Embedding Capture-Magneto-Catalytic Activity into a Nano-Catalyst

Dec 12, 2017 - Using emerging nano-catalysts to investigate the activity of bio-catalysts (protein enzymes, catalytic RNAs, ect.) is increasingly rece...
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Embedding Capture-Magneto-Catalytic Activity into a Nano-Catalyst for the Determination of Lipid Kinase Tao Gao, Chaoli Mu, Hai Shi, Liu Shi, Xiaoxia Mao, and Genxi Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10857 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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ACS Applied Materials & Interfaces

Embedding Capture-Magneto-Catalytic Activity into a Nano-Catalyst for the Determination of Lipid Kinase Tao Gao,†,‡ Chaoli Mu,§ Hai Shi,§ Liu Shi,§ Xiaoxia Mao,†,‡ Genxi Li†,§,* †

Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China ‡ Institute of Biomedical Engineering, School of Communication and Information Engineering, Shanghai University, Shanghai 200444, P. R. China § State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China ABSTRACT: Using emerging nano-catalysts to investigate the activity of bio-catalysts (protein enzymes, catalytic RNAs, ect.) is increasingly receiving attention from material, analytical and biomedical scientists. Here, we have first fabricated a three-in-one nano-catalyst, the nitrilotriacetic acid (NTA)-modified magnetite nanoparticle (NTA-MNPs), to develop an integrated magneto-colorimetric (MagColor) assay for lipid kinase activity, so as to solve the inherent problems in lipid kinase assay. Based on three integrated functions of the NTA-MNPs (capture, magnetic separation and peroxidase activity), catalytic activity of lipid kinase is directly converted to colorimetric signals. Therefore, assay procedure is significantly simplified that one-step and visual detection of lipid kinase activity is possible. Moreover, the whole system responses sensitively in case NTA-MNPs recognize a few numbers of the reaction sites, which efficiently initiates the chromogenic reaction of a large amount of chromogens, thus the detection limit decreases to 6.5 ± 5.8 fM, about three orders of magnitude lower as compared to that of enzyme-linked immune-sorbent assay (ELISA). So, by embedding desired functions into nano-catalysts, the assay for bio-catalysts becomes easy, which may promisingly provide useful tools in biomedical and clinical research in the future. KEYWORDS: magnetic particles, nanocatalyst, biocatalysis, lipid kinase, biosensing To achieve the goal, favorable environment that facilitates lipid kinase activity should be provided. At present, artificial lipid layers have been fabricated both on solid supports and in aqueous solutions to simulate lipid-membrane environment. In previous reports, solid supported lipid membrane was utilized to construct biomimetic sensing surface,10-11 investigate the interaction between peptides and membrane,12 study membrane protein activities,13 among other functions.14-15 Herein, to facilitate the assay of lipid kinase, free-floating liposome has shown unique advantages, which has been widely used as a model of cell membrane. Besides, its microcapsule structure can be fully utilized to facilitate the assay. Firstly, substrates of the kinase (lipid molecules) can be inserted into outside layer of liposome through hydrophobic interaction. The lipid bilayer of liposome can thus provide favorable catalytic environment for lipid kinase. Secondly, the inner part of liposome can be filled with multiple signaling entities, enabling signal enhancement.16-19 So, liposome not only provides favorable catalytic environment for lipid kinase activity, but also acts as a signal amplifier for colorimetric detection. How to transform kinase activity to readable signal is the key issue. The enzyme-based colorimetric signal conversion is an efficient, handy and visible mean that has been used in many bioassays.20-22 So, in this work, an inorganic nano-catalyst, the nitrilotriacetic acid (NTA)-modified

INTRODUCTION In lipid metabolism, lipid kinase is a significant class of enzyme to catalyze the phosphorylation of lipids, both on plasma membrane as well as on the membrane of organelles. Phosphorylation of a lipid can affect its downstream activities in cell signaling, cellular transport, and other important cellular pathways.1-2 Therefore, the assay of lipid kinase activity is critical important in biomedical and clinical research, which may provide insightful information for some related biological issues and diseases.3-4 However, compared to various assay methods of protein kinase, available methods for lipid kinase activity are few, and they are also complicated.5-6 This is mainly caused by (1) amphiphilic property of the lipid molecules that perplexes separation and quantitation tests, and (2) unfavorable catalytic environment that impedes lipid kinase activity. Present methods are thus labor-intensive, time-consuming and instrument-dependent. In a common assay for lipid kinase activity, for instance, the products (phosphorylated lipids) are usually labelled with [γ-32P] group during the phosphorylation reaction, followed by semi-quantitation of the labelled lipids with a phosphor imager or HPLC/MS equipment.7-9 Such an assay requires multi-step operations, sophisticated instruments, and may also produce harmful radioactive contaminants. Therefore, the development of an efficient and simplified assay strategy for lipid kinase is highly required. A

