNAs can be simultaneously detected in one ligation-based PCR reaction. As low as 0.2 fM microRNA can be accurately detected with high specificity.

An anionic fluorescent conjugated polymer (PFPaa), coupled with the metal ion-mediated fluorescence resonance energy transfer, has been used to design a versatile platform for homogeneous detection of protein kinase activity.

A simple, highly sensitive, and dual-readout fluorescent assay is developed for the detection of protein kinase activity based on the specific recognition utility of TiO2-coated Fe3O4/SiO2 magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When the fluorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they can bind specifically to the TiO2 layer of TMSPs by means of phosphate groups, resulting in fluorophore enrichment on the TMSP surfaces. The accumulated fluorophores on the TMSPs are proportional to the kinase activity, and the fluorescence signal readout could be run through either direct fluorescent imaging of the TMSPs or measurement of the fluorescence intensity by simply detaching the fluorescent phosphopeptides into the solution. The TMSPs exhibit extremely high selectivity for capturing phosphorylated peptides over the nonphosphorylated ones, resulting in an ultrahigh fluorescence signal-to-background ratio of 42, which is the highest fluorescence change thus far in fluorescent assays for detection of protein kinase activities. Therefore, the proposed fluorescent assay presents high sensitivity, low detection limit of 0.1 milliunit/μL, and wide dynamic range from 0.5 milliunit/μL to 0.5 unit/μL with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fluorescent assay can simultaneously quantify multiple kinase activities with their specific peptides labeled with different dyes. This new strategy is also successfully applied to monitoring drug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fluorescent assay is very promising in high-throughput screening of kinase inhibitors and in highly sensitive detection of kinase activity, and thus it is a valuable tool for development of targeted therapy, clinical diagnosis, and studies of fundamental life science.

•HCR–CBA provides a practical DNA assay with greatly improved sensitivity.•HCR enables enzyme-free fluorescence signal amplification directly on beads.•Flow cytometry facilitates direct analysis of HCR products-enriched beads.•The stringency design of HCR results in high specificity for DNA detection.A versatile flow cytometric bead assay (CBA) is developed for sensitive DNA detection by integrating the advantages of hybridization chain reaction (HCR) for enzyme-free signal amplification, flow cytometry for robust and rapid signal readout as well as magnetic beads (MBs) for facile separation. In this HCR–CBA, a biotinylated hairpin DNA (Bio-H1) is firstly immobilized on streptavidin-functionalized MBs. Upon the addition of target DNA, each target would hybridize with one Bio-H1 to open its hairpin structure and subsequently initiate a cascade of hybridization events between two species of fluorescent DNA hairpin probes (H1*/H2*) to form a nicked double helical DNA structure, resulting in amplified accumulation of numerous fluorophores on the MBs. Finally, the fluorescent MBs are directly analyzed by flow cytometry. This technique enables quantitative analysis of the HCR products anchored on the MBs as a function of target DNA concentration, and analysis of each sample can be completed within few minutes. Therefore, the HCR–CBA approach provides a practical DNA assay with greatly improved sensitivity. The detection limit of a model DNA target is 0.5 pM (3σ), which is about 3 orders of magnitude lower compared with traditional hybridization methods without HCR. Furthermore, the signal of complementary target can be clearly distinguished from that of single-base mismatched sequences, indicating the high specificity of the HCR–CBA. Moreover, this strategy is also successfully applied to the DNA analysis in complex biological samples, showing great potential in gene analysis and disease diagnosis in clinical samples.

Poor water-solubility and biocompatibility are the biggest obstacles for the bioapplications of upconversion nanophosphors (UCNPs). In this work, a facile solvothermal approach was developed for the one-pot synthesis of monodisperse and water-soluble β-NaYF4 UCNPs by employing poly(acrylic acid, sodium salt) (PAAs) as the capping reagent. The as-prepared UCNPs were thoroughly characterized and a possible reaction mechanism is proposed, which indicates that PAAs plays a crucial role for the shape and phase evolution of the NaYF4 UCNPs. More importantly, each multidentate PAA can tightly bind to the UCNPs surface through multiple –COO− groups, while an abundance of uncoordinated carboxyl groups on the polymer chain extend into the solution, making the UCNPs highly water-soluble. The presence of free carboxyl groups on the surfaces also facilitates the conjugation of the UCNPs with various biomolecules, which paves the way for further bio-applications. As a proof-of-concept model, transferrin was covalently linked to β-NaYF4:Yb,Er to form the transferrin–UCNPs conjugates, which have been successfully applied to the high-contrast fluorescent imaging and detection of HeLa cells. Possessing the advantages of good biocompatibility and inherent high upconversion efficiency, our directly synthesized β-NaYF4 UCNPs exhibit great potential as excellent bio-probes for in vitro or in vivo imaging, detection and diagnosis.

