Inorganic Pyrophosphatase Activity Research Articles

Effect of Near-Freezing Temperature Storage on the Quality and Organic Acid Metabolism of Apple Fruit

Organic acids play critical roles in fruit physiological metabolism and sensory quality. However, the conventional storage of apple fruit at 0 ± 0.1 °C cannot maintain fruit acidity efficiently. This study investigated near-freezing temperature (NFT) storage for ‘Golden Delicious’ apples, and the quality parameters, organic acid content, and malate metabolism were studied. The results indicate that NFT storage at −1.7 ± 0.1 °C effectively maintained the postharvest quality of apple fruit when compared to traditional storage at 0 ± 0.1 °C. Fruit that underwent NFT storage showed a better appearance and lower respiratory rate, ethylene production, weight loss, and malondialdehyde (MDA) content but higher firmness and soluble solids content. Further, fruit after NFT storage contained higher titratable acid (18.75%), malate (51.61%), citrate (36.59%), and succinate (2.12%) content when compared to the control after 250 days. This was achieved by maintaining higher cytosolic NAD-dependent malate dehydrogenase (cyNAD-MDH), phosphoenolpyruvate carboxylase (PEPC), vacuolar H+-ATPase (V-ATPase), and vacuolar inorganic pyrophosphatase (V-PPase) activities that promote malate biosynthesis and accumulation while inhibiting enzyme activity that is responsible for malate decomposition, including phosphoenolpyruvate carboxylase kinase (PEPCK) as well as the cytosolic NAD phosphate-dependent malic enzyme (cyNADP-ME). Further, storage at NFTs maintained a higher expression of malate biosynthesis-related genes (MdcyNAD-MDH and MdPEPC) and transport-related genes (MdVHA and MdVHP) while suppressing malate consumption-related genes (MdcyME and MdPEPCK). The results demonstrate that NFT storage could be an effective application for apple fruit, which maintains postharvest quality and alleviates organic acid degradation.

Phosphorus sorption–desorption changes phosphorus fraction dynamic in a desert revegetation chronosequence

AbstractUnderstanding the characteristics of soil phosphorus (P) sorption and desorption is essential for comprehending P biogeochemical cycling and effectively managing ecosystems in a desert revegetation chronosequence. The present study utilized the Freundlich model and enzymatic activity to characterize the features of P sorption–desorption, and microbial activity, which aims to elucidate the effect of P kinetics and microbial activity on P fractions in the soils of a desert revegetation chronosequence, consisting of 11, 31, 40, 57, and 65 years old revegetated deserts. The findings revealed that the 31 years old soil showed the highest alkaline phosphomonoesterase and phosphodiesterase activities, and the 40 years old displayed the highest inorganic pyrophosphatase activity. In revegetated desert soils, microbial activity changed P sorption–desorption kinetics by decreased or increased the parameters including sorption/desorption energy site, P sorption/desorption ability, and the maximum buffering capacity of P sorption/desorption. And in microbial activity soils of 31–40 years old, P desorption significantly decreased T‐P and A‐P concentrations (p < 0.05). P sorption process and enzyme activity explained 35.10% and 22.20% of P fraction variation, respectively; and P desorption process and enzyme activity explained 48.3% and 22.3% of P fraction variation, respectively. These findings provide valuable insights into the contribution of P kinetics coupled with microbial activity in desert ecosystems, aiding in the effective management of these fragile ecosystems.

Withdrawn.