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magnetite nanoparticle (NTA-MNP), has been newly synthesized as the single transducer. After coordinating Fe(III) ions, NTA-MNPs can specifically bind to phosphate groups23 on liposomes, facilitate magnetic separation, and finally convert the binding event into colorimetric signals based on the intrinsic peroxidase-like activity of MNP.24 Thus a relationship can be established between the kinase activity and the colorimetric signal. Scheme 1 briefly shows the principle of the assay. Lipid kinase catalyzes the phosphorylation of lipid substrates that are previously anchored on liposomes. Phosphorylated liposomes are captured by NTA-MNPs. After magnetic separation of NTA-MNPs, the captured liposomes are split to release encapsulated chromogenic agents (3,3',5,5'-tetramethylbenzidine, colorless TMB), which are catalyzed by NTA-MNPs to form oxidative TMB (blue OxTMB) that gives colorimetric signal readout. So, three functions are integrated into NTA-MNPs: capture, magnetic separation and catalytic reaction, which help to transform the kinase activity into readable signals. Moreover, signal amplification can occur in a very straightforward manner in case NTA-MNPs interact with few numbers of phosphate sites is sufficient on liposomes. Collective behavior of the system is thus fully dependent on the functions of NTA-MNPs. Besides, all procedures of the assay can be performed in one test well, which significantly simplify the assay procedures and reduce human workflow. Therefore, by coupling substrates-encapsulated reactive liposomes with multi-functional NTA-MNPs, one-step and integrated assay for lipid kinase activity has been first developed, which is named as the MagColor assay.

Synthesis of NTA-MNPs. For surface modifications, MNPs were anchored with dopamine (DA) based on the interactions between bidentate enediol ligands of DA and under-coordinated Fe surface sites of MNPs.26-27 3 mL Fe3O4 (4.0 mg mL-1) was mixed with 3 mg DA in a buffer containing 2 mL methanol and 5 mL H2O. After pH value was adjusted to 4.5, the mixture was reacted under sonication for 20 min. Then the modified MNPs were magnetic separated and washed with PBS for three times, and finally suspended in 5 mL PBS. 5 mL 1% glutaraldehyde solution was added to the above solution. After reaction using a shaking table at room temperature for 0.5 h, 3 mg Lys-NTA was added. The mixture was further allowed to react for another 1.0 h. After which, 1 mL NaBH4 (0.1 M) was added to form reductive-amination. Finally, the obtained NTA-MNPs were cleaned and magnetic collected in 10 mM Tris-HCl buffer, pH 6.8. 5 mL FeCl3 solution (1.0 M) was added to the solution obtained in the previous step, and stirred for 1.0 h. Then, the product was separated from the solution with a small permanent magnet and washed with Tris-HCl buffer for two times. The Fe(III)-coordinated NTA-MNPs were stored at 4°C for further use. The obtained nanoparticles were characterized by transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. TEM characterization of MNPs was carried out on a transmission electron microscopy (Hitachi H7650, Japan). FT-IR spectroscopy was characterized on a NEXUS640 infrared spectrometer system (NICOLET, USA). The particle size and density were analyzed on a particle size analyzer (Brookhaven 90Plus, USA) and a particle analyzer system (NanoSight NS 300, Malvern), respectively. Fabrication of R-Liposomes. R-Liposomes were prepared by adding 80 mg L-a-phosphatidylcholine (egg yolk lecithin), 10 mg cholesterol, 3.0 mg sphingosine and 12.5 mg TMB into 20 mL CHCL3 in a round bottomed flask. This mixture was mixed after 30 seconds of vortexing and then blew dry with nitrogen. Lastly, the dry residue was suspended with 20 mL Tris-HCl buffer (20 mM containing 150 mM NaCl, pH 6.5) and sonicated for 30 minutes to obtain the desired composition. The MagColor assay for lipid kinase activity. In each well of the 96-well microplate, 20 µL R-Liposomes and 5 µL desired concentration of SphK1 were added in 25 µL kinase reaction buffer (20 mM Tris-HCl, 500 µM ATP, 50 mM MgCl2, pH 7.4). The solution was incubated at 37 °C for 1.0 h after pipetting mixture. Then, 5 µL Fe(III)-coordinated NTA-MNPs (0.12 mg mL-1) was added, about 4.2 × 106 nanoparticles. After pipetting mixture and incubation for another 5 min, reacted R-Liposomes were magnetically collected on a magnetic separator rack for 96-well plate, and the supernatant was removed. Finally, 100 µL assay buffer (1 mM EDTA, 5 mM citric acid, 10 mM H2O2 and 10% glycerol) was added into the microplate well. 10 µL Tween-20 was added to split the R-Liposome for colorimetric reaction. After incubation at 25 °C for 10 min, 50 µL stop solution (2M H2SO4) was added to terminate the reaction. The mixture in each well was brought to determination at the 450 nm on a microplate reader (M200PRO, Tecan).

