An AIE-based enzyme-activatable fluorescence indicator for Western blot assay:Quantitative expression of proteins with reproducible stable signal and wide linear range

Western blot is a commonly used experimental method to analyze the protein expression.However,the most commonly used chromogenic indicator based on chemiluminescence is limited by narrow linear range and unstable quantitative reproducibility,whereas the recently developed fluorescent indicator suffers from poor detection limit.Herein,we report an enzyme-activatable fluorescence indicator to quantify proteins with reproducible stable signal and wide linear range,through introducing the hydrophilic alkaline phosphatase(ALP)-triggered phosphoric acid moiety into our established aggregation-induced emission(AIE)building block of quinoline-malononitrile(QM).In this strategy,the indicator DQM-ALP disperses well in both aqueous and lipid environments to exhibit initial“off”fluorescence,but when exposing to the ALP-coupled secondary antibody on the PVDF membrane,the specific enzymatic turnover would liberate hydrophobic AIE luminogen(AIEgen)QM-OH to emit strong luminescence,thereby achieving an ideal“off-on”state for sensitively imaging proteins with high signal-to-noise(S/N)ratio.Moreover,benefiting from the excellent signal stability of AIE fluorophore,DQM-ALP indicator exhibits superior quantitative analysis of protein expression with high reproducibility.Upon taking advantage of the AIEgens to reduce high concentration-induced luminance quenching,the linear quantification range is extremely expanded.In contrast with the traditional chemiluminescent indicator,the AIE-based enzymeactivatable indicator DQM-ALP not only greatly improves the signal stability for quantitative reproducibility,but also expands the linear quantification range,and further provides a practical alternative reagent for fluorescence Western blot assay.

Na^+/Ca^(2+) Exchange-mediated La^(3+) Entry in Human Lymphocytes

We determined whether La3+ enter human peripheral blood lymphocytes via Na+/Ca2+ exchanger (measured with fura-2). We first compared the sensitivity of fura-2 with La3+ and Ca2+, the result indicates that the sensitivity of fura-2 for La3+ is much greater than for Ca2+. La3+ forms a 1:1 La3+-fura-2 complex (apparent dissociation constant = 1.7x10(-12) mol/L, pH 7.05). Ouabain-pretreated cells, suspended in Na+-free medium, showed that La3+ can enter human lymphocytes via the Na-i(+)/Ca2+ (La3+)(o) exchanger and is found to be about 10(-12) mol/L in cells exposed to 0.4 mmol/L La3+. Otherwise, the higher concentration (0.1 mmol/L) blocks the Na-i(+)/Ca2+(La3+)(o) exchange-mediated influx of Ca2+, but the lower concentration (0.01 mmol/L) appears to increase Ca2+ entry.

Construction of a dual fluorescence whole-cell biosensor to detect N-acyl homoserine lactones

Detection of N-acyl homoserine lactones （AHLs） is useful for understanding quorum sensing （QS） behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T1-4 to make a dual fluorescent whole- cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components： constitutively expressed enptll：：gfP for indicating host cells, Pahll：：mcherry that produces red fluorescence in response to AHL, and the ahIR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T1-4 （four tandem copies of a transcriptional terminator） were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 × 10^-8-1 × 10^-5 mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wild- type Pseudomonas PhTA125 （an AHL-producing bacterium）, it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a mi tal niche