Mycobacterial inorganic pyrophosphatase (Mt-PPa) play essential role in bacterial in vitro and in vivo survival. This family of proteins reacts on inorganic pyrophosphate to release inorganic phosphate and protect bacteria from pyrophosphate toxicity. Rv3628 (Mt-PPa) encodes inorganic pyrophosphatase protein which is a type I pyrophosphatase with metal ion dependent activity. Apart from the inorganic pyrophosphatase activity, Rv3628 also showed binding with nucleotide tri phosphate (GTP/ATP). The binding of GTP and Rv3628 was analyzed by isothermal calorimetry. However, the GTP binding activity was mild but important to consider, as GTP is the secondary messenger and involved in number of mycobacterial cellular metabolic pathways therefore this interaction might be an important part of signaling cascades. This article thus represents a trilateral approach that considers computational and mutational analysis which was further complemented by virtual screening of natural compounds to find potential drug candidates. Computational analysis showed that Mt-PPa showed extensive interaction with GTP and ATP family genes. Epitope prediction showed the occurrence of amino acid regions in PPa that can act as immunological epitopes. PPa is sensitive to phosphorylation by several kinases that phosphorylate PPa at different Ser/Thr/Tyr sites. Mutational analysis showed W102G, V150G, F44G, I119G, L93F, F3G, F122G, I108G, L32G, M82G, Y17G, L59G, V5G, V26G, I7G, W140D, W140G, W140A, F80G, W140S, L49G, L56G, I9G, V60G, V19G, V92G, L28G, L61G, Y126E and F123G were the top 30 mutational hits and Y126G, Y42G, R30G, E8G, K16G are top mutational hits in active site of Mt-PPa. Mt-PPa activity is temperature and pH sensitive as the mutation in the His 21 and His 86 residues shift the optimal pH. Virtual screening of 700 compounds from herbal ingredient targets (HITs) subset of zinc database showed ZINC000003979028, ZINC000003870413, ZINC000003870412, ZINC000150338758, ZINC0000070450948, ZINC000150338754, ZINC000095098891, ZINC000000119985 and ZINC000005085286 as the top target hits and Mac0182344 and NAV_2729 as the top GTPase inhibitor that would target and hinders Mt-PPa activity. This article is protected by copyright. All rights reserved.

Assay of inorganic pyrophosphatase activity based on a fluorescence “turn–off” strategy using carbon quantum dots@Cu-MOF nanotubes

A highly sensitive and selective sensor for the quantitative assay of inorganic pyrophosphatase (PPase) activity was developed based on a fluorescence “turn–off” strategy. Carbon quantum dots@Cu(II)-based metal–organic framework nanotubes (CQDs@Cu-MOF) with length less than 300 nm and width less than 20 nm were synthesized. CQDs in the nanotubes exhibited weak fluorescence owing to static quenching. The coordination reaction between pyrophosphate ion (PPi) and Cu(II) decomposed CQDs@Cu-MOF and led to the release of CQDs, of which the fluorescence recovered. In the presence of PPase, the hydrolysis of PPi generated phosphate ion (Pi). CQDs@Cu-MOF remained their structural stability and the fluorescence turned off. The fluorescence intensity difference of the mixture of CQDs@Cu-MOF and PPi in the absence and presence of PPase (-ΔF) was proportional to the PPase concentration from 0.1 to 5 mU mL−1 and that from 5 to 50 mU mL−1, and a limit of detection at 0.03 mU mL−1 was obtained. PPase activity in human serum was analyzed using the proposed fluorescence sensor and the recovery values were found to vary from 95.0% to 104 %.

Characterization of an archaeal inorganic pyrophosphatase from Sulfolobus islandicus using a [31P]-NMR-based assay

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 μmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

In situ Cu2+-triggered wavelength-tunable fluorescent sensor for highly sensitive sensing inorganic pyrophosphatase activity and its logic gate application

