A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu2+ to form stable PPi/Cu2+ complex and Cu2+ in the complex cannot be reduced to Cu+. The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu2+ from the stable PPi/Cu2+ complex, and the free Cu2+ is easily reduced to Cu+ by sodium ascorbate. Then Cu+ catalyzes the cyclization reaction between the specially designed 5′-azide and 3′-alkyne tagged padlock probes through Cu+ catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

•We have proposed a simple, high selective and sensitive fluorescent biosensor for ALP.•The biosensor combines the high selectivity and the click reaction and the high sensitivity of the fluorescence detection.•The biosensor has been successfully applied to detect ALP in serum samples with satisfied results.Alkaline phosphatase (ALP) plays an important role in phosphate metabolism processes; deviation from its normal level may indicate different kinds of diseases, so it is highly necessary to develop some simple and sensitive methods to monitor the ALP level. In this study, a simple, high selective, and sensitive fluorescent biosensor has been proposed for ALP activity determination. The Cu(II)-dependent DNAzyme (Cu-Enzyme) are divided into two parts: Cu-Enzyme 1 and Cu-Enzyme 2, and labelled with alkyne and azido groups, respectively. The Cu-substrate (Cu-Sub) is labelled with a FAM fluorophore (6-carboxyfluorescein) at the 3′-end and an additional quencher (BHQ1) at the 5′-end. The 5′-end of Cu-Enzyme 1 is labelled with BHQ1 as well. The hybridization of the Cu-Enzyme 1 and Cu-Enzyme 2 with Cu-Sub strand results in the low background fluorescence signal because the fluorescence from FAM is quenched. The addition of ALP can hydrolyze AA-P into AA, which can reduce Cu(II) into Cu(I) and in turn catalyze the cycloaddition of Cu-Enzyme 1 and Cu-Enzyme 2 to form a modified Cu-Enzyme. Then the modified Cu-Enzyme catalyzes the cleavage of the Cu-Sub strands into two pieces. One piece containing FAM fluorophore can easily diffuse into solution and give off a strong fluorescence signal. The enhanced fluorescent intensity has a linear relationship with the ALP concentration in the range of 0.36–54.55 U L−1 with the detection limit of 0.14 U L−1 (S/N = 3). The proposed biosensor has been successfully applied to detect ALP in serum samples with satisfied results.

A fluorescent aptasensor for the Ochratoxin A (OTA) assay, which combines the high efficiency of hyperbranched rolling circle amplification (HRCA) and the specific recognition of the aptamer has been developed. OTA can bind with the aptamer with high affinity, which hinders hybridization between the aptamer and capture probe DNA (CDNA). Then the free CDNA hybridizes with the padlock probe and initiates a HRCA reaction. The HRCA products contain large amounts of double strand DNA (dsDNA), which can then combine with SYBR Green I to produce a fluorescence signal. The fluorescence intensity of the system has a linear relationship with the logarithm of the OTA concentration in the range of 4 fg mL−1 to 400 pg mL−1 and a limit of detection (LOD) of 1.2 fg mL−1 is obtained. The fluorescent aptasensor was applied to detect OTA in corn and oat samples with satisfying results.

The adsorption of mixed solutions containing an anionic polyelectrolyte, carboxymethylchitosan (CMCH), and cationic gemini surfactants, alkanediyl-bis-(dimethyldodecyl-ammonium bromide) (C12-s-C12, s = 2, 6, 12), has been investigated by surface tension method. The oppositely charged polyelectrolyte and the surfactants co-adsorb at the surface to form highly surface-active complexes. Combining the surface tension data with the Gibbs equation, it is referred that the surface layers of the mixed solutions have the multi-level structure, which includes the sublayers beneath an outermost layer. The gemini surfactant spacer with different length takes different conformations in the surface layers. The salt (NaBr) effects on the adsorption of the mixtures have also been studied. The spacer length of C12-s-C12 influences the responses of CMCH/C12-s-C12 mixtures to the salt effects. The comprehensive salt effects depend on the competition between the salt-enhancing effect and the salt-weakening effect.

A fluorescent aptasensor for the Ochratoxin A (OTA) assay, which combines the high efficiency of hyperbranched rolling circle amplification (HRCA) and the specific recognition of the aptamer has been developed. OTA can bind with the aptamer with high affinity, which hinders hybridization between the aptamer and capture probe DNA (CDNA). Then the free CDNA hybridizes with the padlock probe and initiates a HRCA reaction. The HRCA products contain large amounts of double strand DNA (dsDNA), which can then combine with SYBR Green I to produce a fluorescence signal. The fluorescence intensity of the system has a linear relationship with the logarithm of the OTA concentration in the range of 4 fg mL−1 to 400 pg mL−1 and a limit of detection (LOD) of 1.2 fg mL−1 is obtained. The fluorescent aptasensor was applied to detect OTA in corn and oat samples with satisfying results.