Highly sensitive detection of protein kinase activity using upconversion luminescent nanoparticles

Abstract

An upconversion nanophosphor (UCNP)-based highly sensitive assay for the detection of protein kinase activity is developed with the assistance of Zr⁴⁺-functionalized magnetic beads.

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Near infrared (NIR)-to-visible upconversion (UC) nanophosphors (UCNPs) are a special class of luminescent materials which are capable of emitting intense visible emissions by absorbing two or multiple NIR photons.¹ Compared with conventional down conversion fluorescent materials such as organic dyes and semiconductor quantum dots (QDs), UCNPs exhibit many distinct advantages, such as high chemical stability, high resistance to photobleaching or blinking, low toxicity, large Stokes shift, and deep-penetration in tissues as well as minimum photodamage to living organisms due to the use of NIR illumination. On the other hand, background interference such as autofluorescence or Rayleigh scattering is particularly a concern for conventional fluorescent bioassays using ultraviolet (UV) or high energy visible light excitation. Fascinatingly, NIR illumination of UCNPs does not lead to autofluorescence of biomolecules and the light scattering can also be effectively suppressed, which can greatly enhance the signal-to-background ratios. Therefore, these exceptional optical properties make NIR-to-visible UCNPs extremely attractive as fluorescent tags for biosensing and bioimaging.² For example, UCNPs have been successfully applied as direct luminescent reporters in many heterogeneous bioassays, such as detection of DNA hybridization^3a or immunoassays.^3b,c Compared with heterogeneous assays, more intense attention has been focused on the fabrication of UCNPs-based homogeneous bioassays by luminescence resonance energy transfer (LRET) technique.^3d To date, by using UCNPs as the excellent LRET donors and organic dyes, gold nanoparticles or graphene oxide as versatile LRET acceptors, great progress has been made for the detection of various biomolecules or small molecules such as DNA,^3e proteins,^3f glucose,^3g glutathione,^3h ATP,³ⁱ cyanide anion,^3j oxygen^3k and so on.

Protein kinases (PKs)-catalyzed protein phosphorylation plays critical regulatory roles in a majority of biological processes including cell proliferation, differentiation, and apoptosis.⁴ It is estimated that up to 30% of all human proteins are modified by kinase activity. The aberration of PKs activity and the subsequent abnormal protein phosphorylation states have been proved to be closely associated with many human diseases including Alzheimer's diseases and cancers.⁵ So PKs have become important molecular targets for drug therapy and the screening of PKs inhibitors as potential drugs is always a hot topic. In this regard, highly sensitive detection of PKs activity is not only valuable to provide insights regarding the fundamental biochemical process of diseases but also essential to the PKs-targeted drug discovery and therapies.

Up to now, many techniques have been utilized for PKs analysis, including radioactive methods,⁶ fluorescent methods,⁷ electrochemical assays,⁸ magnetic resonance imaging (MRI) approach,⁹ and mass spectrometry-based methods.¹⁰ Among these kinase assays, fluorescence technique is particularly attractive due to its easy operation, design flexibility, and high-throughput capability. However, the existing fluorescent kinase assays, which typically use organic dyes⁷ or QDs¹¹ as the signal reporting tags, also have many intrinsic limitations. Organic fluorophores suffer from a number of known drawbacks such as weak photostability and broad emission bands. QDs are also limited by their inherent toxicity and chemical instability. In addition, the excitation of dyes or QDs generally needs high energy light, which will result in strong background fluorescence signal and thus limit the detection sensitivity. In light of the unique anti-Stokes photoluminescence (PL) properties of UCNPs, we envision that the inherent problems of conventional fluorescent kinase assays can be elegantly solved by using NIR-to-visible UCNPs as the fluorescent labels. Towards this aim, an UCNPs-based highly sensitive PKs assay has been developed in this work.

The design principle of the UCNPs-based kinase assay is illustrated in Fig. 1 by using cAMP-dependent protein kinase A (PKA) as a proof-of-concept target. β-NaYF₄:Yb,Er (∼30 nm in diameter), one of the most efficient NIR-to-visible UCNPs known to date, is synthesized according to our previous work¹² and is used as the signal reporter for kinase analysis. As depicted in Fig. 1, in the presence of PKA, the γ-phosphoryl of ATP will be transferred to the hydroxyl group of serine (S) in the biotinylated peptide (biotin–LRRASLG), which is used as the PKA-specific substrate. Zr⁴⁺-functionalized magnetic beads (ZrMBs) are prepared for highly selective capture and enrichment of phosphopeptides with facile magnetic separation. It has been proved that Zr⁴⁺ can selectively bind with phosphate groups on proteins or peptides.^7c,8c Therefore, when the biotin–peptides are phosphorylated by PKA, the phosphopeptides will be specifically bind on the ZrMBs while the unphosphorylated peptides will not. After magnetic separation of unbound peptides, avidin–UCNPs conjugates are used to recognize the biotin–peptides accumulated on the ZrMBs through the strong affinity between avidin and biotin. As such, the amount of UCNPs anchored on the ZrMBs will be proportional to the PKA activity. Our previous work has demonstrated that NH₃·H₂O can effectively break down the complexing interaction between phosphate group and metal ions.^7b So in this work, the UCNPs–avidin/biotin–peptides complexes can be dissociated from ZrMBs surface into NH₃·H₂O solution. By recording the UC luminescence signal of the dissociated UCNPs solutions, quantitative analysis of PKA activity can be realized.

Fig. 1 Schematic illustration of the UCNPs-based protein kinase assay.