As inorganic pyrophosphatase (PPase) gets involved in the regulation of various pathophysiological procedures, rapid and effective detection of its activity is of great value for monitoring and managing PPase-related diseases. Herein, an in situ Cu2+-triggered wavelength-tunable fluorescent sensor for high sensitivity monitoring of PPase activity is constructed. The proposed sensor is based on the principle of Cu2+-triggered polymerization of dopamine (DA) and m-phenylenediamine (MPD), the stronger coupling effect of pyrophosphate ion (PPi) and Cu2+, and the effective hydrolysis of PPase to PPi. With the change of substituents of MPD, the wavelength of fluorescence polymer nanoparticles (FPNPs) exhibits tunable capacity from 535 nm to 565 nm. Compared with the traditional detection method, as developed sensor shows high sensitivity to PPase with a low detection limit of 0.333 mU/mL in green channel and 0.033 mU/mL in orange channel. Additionally, the sensor realizes PPase sensing in the diluted human serum with excellent recovery in both green and orange models. Furthermore, this sensor is also applicable for inhibition assay of PPase (NaF: IC50 of 134.7 μM) and logic gates construction. Summarily, as prepared sensor provides one better way for monitoring PPase in diagnostic as well as drug screening.

Insights into the functional divergence of the haloacid dehalogenase superfamily from phosphomonoesterase to inorganic pyrophosphatase

The evolution of enzyme catalytic structures and mechanisms has drawn increasing attention. In this study, we investigate the functional divergence from phosphomonoesterase to inorganic pyrophosphatase in the haloacid dehalogenase (HAD) superfamily. In this study, a series of models was constructed, and calculations were performed by using density functional theory with the B3LYP functional. The calculations suggest that in most HAD members, the active-site structure is unstable due to the binding of the substrate inorganic pyrophosphate (PPi), and reactions involving PPi cannot be catalyzed. In BT2127, which is a unique member of the HAD superfamily, the Mg2+-coordinating residues Asn172 and Glu47 play a role in stabilizing the active-site structure to adapt to the substrate PPi by providing much stronger coordination interactions with the Mg2+ ion. The calculation results suggest that Asn172 and Glu47 are crucial in the evolution of the inorganic pyrophosphatase activity in the HAD superfamily. Our study provides definitive chemical insight into the functional divergence of the HAD superfamily, and helps in understanding the evolution of enzyme catalytic structures and mechanisms.

Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities.

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium’s composition, whose components are involved in multiple equilibria.

Aggregation-Induced Emission of Au/Ag Alloy Nanoclusters for Fluorescence Detection of Inorganic Pyrophosphate and Pyrophosphatase Activity.

The development of sensitive and accurate detection of inorganic pyrophosphate (PPi) and pyrophosphatase activity (PPase) is important as they play vital roles in biological systems. However, it is still not satisfactory for most of the analytical methods for PPi and PPase because of their Cu2+-dependence and poor accuracy. Although the metal ion triggered aggregation-induced emission (AIE) of metal nanoclusters (NCs) offers a new approach to design a Cu2+-free strategy for the accurate determination of PPi and PPase recently, current methods are all focused on utilizing pure metal NCs. Alloy NCs incorporating the advantages of diverse metal usually can achieve improved behaviors in the application, such as enhanced sensitivity and stability. In this work, glutathione stabilized alloy Au/Ag NCs were synthesized via a simple method and used for the fluorescence detection of PPi and PPase based on a Zn2+-regulated AIE strategy. The controlled release of Zn2+ by PPi and PPase could regulate the AIE of Au/Ag NCs and be employed to response PPi concentration and PPase activity. This method processes simple procedure, high sensitivity and stability, and low toxicity. In addition, we also studied the AIE behaviors of this Au/Ag NCs and offer some fundamental understanding of the AIE properties of water-soluble alloy NCs. This study not only provides a straightforward and new approach for PPi and PPase determination but a basis for further study on the AIE properties of alloy NCs and their application.

Biological properties and roles of a Trichinella spiralis inorganic pyrophosphatase in molting and developmental process of intestinal larval stages

Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg2+, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.

