Enzymatic Hydrogelation-Induced Fluorescence Turn-Off for Sensing

Letter pubs.acs.org/ac

Enzymatic Hydrogelation-Induced Fluorescence Turn-Off for Sensing Alkaline Phosphatase in Vitro and in Living Cells Ling Dong,†,‡ Qingqing Miao,† Zijuan Hai,† Yue Yuan,† and Gaolin Liang*,† †

CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China ‡ Department of Chemistry and Chemical Engineering, Hefei Normal University, Hefei, Anhui 230061, China S Supporting Information *

ABSTRACT: Alkaline phosphatase (ALP)-catalyzed hydrogelation has been extensively explored and found wide applications. Spectroscopic and electrochemical approaches are commonly employed for the detection of ALP activity. Herein, by rational design of a fluorescence probe FmocK(FITC)FFYp (P1) (where FITC is fluorescein), we incorporated sol−gel transition with fluorescence “turn-off” and developed a new method for quantitative sensing ALP activity in vitro and in living cells. Under the catalysis of ALP, P1 was converted to hydrogelator Fmoc-K(FITC)FFY (1) which self-assembles into nanofibers to form Gel I. Accompanying this sol−gel transition, the fluorescence emission of P1 was turned off. Our assay was employed to detect ALP activity over the range of 0−2.8 U/mL with a limit of detection (LOD) of 0.06 U/mL. ALP-inhibitor-treated cell imaging indicated that P1 could be applied for sensing ALP activity in living cells. Our method provides a new option for real time and quantitative sensing ALP activity in vitro and even in living cells.

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applications in inhibitor screening, drug delivery, control of cell fate, metal ion detection, etc.16,21−26 In turn, people could use this enzymatic sol−gel transition for ALP activity detection with the naked eyes.27 However, to the best of our knowledge, there has been no report on incorporating fluorescence with hydrogelation for sensing ALP activity in vitro and in living cells. Inspired by these pioneering studies mentioned above, as shown in Figure 1, we rationally designed a fluorescein (FITC)based precursor Fmoc-K(FITC)FFYp (P1). Dephosphoryla-

lkaline phosphatase (ALP), the important enzyme of the antagonistic phosphatase/kinase pair which is responsible for the dephosphorylation process of proteins, nucleic acids, or small molecules in biological organisms, can be found in a variety of tissues such as intestine, liver, bone, kidney, and placenta.1−4 Disorder of the ALP level leads to the occurrence of several severe diseases such as dynamic bone disease, hepatitis, prostatic cancer, diabetes, etc.5−8 To date, a large number of spectroscopic methods (e.g., colorimetric, fluorometric, and surface-enhanced Raman scattering (SERS)) and electrochemical approaches have been reported for detection of ALP activity.9−14 Due to their operational simplicity and high spatial temporal sensitivity, fluorometric methods using organic dyes,15,16 fluorescent polymers,9,17 inorganic quantum dots (QDs), or noble metal nanoclusters18−20 as indicators for ALP detection have been extensively explored. Besides biomacromolecules, phosphate groups of synthetic small molecules also can be efficiently removed by phosphatases. Dephosphorylation of small molecules by phosphatases results in the decrease of their hydrophilicity and thereby triggers the self-assembly of the amphiphilic products. Supramolecular interactions (e.g., π−π interactions, hydrogen bonding, and charge interactions) among the amphiphilic molecules usually confer the formation of threedimensional (3D) fibrous networks to form supramolecular hydrogels. Compared with vigorous physical or chemical hydrogelation, this phosphatase-catalyzed hydrogelation is mild and more biocompatible and has shown promising © XXXX American Chemical Society

Figure 1. Schematic illustration of fluorescence “turn-off” for sensing ALP activity. Received: May 1, 2015 Accepted: June 23, 2015

