Detection of Mercury Ion by Infrared Fluorescent Protein and Its

Feb 16, 2011 - Mercury is a highly hazardous and widespread pollutant with bioaccumulative properties. Novel approaches that meet the criteria of desi...
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Detection of Mercury Ion by Infrared Fluorescent Protein and Its Hydrogel-Based Paper Assay Zhen Gu,*,†,§,^ Muxun Zhao,†,§ Yuewei Sheng,‡ Laurent A. Bentolila,‡,§ and Yi Tang*,†,‡,§ †

Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry and Biochemistry, and §California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California 90095, United States

bS Supporting Information ABSTRACT: Mercury is a highly hazardous and widespread pollutant with bioaccumulative properties. Novel approaches that meet the criteria of desired selectivity, high sensitivity, good biocompatibility, and low background interference in natural settings are continuously being explored. We herein describe a new strategy utilizing the combination of infrared fluorescent protein (IFP) and its chromophore as an infrared fluorescence probe for mercury ion (Hg(II)) detection. Hg(II) has been validated to have specific binding affinity to a cysteine residue (C24) of IFP, thereby inhibiting the conjugation of IFP chromophore biliverdin (BV) to C24 and “turning off” the infrared emission of IFP. The IFP/BV sensor has high selectivity toward Hg(II) among other metal ions over a broad pH range. The in vitro detection limit was determined to be less than 50 nM. As a genetically encoded probe, we demonstrate the IFP/BV sensor can serve as a tool to detect Hg(II) in living organisms or tissues. Moreover, we have exploited a protein-agarose hydrogel-based paper assay to immobilize IFP for detection of Hg(II) in a portable and robust fashion.

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ercury pollution is highly hazardous and widespread, resulting in serious environmental and health issues.1 Mercury ions (Hg(II)) can readily penetrate through biological membranes and cause severe damage to the brain, nervous system, kidneys, heart, and endocrine system.1 Therefore, the exploration of new approaches for detecting Hg(II), particularly those that can be applied to living cells, has attracted intense attention.2 Fluorescence spectroscopy serves as a powerful tool for detecting trace levels of analytes.2,3 To date, a number of fluorescent probes for sensing Hg(II) have been developed, including small molecules,4 synthetic polymers,5 foldamers,6 biomolecules,7 and nanoparticles.8 Despite those advances, creating new strategies that can have selectivity, optical sensitivity, biocompatibility, and low background interference in natural settings remains challenging. Herein, we demonstrate that the infrared fluorescent protein (IFP),9 which is the first genetically encoded infraredemitting probe, can be utilized for Hg(II) detection. An IFPhydrogel-based paper assay has also been developed for probing Hg(II) through a convenient and high-sensitive fashion. IFP is engineered from a bacteriophytochrome (DrBphP)10 with cofactor biliverdin (BV) as the chromophore (Figure 1a). IFP is of particular value to bioimaging, because its emission wavelength of ∼708 nm ensures low interference absorbance and high tissue penetration.11 Neither IFP itself nor BV emits the infrared fluorescence. BV is spontaneously and irreversibly attached to IFP via a thioether linkage between a cysteine residue (C24) and the A-pyrrole ring C3 vinyl side chain of BV.9,10 Upon the covalent attachment to IFP, the D ring of BV is rotated 44 r 2011 American Chemical Society

away from the coplanar A, B, and C rings and is rigidly stabilized by the engineered residues surrounding D ring (Figure 1bi). The rotation of BV leads to a partial decoupling of the π conjugation system,10 and the stabilization confines the nonradiative decay,9 resulting in the emission of the infrared fluorescence from the IFP-BV complex with a quantum yield of 0.07 and an extinction coefficient of >90 000 M-1cm-1 as reported9 (Figure 1bi). Exposed cysteine residues on proteins are known to selectively bind to Hg(II).12 As shown in Figure 1b, we hypothesized and further validated that the competitive binding of Hg(II) to C24 can block BV conjugation and “turn off” the IR signal, a switch that can be useful for Hg(II) detection.