Impact of drying–rewetting cycles and organic amendments on phosphorus speciation of paddy soil

Little is known about the effects of drying–rewetting cycles on phosphorus (P) speciation in paddy soils with organic amendments incorporated. In this study, the responses of microbial biomass P (MBP), alkaline phosphatase activity, inorganic pyrophosphatase activity, soluble P, available P (AP), organic P, total P (TP), organic carbon (C), and C:P ratio to two types of the organic amendment (Azolla compost and rice straw) under drying–rewetting cycles were evaluated in a 60-day incubation experiment. The experimental treatments follow: amendment factor at three levels of (i) unamended control (NT), (ii) soil amended with Azolla compost (AC), and (iii) soil amended with rice straw (RS); and irrigation regimes factor at three levels of (i) constant soil moisture in a waterlogged state, (ii) mild drying–rewetting (MDR) (moisture reduced to 100% water-holding capacity (WHC) at alternate periods), and (iii) severe drying–rewetting (SDR) (moisture reduced to 70% WHC at alternate periods). Soil drying–rewetting increased the contents of AP and soluble P and the activities of alkaline phosphatase and inorganic pyrophosphatase, but decreased contents of organic P and organic C. The contents of AP and TP in organic treated soils were significantly higher than those in non-treated soils (P < 0.01). Application of AC and RS amendments increased organic P content (45% and 46%, respectively) and also further intensified the increase in AP content (63% and 37%, respectively), soluble P content (3.7- and 2.7-fold, respectively), and MBP content (6.0- and 1.6-fold, respectively) compared to control. Rice straw addition combined with MDR also increased the C:P ratio 30 days after incubation. The MBP as a microbial factor had the highest correlation with other measured indices. The MDR along with organic matter (RS and AC) caused a continuous increase in the concentration of P species. In short, the best soil quality in terms of P supply in paddy soils was achieved by regulating soil drying–rewetting cycles along with the use of appropriate timing of organic amendments.

Single-strand DNA-scaffolded copper nanoclusters for the determination of inorganic pyrophosphatase activity and screening of its inhibitor.

A fluorescence method for the determination of inorganic pyrophosphatase (PPase) activity has been established based on copper nanoclusters (CuNCs). The polythymine of 40 mer (T40) acts as a template for the reduction reaction from Cu2+ to Cu0 by ascorbic acid (AA). This reaction leads to the formation of fluorescent CuNCs with excitation/emission peaks at 340/640nm. However, the higher binding affinity between inorganic pyrophosphate (PPi) and Cu2+ hinders the effective formation of CuNCs. This shows low fluorescence intensity. PPase catalyzes the hydrolysis of PPi into Pi during which free Cu2+ ions are produced. This facilitates the formation of fluorescent CuNCs. Thus, the fluorescence intensity was restored. The fluorescence enhancement of the system has a linear relationship with PPase activity in the range 0.3 to 20mU·mL-1, and the detection limit is0.2mU·mL-1. The relative intensity (I/I0) at 640nm for the analytical solution versus system is also employed to screen the inhibitor for PPase with high efficiency. Graphical abstract Schematic representation of a fluorescent assay for the determination of inorganic pyrophosphatase activity and screening its inhibitor based on single-strand polythymine-scaffolded copper nanoclusters.

An evaluation of selected chemical, biochemical, and biological parameters of soil enriched with vermicompost