Fig. 2a shows the PL spectra of the proposed kinase sensing system in the presence of different activities of PKA. β-NaYF₄:Yb,Er UCNPs display emissions centred at 545 nm and 658 nm under the excitation of a 980 nm laser, which are assigned to the ⁴S_3/2 → ⁴I_15/2 and ⁴F_9/2 → ⁴I_15/2 transition of Er³⁺, respectively. The plot of PL intensity at 545 nm with PKA activity is shown in Fig. 2b. One can see that as low as 0.05 mU μL⁻¹ PKA can be clearly discriminated from the blank control, and the PL intensities increase gradually with increasing concentrations of PKA from 0.05 mU μL⁻¹ to 0.2 U μL⁻¹, crossing a wide working range of more than three orders of magnitude. The detection limit of PKA is estimated to be 0.02 mU μL⁻¹ (3σ, n = 11). To date, most of the highly sensitive methods for PKA detection are established by using electrochemical or fluorescence techniques. The detection limits of these assays for PKA are generally in the range of 0.1–0.5 mU μL⁻¹.^7b,c,8c–e Therefore, one can see that the sensitivity of the UCNPs-based kinase assay is much higher or at least comparable to those known most sensitive kinase assays. Furthermore, for comparison with the UCNPs labels, control experiments were performed by using fluorescein-labeled peptides (FITC–LRRASLG) as the substrate for detection of PKA activity, and the results are shown in Fig. S4.† As can be seen from Fig. S4,† the lowest PKA activity that can be detectable is ∼0.5 mU μL⁻¹. These results clearly demonstrate the advantage of UCNPs bio-labels for PKA analysis, which shows about 1 order of magnitude higher sensitivity than that by using fluorescein dye.

Fig. 2 (a) UC PL spectra of the proposed assay system in the presence of varying concentrations of PKA under the excitation of a 980 nm laser; (b) corresponding plot of PL intensity at 545 nm with PKA activity. Error bars are estimated from the standard deviation of three repetitive measurements.

Now that PKs inhibitors may become potential targeted drugs for disease therapy, the screening of kinase inhibitor is clinically important. The feasibility of the proposed method in inhibition assay is evaluated by mixing PKA with varying concentrations of H-89, an efficient inhibitor of PKA. As shown in Fig. 3a, with the fixed concentration of PKA, the PL signal decreases gradually as the concentration of H-89 increases, revealing that the more H-89 added, the higher inhibition of PKA activity. The relationship between PL intensity at 545 nm and H-89 concentration is plotted in Fig. 3b as a sigmoidal profile, from which the IC₅₀ value of H-89 is determined to be 106 nM. This value is consistent with those reported in literatures.^7c,11a

Fig. 3 (a) UC PL spectra of UCNPs-based kinase assay system in the presence of different concentrations of H-89. The concentration of PKA is fixed at 0.02 U μL⁻¹; (b) the relationship between PL intensity at 545 nm and H-89 concentration. The error bars represent standard deviation of three replicates for each data point.

Furthermore, since PKs activities are highly regulated in cells, we testified whether the UCNPs-based method could be applied for PKA detection in cell lysates. In this study, forskolin combined with IBMX, which can effectively increase the intracellular level of cAMP, are used to stimulate the activation of cAMP-dependent PKA in Hela cells. The PKA activities in cell lysates with/without drug stimulation are all examined by the proposed assay. The results are shown in Fig. 4, where I represents the PL intensity (545 nm) produced by the cell lysates after kinase reaction, while I₀ is the PL intensity of blank control without adding cell lysate or PKA. As can be seen from Fig. 4 that compared to the blank control, the UC PL signal is almost unaffected by the cell lysate without drug stimulation. On contrast, the lysates of stimulated cells all arouse obviously increased PL signals, and the response increases with increasing concentration of stimulants, suggesting that forskolin/IBMX have successfully activated PKA in cells. In addition, if H-89 was pre-mixed with the drug-stimulated cell lysate, the PL signal would decrease sharply again, indicating that the observed UC PL responses are indeed generated by PKA-induced peptides phosphorylation but not other factors. These results demonstrate that the UCNPs-based assay is suitable for in vitro detection of cell kinases in complex biological samples.

Fig. 4 Relative PL responses of the proposed assay for the detection of PKA in different cell lysates. (a) Blank control without any cell lysate; (b) cell lysate without drug stimulation; (c) cell lysate stimulated with 10 μM forskolin/20 μM IBMX; (d) cell lysate stimulated with 50 μM forskolin/100 μM IBMX; (e) cell lysate stimulated with 50 μM forskolin/100 μM IBMX and then premixed with 5 μM H-89. The total protein concentration of all the Hela cell lysates are all controlled to be 10 μg mL⁻¹. Error bars are estimated from the standard deviation of three repetitive measurements.

In conclusion, a UCNPs-based protocol is developed for highly sensitive detection of protein kinase activity with the assistance of ZrMBs. This versatile approach has several advantageous features. On one hand, the Zr⁴⁺ layer of ZrMBs exhibits high selectivity for recognizing and enriching phosphopeptides over the unphosphorylated ones, and the magnetic characteristic of ZrMBs facilitates simple purification and washing operations. On the other hand, the application of UCNPs as the signal reporters greatly reduces the autofluorescence and light scattering interferences. In addition, to date, β-NaYF₄:Yb,Er is known to be one of the most efficient NIR-to-visible UCNPs. So their strong UC emission can guarantee the high detection sensitivity. Therefore, by integrating these distinct advantages of UCNPs and ZrMBs, excellent analytical performance of the proposed method is achieved for PKA analysis, showing great potential in PKs-related disease diagnosis and drug discovery.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (20925519, 91127035, 21335005) and Program for Changjiang Scholars and Innovative Research Team in University (IRT 1124).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra00470a

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