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DOI: 10.1021/acs.analchem.5b01657 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry tion of P1 by ALP yields the amphiphilic hydrogelator FmocK(FITC)FFY (1) which self-assembles into nanofibers to form hydrogel Gel I. The “dispersion-to-aggregation” state of FITC accompanying the sol−gel transition is displayed along with a fluorescence “turn-off”, making it possible for naked-eye readout of ALP activity in physiological conditions (i.e., buffers at pH 7.4) or in living cells. We began the study with the synthesis of probe P1, as shown in the Supporting Information (Scheme S1 and Figures S1− S3). After obtaining P1, we used ALP to trigger the hydrogelation and a UV lamp to record the fluorescence “turn-off”. In brief, 10 mg of P1 was well dissolved in 1 mL of phosphate-buffered saline (PBS, pH 7.4) to prepare the 1 wt % clean, transparent, yellow solution (Figure 2A). Excitation of

Figure 3. (A) Dynamic frequency of storage modulus (G′) and the loss modulus (G″) of Gel I at 1 wt %. Rheological measurements were conducted at 25 °C and strain of 10%. (B) TEM image of Gel I at 1 wt %.

the internal networks in Gel I. Figure 3B indicates that Gel I is composed of long and flexible nanofibers with an average diameter of 10 ± 1.8 nm. As described above, under the catalysis of ALP, P1 could be converted to 1 and self-assembled into Gel I, and its fluorescence was efficiently quenched after Gel I formation. After optimizing the detection condition (Figure S7, Supporting Information), we could use P1 for quantitative analysis of ALP activity. 288 μM P1 in Tris-HCl (50 mM, pH 8.0) buffer containing 1 mM MgCl2 was added with different amounts of ALP ranging from 0 to 2.8 U/mL, and their fluorescence spectra were recorded after 3 h of incubation at 37 °C. As shown in Figure 4A, the fluorescence intensity of P1 solutions

Figure 2. Photographic images of P1 solution at 1 wt % in PBS (A) and corresponding Gel I obtained by treating the 1 mL P1 solution with 200 U of ALP and incubation at 37 °C for 12 h (C). Corresponding fluorescent images of the above P1 solution (B) and Gel I (D) under a UV lamp. Excitation: 365 nm.

this P1 solution by 365 nm light from a UV lamp showed very strong green fluorescent emission, as shown in Figure 2B. After addition of 200 units of ALP and 12 h of incubation at 37 °C, the above solution changed to a transparent, yellow hydrogel (Figure 2C). High performance liquid chromatography (HPLC) analysis indicated that 72% of P1 was converted to its dephosphorylation product 1 by ALP under this condition (Figures S4 and S5, Supporting Information). Interestingly, the location of Gel I with the same UV lamp did not show observable green fluorescence as before (Figure 2D). This indicated that the self-assembly of FITC fluorophore in the hydrogel produced the “aggregation-induced-quenching” (AIQ) of its fluorescence, as we proposed. To evaluate the viscoelastic properties of the obtained Gel I, we first used dynamic strain sweep to determine the proper condition for the dynamic frequency sweep. As shown in Figure S6, Supporting Information, for Gel I at 1 wt %, its storage modulus (G′) and loss modulus (G″) values exhibit a weak dependence from 1% to 100% of strain (with G′ dominating G″), indicating that the sample is hydrogel. After setting the strain amplitude at 10% (within the linear response regime of strain amplitude), we used dynamic frequency sweep to study Gel I. As shown in Figure 3A, G′ and G″ values of Gel I increase with the increase of frequency from 0.1 to 10 Hz. The values of G′ are ten times larger than those of G″ in the whole range (0.1−10 Hz), suggesting that the hydrogels are fairly tolerant to external force. After rheology tests, we then conducted transmission electron microscopy (TEM) to study

Figure 4. (A) Fluorescence spectra of P1 (288 μM, λex = 465 nm) in Tris-HCl (50 mM, pH 8.0) buffer in the presence of various concentrations of ALP (0, 0.8, 1.2, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, or 2.8 U/ mL) at 37 °C for 3 h. (B) Fitted calibration curve of the fluorescence intensity at 530 nm in (A) as a function of ALP concentration.

decreases with the increase of ALP concentration. By correlating the fluorescence emission at 530 nm with the concentration of ALP, we obtained a calibration curve for the determination of ALP in vitro. As shown in Figure 4B, a linear relationship between the fluorescence emission at 530 nm and ALP concentration (Y = 1821 − 441 × X, R2 = 0.996) was obtained over the range of 0−2.8 U/mL. The limit of detection B