The aim of this study was to assess the changes in chemical and microbial properties and enzymatic activity of soil enriched with vermicompost derived from household waste. The vermicompost was tested in the rhizosphere of Larix decidua seedlings cultivated in 10-L pots in: (i) nursery soil (as the control), (ii) soil with 10% v/v vermicompost, and (iii) with 20% v/v vermicompost. The impact of vermicompost was assessed in terms of soil C/N ratio; bacterial, fungal, and nematode counts; and enzymatic activity. It was found that vermicompost increased the C/N ratio from 21 to 32, as well as the content of nitrate from 78 to 134 mg kg−1, of ammonium from 14 to 139 mg kg−1, of phosphorus from 92 to 521 mg kg−1, and of potassium from 142 to 1912 mg kg−1, compared with the control soil. The abundance of beneficial bacteria was increased (from 8.61 × 107 to 37.9 × 107), along with decreases in microbiological ratios of fungi and bacteria (e.g. fungi/Bacillus from 0.18818 to 0.00425). A significant 2- to 4-fold increase was observed compared with the control in the number of beneficial nematodes belonging to bacterivorous, fungivorous, and predatory groups with no change in the abundance of plant-parasitic nematodes. Addition of vermicompost brought about a change in soil enzyme activity. Vermicompost reduced the activity of alkaline phosphatase only. Both doses of vermicompost led to an increase in the activity of acid phosphatase, inorganic pyrophosphatase, dehydrogenases, β-glucosidase, and urease. Only the higher dose had an effect on increasing the activity of o-diphenol oxidase and proteases. No significant change was observed for nitrate reductase. Also, the presence of antibiotics produced by bacteria was detected depending on the dose of vermicompost, e.g. iturin (ituC) and bacillomycin (bmyB) were found in soil with a dose of 20% v/v vermicompost. Overall, vermicompost produced from household waste can be an excellent organic fertilizer for larch forest nurseries.

Effect of Structure Variations in the Inter-subunit Contact Zone on the Activity and Allosteric Regulation of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase. In Ec-Mt-PPase, the missing part of the polypeptide chain was compensated with a fragment of PPase from Escherichia coli (Ec-PPase). In R14Q-Mt-PPase, a point mutation was introduced to the contact interface between the two trimers of the hexamer. Both modifications significantly improved the catalytic activity of the enzyme and abolished its inhibition by the cofactor (Mg2+ ion) excess. Activation of Mt-PPase by low (~10 μM) concentrations of ATP, fructose-1-phosphate, L-malate, and non-hydrolyzable substrate analogue methylene bisphosphonate (PCP) was observed. At concentrations of 100 μM and higher, the first three compounds acted as inhibitors. The activating effect of PCP was absent in both mutant forms, and the inhibitory effect of fructose-1-phosphate was absent in Ec-Mt-PPase. The effects of other modulators varied only quantitatively among the mutants. The obtained data indicate the presence of allosteric sites in Mt-PPase, which are located in the zones of inter-subunit contact or associated with them.

Monitoring inorganic pyrophosphatase activity with the fluorescent dizinc(ii) complex of a macrocycle bearing one dansylamidoethyl antenna.

The dizinc(ii) complexes of L were used for the recognition of anions by fluorescence spectroscopy (L is a heteroditopic hexaazamacrocycle with two diethylenetriamine coordination heads with 2-methylpyridyl and dansylamido ethyl arms, and m-xylyl spacers). The protonation of L and stability constants of its zinc(ii) complexes were determined in aqueous solution, at 298.2 ± 0.1 K and I = 0.10 ± 0.01 M in KNO3. At a 2 : 1 Zn2+/L ratio, the dinuclear complexes clearly dominate. The ligand alone does not display fluorescence changes upon increasing the pH value, but in the presence of Zn2+ the emission reaches a maximum at pH ≅ 7.5, at which 95% of the ligand is in the dinuclear complex form. The emission appears concomitantly with the [Zn2H-1L]3+ species formation, which supports that the latter complex corresponds to the metal-promoted deprotonation of dansylamide NH. The [Zn2H-1L]3+ complexes were used for the recognition of phosphate and polyphosphate anions in aqueous solution buffered at pH 7.5 with 2 mM PIPPS, at 298.2 K. The binding of anions causes a decrease of the emission. The association constant determination revealed that HPPi3- is the strongest bound anion (log Kapp = 5.57), followed by HATP3- (two times weaker), and the remaining anions show lower binding constants, with HPO42- having the weakest uptake by the receptor. The observed selectivity of the [Zn2H-1L]3+ receptor for PPi in relation to HPO42-, and the fact that the formation of the [Zn2H-1L]3+ complex is not disturbed by the presence of Mg2+, allowed monitoring of the PPi hydrolysis by using inorganic pyrophosphatase in real-time.