EXPERIMENTAL SECTION Material and Regents. Nα,Nα-Bis(carboxymethyl)-L-Lysine (Lys-NTA), FeCl3•6H2O, L-a-phosphatidylcholine (egg yolk lecithin), cholesterol and 3,3',5,5'-Tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich. Ethylene glycol and NaAc were purchased from Nanjing Chemical Reagents. Other chemicals were purchased from Sigma-Aldrich and were used as received. Recombinant human sphingosine kinase 1 (EC 2.7.1.91) was purchased from Sino Biological Inc. Sphingosine kinase 1 ELISA Kit (ABIN420321) was purchased from 4A Biotech Co., Ltd. All solutions were prepared with deionized water that was purified with a Milli-Q purification system (Bedford, MA), to a resistance of 18.2 MΩ cm before use. Synthesis of MNPs. Fe3O4 MNPs with diameters of approximately 140 nm were prepared according to a solvothermal method with slight modification.25 0.68 g FeCl3·6H2O was added in 20 mL ethylene glycol to form a clear solution, followed by adding 1.8 g NaAc and 0.5 g polyethylene glycol. The solution was stirred vigorously for 1.0 h, and then sealed in a teflonlined stainless-steel autoclave. The autoclave was heated to 200°C and maintained for 24 h, and allowed to cool to room temperature slowly. The black products were washed several times with ethanol and dried at 90°C for 30 min. The obtained samples were characterized on a Brucker D8-advance X-ray powder diffractometer (XRD) with Cu Kα radiation (λ = 1.5418 Å), scanning angle from 10° to 70°. B

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Scheme 1. Schematic illustration shows the principle and assay procedures of the MagColor assay for lipid kinase activity.

fitted to the Michaelis–Menten model. Increased Km value may indicate affinity loss after NAT modification (Table 1). However the activity loss would not influence its application in catalytic transformation for quantitative signal readout. Therefore, NTA-MNPs with peroxidase-like activity have been synthesized, which can be satisfactorily used for signal transduction in the assay for lipid kinase activity. Table 1. Kinetic parameters of MNPs before and after the modification of NTA.

RESULTS AND DISSCUSION Synthesis and characterizations of NTA-MNPs. Fe3O4 MNPs with peroxidase activity were synthesized according to a previous literature.20 MNPs were then functionalized with NTA by using dopamine as the linker, the synthetic method is described in the experimental section. The morphology, size and activity of NTA-MNPs have been characterized. In Figure 1, TEM image reveals the mean particle size is 140 ± 15 nm (Figure 1B). Dynamic light scattering (DLS) analysis further indicates the mean hydrophobic diameters (HD) for MNPs and NTA-MNPs are 282 ± 11 nm and 811 ± 98 nm respectively, larger compared to the TEM observation as a consequence of aggregation. Zeta potentials of MNPs showed a negative move from -23.1 mV to -44.0 mV after the linkage reaction (Figure 1C), which indicates the successful linkage of negatively charged NTA moieties on MNPs surface. XRD analysis in Figure 1D has confirmed the production of Fe3O4 MNPs, the pattern of which can be easily indexed to Fe3O4 (JCPDS 75-1609). FT-IR spectra show the characteristic peaks of DA (1490, 1636 and 1240 cm−1, corresponding to the bending vibration of N-C, aromatic C-C and the aryl oxygen stretching vibration respectively) and NTA (1640 cm−1 and 1410 cm-1, corresponding to amide bond; the peak groups at 2980, 2930 and 2890 cm−1, originating from the symmetric and asymmetric stretch of carboxylate groups). FT-IR analysis further indicated the successful modifications of the MNPs. Afterwards, the peroxidase activity of NTA-MNP was tested. An average 17% of the activity was lost as a consequence of surface coverage of NTA moieties (Figure 1F). We demonstrated this change by determination of apparent steady-state kinetic parameters for the reaction. The data were