A new enzyme-free signal amplification-based assay for microRNA (miRNA) detection is developed by using hybridization chain reaction (HCR) coupled with a graphene oxide (GO) surface-anchored fluorescence signal readout pathway. MiRNAs can efficiently initiate HCR between two species of fluorescent hairpin probes. After HCR, both of the excess hairpin probes and the HCR products will be anchored on the GO surface. The fluorescence of the hairpin probes can be completely quenched by GO, whereas the HCR products maintain strong fluorescence. Therefore, integrating HCR strategy for signal amplification with selective fluorescence quenching effects of GO provides a versatile miRNA assay.Keywords: enzyme-free; fluorescence imaging; fluorescence sensor; graphene oxide; hybridization chain reaction; MicroRNA;

Graphene oxide can act as an ultrahighly efficient quencher for upconversion nanophosphors and thus, an extraordinarily sensitive biosensing platform is constructed.

β-NaYF4:Yb,Er upconversion nanophosphor (UCNP) is known as one of the most efficient NIR-to-visible upconversion materials, which shows great potential in bioanalytical chemistry and bioimaging. However, its applications are greatly limited due to its low water dispersibility and thus poor biocompatibility. In this paper, poly(acrylic acid) (PAA)-based ligand exchange strategies are carried out to modify oleic acid-capped hydrophobic β-NaYF4:Yb,Er UCNPs into hydrophilic ones. After efficient surface modification, the presence of free carboxylic acid groups on the surfaces of UCNPs results in high solubility in water, and also allows further conjugation with NH2-containing biomolecules. Facilitated by the covalent linkage between the -COOH groups on UCNPs surfaces and -NH2 groups in antigen/antibody, a sensitive immunosensor is constructed by using PAA-functionalized β-NaYF4:Yb,Er UCNPs as biolabels. Through monitoring the upconversion fluorescence intensity or fluorescent imaging of the final immunocomplexes, high sensitivity is achieved for the proposed immunoassay and as low as 0.1 ng/mL goat anti-human immunoglobulin G (IgG) can be detected, which suggests that PAA-modified UCNPs may serve as an ideal candidate for use as bioanalysis and bioimaging probes.

An anionic fluorescent conjugated polymer (PFPaa), coupled with the metal ion-mediated fluorescence resonance energy transfer, has been used to design a versatile platform for homogeneous detection of protein kinase activity.

Poor water-solubility and biocompatibility are the biggest obstacles for the bioapplications of upconversion nanophosphors (UCNPs). In this work, a facile solvothermal approach was developed for the one-pot synthesis of monodisperse and water-soluble β-NaYF4 UCNPs by employing poly(acrylic acid, sodium salt) (PAAs) as the capping reagent. The as-prepared UCNPs were thoroughly characterized and a possible reaction mechanism is proposed, which indicates that PAAs plays a crucial role for the shape and phase evolution of the NaYF4 UCNPs. More importantly, each multidentate PAA can tightly bind to the UCNPs surface through multiple –COO− groups, while an abundance of uncoordinated carboxyl groups on the polymer chain extend into the solution, making the UCNPs highly water-soluble. The presence of free carboxyl groups on the surfaces also facilitates the conjugation of the UCNPs with various biomolecules, which paves the way for further bio-applications. As a proof-of-concept model, transferrin was covalently linked to β-NaYF4:Yb,Er to form the transferrin–UCNPs conjugates, which have been successfully applied to the high-contrast fluorescent imaging and detection of HeLa cells. Possessing the advantages of good biocompatibility and inherent high upconversion efficiency, our directly synthesized β-NaYF4 UCNPs exhibit great potential as excellent bio-probes for in vitro or in vivo imaging, detection and diagnosis.

Graphene oxide can act as an ultrahighly efficient quencher for upconversion nanophosphors and thus, an extraordinarily sensitive biosensing platform is constructed.

Using the ribonucleotide-modified DNA probes, multiple microRNAs can be simultaneously detected in one ligation-based PCR reaction. As low as 0.2 fM microRNA can be accurately detected with high specificity.