Cu(II)-Regulated On-Site Assembly of Highly Chemiluminescent Multifunctionalized Carbon Nanotubes for Inorganic Pyrophosphatase Activity Determination.

A novel signal-on chemiluminescence (CL) assay for pyrophosphatase (PPase) activity determination was innovatively developed based on the Cu(II)-regulated on-site assembly of highly chemiluminescent Cu(II), N-(aminobutyl)-N-(ethylisoluminol) (ABEI), gold nanodot, and chitosan multifunctionalized carbon nanotubes (Cu(II)/ABEI-Au/cs-CNTs). First, ABEI-functionalized gold nanodots (ABEI-Au) were assembled on the surface of chitosan-modified carbon nanotubes (cs-CNTs) via the reduction of HAuCl4 with ABEI in a cs-CNT suspension to form ABEI-Au/cs-CNTs. Then, it was found that the catalyst Cu(II) can be selectively, efficiently, and quickly adsorbed onto ABEI-Au/cs-CNTs via the high-affinity interactions between Cu(II) and cs-CNTs to form novel hybrid nanomaterials Cu(II)/ABEI-Au/cs-CNTs. The CL intensity of Cu(II)/ABEI-Au/cs-CNTs was enhanced by about 2 orders of magnitude compared with that of ABEI-Au/cs-CNTs. Furthermore, it was found that in the presence of pyrophosphate ions (PPi), PPi could coordinate with Cu(II) to form a stable PPi-Cu(II) complex and block the assembly of Cu(II)/ABEI-Au/cs-CNTs. After the addition of PPase, PPase could catalyze the hydrolysis of PPi into Pi and release Cu(II) from the PPi-Cu(II) complex. The released free Cu(II) could trigger the on-site assembly of highly chemiluminescent Cu(II)/ABEI-Au/cs-CNTs, resulting in an enhanced CL intensity. The enhanced CL intensity had a good linear relationship with the activity units of PPase ranging from 0.025 to 0.5 U, with a detection limit of 9 mU. The method was employed to monitor the PPase inhibitor efficiently. Cu(II)/ABEI-Au/cs-CNTs with excellent CL may also find more applications in the development of novel CL analytical methods.

Target-mediated surface chemistry of gold nanorods for breaking the low color resolution limitation of monocolorimetric sensor

A long-term limitation of low color resolution in the monocolorimetric sensors seriously restricts its extending application, especially for the traditional organic dyes-based monocolorimetric sensors. Herein, we explore a simple target-mediated surface chemistry-based modulation for regulating the surface of gold nanorods (AuNRs), resulting in a successful multicolorimetric sensor for inorganic pyrophosphatase (PPase) activity. It operates on the principle that PPase modulates the release of Fe3+ free from the ferric-pyrophosphate complex (Fe3+-PPi) through a specific target-catalyzed hydrolysis process, leading to a better mediation for the unique chemical redox reaction between Fe3+ and I−. Subsequently, I− is oxidized into I2, which as a moderate oxidant further regulates the surface of AuNRs due to the rapidly etching reaction between I2 and AuNRs. As a result, the resultant AuNRs surface change further leads to a blue-shift longitudinal LSPR effect of AuNRs, along with a naked-eye-detectable rainbow-like multicolor signal readout. Under optimal conditions, the proposed multicolorimetric sensor exhibits an affordable sensing performance for PPase activity in the range from 1.5 U L−1 to 9 U L−1 with a detection limit of 0.8 U L−1 (S/N = 3). To this point, our strategy not only provides a promising way for breaking the low color resolution limitation of the traditional monocolorimetric sensors, but also broadens the applicability of AuNRs etching-based multicolorimetric sensors.