[E] (M) MNPs

4.92 10-12

×

NTA-MNPs

4.59 10-12

×

Km (mM)

Vmax s-1)

(M

0.11

1.45×10-7

2.94×104

0.13

1.11×10-7

2.43×104

Kcat (s-1)

[E] is the MNP (or MNP-NTA) concentration, Km is the Michaelis constant, Vmax is the maximal reaction velocity, and Kcat is the catalytic constant, where Vmax = Kcat [E].

Fabrication of the reactive liposome. Then, the liposome used for lipid kinase assay was fabricated. The outside layer of liposome was anchored with the substrates (lipid molecules) of lipid kinase, and the inner space was filled with TMB as the colorimetric signal reporters. In this way, the fabricated liposome (R-Liposome) can react with lipid kinase and NTA-MNPs sequentially. Characterizations of R-Liposome are shown in Figure 2. TEM image shows that the size distribution of R-Liposomes is about 190 ± 44 nm (Figure 2A and 2B). To reveal the encapsulation efficiency of TMB in C

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R-Liposome, colorimetric reactions were conducted by adding NTA-MNPs into liposome, R-Liposome and split R-Liposome in the assay buffer. After reaction, digital photographs in Figure 2C show that an obvious chromogenic reaction occurred for split R-Liposome, which was not observed for empty liposome. The result revealed the successful encapsulation of TMB molecules in liposome. Besides, limited colorimetric reaction was observed for intact R-Liposome, indicating relatively good stability of R-Liposome. Therefore, the above results have indicated successful fabrication of R-Liposome.

Feasibility tests of the MagColor assay. In order to test this new assay strategy for lipid kinase activity, we have investigated a critical lipid kinase in cell signaling, sphingosine kinase 1 (SphK1).28-29 In the absence of SphK1, R-Liposomes and NTA-MNPs are individually dispersed. While after catalytic reaction of SphK1, NTA-MNPs can bind to the phosphorylated R-Liposomes based on the affinity interaction between Fe(III)-NTA moieties and phosphorylated sites. This indicates that the phosphorylated R-Liposomes can be efficiently captured by NTA-MNPs.

Figure 1. Synthesis and characterizations of NTA-MNPs. (A) Schematic illustration shows NTA modifications on the surface of MNP. (B) TEM image of NTA-MNPs. The insert histogram shows size distribution of the NTA-MNPs, n = 128. (C) XRD analysis of the synthesized MNPs (top) and corresponding indexing of XRD patterns (down). (D) Zeta potentials of the naked (left) and NTA-bound MNPs (right) in PBS buffer at pH 7.4. Dynamic light scattering (DLS) analysis showing the mean hydrophobic diameters of MNPs before (left) and after (right) the modification of NTA moieties. (E) FT-IR analysis of the sequential modification of MNPs, (a) dopamine modified MNPs and (b) NTA modified MNPs. (F) Relative peroxidase activity of MNPs and NTA-MNPs, corresponding digital photograph show the colorimetric reaction of the synthesized NTA-MNPs.

D

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ACS Applied Materials & Interfaces Figure 2. Characterizations of the fabricated R-Liposomes. (A) Typical TEM images of the fabricated R-Liposomes. (B) Corresponding histogram shows the size distribution of R-Liposomes in TEM image, 190 ± 44 nm. The scale bar is 200 µm. (C) Reactive activity of the fabricated R-Liposomes. Photograph shows the colorimetric reaction of liposome, R-Liposome and split R-Liposome in the presence of NTA-MNPs in assay buffer. Additionally, colorimetric assay was performed to reveal the catalytic activity of SphK1, as Figure 3C shows that an obvious chromogenic reaction occurs in the presence of enzyme. On the contrary, almost no colorimetric reaction was observed when we solely added SphK1 or R-Liposome in the assay system. Corresponding colorimetric signals were recorded after the reaction was terminated with stop solution (Figure 3D). These observations reveal that this new assay strategy can be translated into a working colorimetric assay for the detection of lipid kinase activity. Optimization of assay conditions. To optimize assay conditions for the SphK1 activity, experiments were performed using different concentrations of sphingosine and TMB. The final colorimetric signals were recorded to reveal the influence of these fabrication molecules when using different concentrations. As is shown in Figure 4, with the increased concentrations of sphingosine, the signal intensity increases and reaches a plateau after 0.5 mM. So the concentration of sphingosine in the solution used for preparation of the sphingosine-anchored liposomes is 0.5 mM. Additionally, excess TMB may cause insolubility and instability of liposomes, so the concentration of TMB was optimized to a final concentration of 2.5 mM in the solution used for R-Liposome preparation. Besides, the TMB entrapment efficiency was about 56.4%, according to colorimetric signals (O.D. values) of the TMB respectively in the filtration collected solution and the original preparation solution (Figure 4C). The function used to calculate the entrapment efficiency is as follows: O.D.total -O.D.out Entrapment efficiency ሺ%ሻ= ×100% O.D.total