DOI: 10.1021/acs.analchem.5b01657 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

hydrogelator 1 which self-assembles into nanofibers to form Gel I. Accompanying this sol−gel transition, the fluorescence emission of P1 was turned off. Rheological tests were employed to study the viscoelastic properties of Gel I, and the TEM image indicated that Gel I was composed of fibrous nanostructures with an average diameter of 10 nm. In vitro quantitative analysis indicated that P1 could be used to detect ALP concentrations within a linear range of 0−2.8 U/mL with a LOD of 0.06 U/mL. Cell imaging studies indicated that ALPinhibitor-pretreated LoVo cells have 1.7-fold fluorescence emission of those directly incubated with P1, suggesting P1 could be used for sensing ALP activity in living cells. Our method of incorporating sol−gel transition with fluorescence “turn-off” provides people with a new option for real time sensing ALP activity in vitro and in living cells.

(LOD) of ALP of this assay was 0.06 U/mL (S/N = 3). Timecourse fluorescent spectroscopic analyses indicated that the minimum incubation time of this assay was about 2 h (Figure S8, Supporting Information), which is comparable to those of reported methods (Table S3, Supporting Information). Selectivity study of P1 to ALP over other enzymes (e.g., glucose oxidase, caspase-3, thrombin, or exonuclease) indicated that our assay is specific for ALP detection (Figure S9, Supporting Information). Before applying P1 for cellular imaging, we examined its cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure S10, Supporting Information, 99% of human colorectal cancer LoVo cells survived at P1 concentration of 160 μM for 12 h, suggesting that 80 μM P1 for following cell imaging would not induce cytotoxicity to the cells. To test the stability of P1 for cell culture, we incubated P1 in DMEM medium with (or without) fetal bovine serum (FBS) for 12 h at 37 °C. HPLC traces indicated that P1 was very stable during the observation time (Figure S11, Supporting Information). Interestingly, after incubation of 1 mg of P1 with the cell lysate of 8 × 107 LoVo cells overexpressing ALP for 3 h at 37 °C, we found that P1 was almost converted to 1 which self-assembled into nanofibers (Figure S12, Supporting Information). Encouraged by the data described above, we applied P1 for sensing ALP activity in living cells. As shown in Figure 5, after 3 h of incubation with

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ASSOCIATED CONTENT

* Supporting Information S

Additional experimental details as described in text, selectivity test, stability study, MTT assay, HPLC monitoring of the reactions of P1 with ALP or cell lysate, HR-MS, and NMR spectra. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.analchem.5b01657.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology, Hefei Normal University (2015QN08), and the National Natural Science Foundation of China (Grants 21175122 and 21375121).

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Figure 5. Differential interference contrast (DIC) images (left), fluorescence images (middle, EGFR channel), and merged images (right) of LoVo cells incubated with 80 μM P1 (top row) or LoVo cells pretreated with 0.8 mM L-phenylalanine in serum-free medium for 30 min and then incubated with 80 μM P1 (bottom row) at 37 °C for 3 h and washed with PBS buffer for three times prior to imaging. Scale bar: 20 μm.

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80 μM P1 in serum-free culture medium and washing with PBS for three times, weak fluorescent emission from LoVo cells could be observed. However, when the cells were pretreated with 0.8 mM L-phenylalanine (L-Phe, one of the inhibitors of ALP) for 30 min prior to incubation with P1, we observed a strong fluorescence emission from the cells. This suggests that it is ALP that induces the fluorescence “turn-off” of P1 in LoVo cells. Quantitative analysis indicated that L-Phe-treated LoVo cells have 3.9-fold of fluorescence intensity (FL) over that of background while that of untreated LoVo cells is only 2.3-fold of background (Figure S13, Supporting Information). All these results suggest that P1 could be used for sensing ALP activity in living cells. In summary, by rational design, we developed a fluorescence probe P1 for quantitative sensing ALP activity in vitro and in living cells. Under the catalysis of ALP, P1 was converted to C

DOI: 10.1021/acs.analchem.5b01657 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.5b01657 Anal. Chem. XXXX, XXX, XXX−XXX