Zinc ion-triggered aggregation induced emission enhancement of dual ligand co-functionalized gold nanoclusters based novel fluorescent nanoswitch for multi-component detection

Herein, a zinc ion (Zn2+)-triggered aggregation induced emission enhancement (AIEE) fluorescence “on-off-on” nanoswitch was fabricated for inorganic pyrophosphate (PPi) and inorganic pyrophosphatase (PPase) activity detection. Dual ligand functionalized Au NCs were utilized as the substrate of the AIEE nanoswitch. The introduction of Zn2+ can cause Au NCs aggregated along with the enhanced fluorescence. After the addition of PPi, aggregated Au NCs disaggregated along with decreased fluorescence due to the competitive combination between PPi and Zn2+ (on-off). When PPase was introduced, PPi was hydrolyzed and release Zn2+, resulting in aggregated Au NCs along with enhanced fluorescence again (off-on). On the basis of this, highly selective and sensitive detection PPi (liner range from 0.1 to 300 μM) and PPase activity (liner range from 0.1 to 10 mU) can be achieved. The detection limits are 0.04 μM for PPi and 0.03 mU for PPase, respectively. Furthermore, the as-prepared Zn2+-triggered AIEE nanoswitch was successfully used for quantitative analysis of PPase activity in human serum with satisfactory spiked recoveries, and applied for the inhibitors screening.

Fluorometric determination of the activity of inorganic pyrophosphatase and its inhibitors by exploiting the peroxidase mimicking properties of a two-dimensional metal organic framework.

A copper(II)-based two-dimensional metal-organic framework with nanosheet structure (CuBDC NS) that possesses peroxidase (POx) mimicking activity was prepared. In the presence of hydrogen peroxide, the system catalyses the oxidation of terephthalic acid to a blue-fluorescent product (excitation = 315nm; emission = 425nm). Pyrophosphate has a very strong affinity for Cu2+ ionand blocks the POx-mimicking activity of theCuBDC NS. If, however, inorganic pyrophosphatase is present, the POx mimicking activity is gradually restored because pyrophosphate is hydrolyzed. The findings were used to design a method for the determination of the activity of inorganic pyrophosphataseby fluorometry. Fluorescence increases linearly in the 1-50mU·mL-1inorganic pyrophosphatase activity range. The limit of detection is 0.6mU·mL-1 (S/N= 3). Graphical abstract A copper(II)-based two-dimensional metal-organic framework (CuBDC NS) is described that possesses POx-mimicking activity. Inorganic pyrophosphate (PPi) was hydrolyzed to phosphate in the presence of inorganic pyrophosphatase (PPase). Hence, it cannot coordinate with Cu2+ in CuBDC NS, its structure was well-conserved to catalyses the oxidation of terephthalic acid (H2BDC) to produce a blue fluorescent product (oxBDC) in the presence of hydrogen peroxide (H2O2).

Copper nanoclusters reversible switches based on ions-triggered for detection of inorganic pyrophosphatase activity

In this work, copper nanoclusters (CuNCs) were synthesized via a one-step method using glutathione as the reducing agent and stabilizer, then characterized by means of various methods. The prepared CuNCs with the average diameter of 2.1 nm had excitation and emission wavelengths at 360 and 615 nm. Further, it was found that Al3+ could trigger the aggregation of CuNCs causing a remarkable fluorescence enhancement of the system. However, owing to the stronger coordination between Al3+ and pyrophosphate (PPi), CuNCs/Al3+ was disaggregated with the addition of PPi, resulting in a significant fluorescence quench of system. When PPase was present, PPi was consumed by PPase, which inhibited the disaggregation of CuNCs. Based on the principle of Al3+ and PPi inducing aggregation/disaggregation of CuNCs accompanied by fluorescence enhancement/quenching, a novel method was established for fluorescence "turn-on-off-on" detection of PPase activity. The detection limit of PPase was determined to be 1.3 mU mL−1 in the linear range of 3.0–40.0 mU mL−1. The established method was used to detect the positive samples of rat serum with satisfactory spiked recoveries, and applied for the screening of inhibitors.