Figure 3. Feasibility test of the MagColor assay. Schematic illustrations(A) and corresponding TEM images of the states of R-Liposomes (green dashed-line circle) and NTA-MNPs (red dashed-line circle) before (B) and after (C) the catalytic reaction of SphK1. The scale bar is 200 µm. (D) Optical photograph showing the colorimetric reaction using different components of the assay system. Top to down: 1) SphK 1, 2) R-Liposome, 3) SphK 1 and R-Liposome, and 4) inactivated SphK 1 and R-Liposome. (E) Histogram columns showing relative optical densities of the reaction mixture at 450 nm, which indicates the reaction activity of the assay system under different conditions. Error bars are obtained from three independent experiments.

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Figure 4. Assay condition optimizations. (A) The influence of the substrate (sphingosine, Sph) on colorimetric signal responses. (B) The influence of TMB concentration on the fabrication of R-Liposome. (C) TMB entrapment efficiency test. Histogram shows the colorimetric signals (%) generated by TMB in the filtration collected solution and the original preparation solution.

Assay for lipid kinase activity. Based on the principle of the assay, our intention to illustrate liposome-based signal amplification relies on thousands of signal entities in R-Liposomes, which ensures high sensitivity because only a few phosphate sites are required for magnetic collection of the phosphorylated R-Liposomes. To confirm sensitivity of the assay and the ability to provide unprecedented response at low kinase activity level, a comparison has been made between the MagColor assay and a sandwich immunoassay (ELISA). SphK1 was used as the target analyte in the sandwich immunoassay on a 96-wells ELISA plate.

Table 2. A comparison of assay methods for lipid kinase.

Techniques

Types of Kinases

ELISA30

e.g. SpHK1, PI3K)

0.31 ng/mL (~7.3 pM)

Radiolabeling and phosphor-imaging5,

e.g. PIK-93

0.048 µM (IC50)

Electrochemistry32-33

e.g. SpHK1

Colorimetric (this work)

e.g. SpHK1

7, 31

assay

Lipid

Manageability of the Assay Procedure

Detection Range

Sensitivity of the Assay

0.31-20 ng/mL

Medium

-

Low (semi-quantitative)

2.33 pmol/min/mg

0.01-12 nmol/min/mg

High

Medium

5.6 fM

0.14-230 pM

High

Simple

Limit of Detection

F

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Complicated

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ACS Applied Materials & Interfaces using of NTA-MNPs has significantly simplified assay procedures. Additionally, the whole system responses sensitively in case NTA-MNPs interact with a limited number of phosphate sites on R-Liposomes. The subsequent release of thousands of TMB molecules and the colorimetric reaction catalyzed by NTA-MNPs can help to amplify the quantitative signal, and sensitive results are ensured. So, without involvement of target amplification, detection limit of the assay can reach 6.5 ± 5.8 fM, equal to 14.3 ± 12.8 fmol min-1 in 50 µL reaction volume, which has been improved by three orders of magnitude as compared with that of ELISA. Therefore, based on the integrated functions of NTA-MNPs, our proposed MagColor assay has shown advantages of simplicity and high sensitivity, which enables it to be further extended to sensitive and simple detection of lipid phosphorylation and other signaling kinases, and finding promising applications in biomedical research in the future.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: +86-25-83593596. ORCID Genxi Li: 0000-0001-9663-9914 Notes The authors declare no competing financial interest.

Figure 5. MagColor assay for lipid kinase activity. (A) Photographs show the ELISA assay (up) and the MagColor assay (low) for SphK1 at different concentrations. The concentrations in each row are the same. (B) Corresponding calibration plots for SphK1 concentration in samples.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant No. 21235003), the National Postdoctoral Program for Innovative Talents (Grant No. BX201600098) and the China Postdoctoral Science Foundation funded project (Grant No. 2017M611532).

Photographs in Figure 5A and 5B show the colorimetric responses of the two assay methods when different concentrations of SphK1 were used. An obvious response can be detected in MagColor assay when the SphK1 concentration is at femtomolar level. However, the ELISA assay gives a negative response at the same concentration. The observation indicates high sensitivity of the MagColor assay method. Detection limit of the MagColor assay was calculated to be 6.5 ± 5.8 fM based on the non-linear fitted calibration curve by using the Origin 8.5 software. It is three orders of magnitude lower than that of ELISA assay (7.31 ± 4.12 pM). According to instructions of the commercially provided SphK1 protein (1028 nmol min-1mg-1), the detection limit of SphK1 activity was calculated to be 14.3 ± 12.8 fmol min-1 in 50 µL reaction volume. Besides, a linear detection range from 0.37 pM to 0.23 nM was established, y = 0.43 + 0.21x, R2 = 0.99. Based on the equation, the detection limit was estimated to be 0.14 ± 0.73 pM. So the applicable range of quantification is from 0.14 pM to 0.23 nM. To be noted, the MagColor assay is very easy to operate, where all required components are added into one well of the microplate to obtain the final colorimetric signal, which is significantly simplified when compared to that of ELISA. So, in consideration of its sensitivity and easy operation, the MagColor assay shows many advantages over present methods used for lipid kinase activity (Table 2).

REFERENCES (1) Cantley, L. C. The Phosphoinositide 3-Kinase Pathway. Science 2002, 296, 1655-1657. (2) Vanhaesebroeck, B.; Stephens, L.; Hawkins, P. PI3K Signalling: The Path to Discovery and Understanding. Nat. Rev. Mol. Cell. Bio. 2012, 13, 195-203. (3) Wymann, M. P.; Schneiter, R. Lipid Signalling in Disease. Nat. Rev. Mol. Cell. Bio. 2008, 9, 162-176. (4) Zhao, Y. T.; Kalari, S. K.; Usatyuk, P. V.; Gorshkova, I.; He, D. H.; Watkins, T.; Brindley, D. N.; Sun, C. D.; Bittman, R.; Garcia, J. G. N., Berdyshev, E. V. Natarajan, V. Intracellular Generation of Sphingosine 1-Phosphate in Human Lung Endothelial Cells - Role of Lipid Phosphate Phosphatase-1 and Sphingosine Kinase 1. J. Biol. Chem. 2007, 282, 14165-14177. (5) Knight, Z. A.; Feldman, M. E.; Balla, A.; Balla, T.; Shokat, K. M. A Membrane Capture Assay for Lipid Kinase Activity. Nat. Protoc. 2007, 2, 2459-2466. (6) Zhao, X. H.; Varnai, P.; Tuymetova, G.; Balla, A.; Toth, Z. E.; Oker-Blom, C.; Roder, J.; Jeromin, A.; Balla, T. Interaction of Neuronal Calcium Sensor-1 (NCS-1) with Phosphatidylinositol 4-Kinase Beta Stimulates Lipid Kinase Activity and Affects Membrane Trafficking in COS-7 Cells. J. Biol. Chem. 2001, 276, 40183-40189. (7) Yanamandra, M.; Kole, L.; Giri, A.; Mitra, S. Development of Phosphocellulose Paper-Based Screening of Inhibitors of Lipid Kinases: Case Study with PI3K Beta. Anal. Biochem. 2014, 449, 132-138. (8) Christoforidis, S.; Miaczynska, M.; Ashman, K.; Wilm, M.; Zhao, L. Y.; Yip, S. C.; Waterfield, M. D.; Backer, J. M.; Zerial, M. Phosphatidylinositol-3-OH Kinases Are Rab5 Effectors. Nat. Cell Biol. 1999, 1, 249-252.

CONCLUSION In conclusion, a function-integrated nano-catalyst (NTA-MNP) has been fabricated to develop the MagColor assay for one-step and colorimetric detection of lipid kinase activity. The application of R-Liposomes has provided lipid kinase with favorable catalytic environment, and the skillfully G

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