’ EXPERIMENTAL SECTION Materials. The pENTR1A and pcDNA3 plasmids encoding the sequence of IFP1.49 were generously provided by Prof. Roger Y. Tsien (University of California, San Diego, La Jolla, CA). All chemicals were purchased from Sigma-Aldrich unless otherwise specified and were used as received. Biliverdin hydrochloride was purchased from Frontier Scientific. The culture-well chambered cover glass (50-well slide, with a diameter of 3 mm and a depth of 1 mm for each well) for detection assays was purchased from Received: December 12, 2010 Accepted: January 30, 2011 Published: February 16, 2011 2324

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Figure 1. IFP-mediated Hg(II) detection mechanism. (a) Structures of infrared-fluorescent protein (IFP) (simulated on the basis of DrCBD [Protein Data Bank (PDB) ID: 1ztu] by Swiss-model server) and biliverdin IXR (BV). (b) Schematic of the proposed mechanism of detecting Hg(II) by IFP. (c) Electrospray-ionization mass spectral based analysis: (i) IFP only; (ii) IFP added with BV (molar ratio: 1:2); (iii) IFP added with Hg(II) (molar ratio: 1:1); (iv) IFP added with Hg(II) (molar ratio: 1:4); (v) IFP added with Hg(II), and subsequent BV (molar ratio: 1:1:2); (vi) IFP added with a mixture of Hg(II) and BV (molar ratio: 1:1:2); (vii) IFP C24A mutant added with Hg(II) or BV. (See details in Figure S2 and S3, Supporting Information.)

Electron Microscopy Science. The tested metal salts included Pb(NO3)2, Fe(NO3)3, Mn(NO3)2, Cd(NO3)2, Ce(NO3)3, Cd(NO3)3, MgSO4, CuSO4, NiSO4, KCl, CoCl2, PdCl2, CaCl2, ZnCl2, and HgCl2. The deionized water was prepared by a Millipore NanoPure purification system (resistivity higher than 18.2 MΩ 3 cm-1). The filter paper used for the protein-gel-based assay was purchased from Whatman (ashless, diameter: 150 mm). Instruments. Protein concentration was determined by a Thermo Scientific NanoDrop 2000C Spectrophotometer. UV spectra were obtained by a BeckmanCoulter DU640 UV-vis spectrometer. Fluorescence spectra were recorded on Spectrofluorimeter-SPEX. Fluorescence intensity of a 96-well plate was measured by Spectra MAX GEMINI XS (Molecular Devices). SEM images were obtained with JEOL JSM-6700F FE-SEM. Fluorescence images of HEK-293 cells were obtained with a Yokogawa spinning-disk confocal microscope (Solamere Technology Group, Salt Lake City, UT) on a Nikon eclipse Ti-E Microscope equipped with a 60  1.49 Apo TIRF oil objective and a Cascade II: 512 EMCCD camera (Photometrics). The storage modulus and loss modulus of hydrogel were tested using an AR2000 rheometer (TA Instruments, New Castle, DE) under constant strain of 0.05 and frequency from 0.1 to 10 Hz. The pictures of droplets on the substrate of filter paper or gel paper were captured with a CMOS camera together with an illuminator. Far-UV circular dichroism (CD) spectra of protein samples were performed by a JASCO J-715 circular dichroism spectrometer, at 20 C in a buffer containing 100 mM KH2PO4/ K2HPO4, pH 7.4, with 20 μM protein. Determination of Protein Mass. Mass analysis of native proteins or covalent conjugation with BV/Hg(II) was carried out by the size exclusion chromatography-electrospray ionization mass spectrometry (SEC-ESI-MS). Samples were acidified with the addition of formic acid (1/1; v/v) immediately prior to SEC-

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ESI-MS. SEC-ESI-MS was carried out in CHCl3/MeOH/0.1% aqueous formic acid (4/4/1; v/v/v) using a Super SW 2000 column (4.6  300 mm, Tosoh Bioscience, Montgomeryville, PA, USA) at 0.250 mL/min and 40 C. ESI-MS was performed via a triple quadrupole instrument (API III, Applied Biosystems). An m/z 600-2200 range was scanned (positive mode, orifice 90 V, 0.3 Da step size, 5.61 s/scan). Data were processed and analyzed using MacSpec 3.3, Hypermass, and BioMultiview 1.3.1 software (Applied Biosystems). Calculated molecular weights were obtained by ProtParam at the proteomic server of the Swiss Institute of Bioinformatics (http://ca.expasy.org/). Infrared Fluorescence Images. Infrared fluorescent images shown in Figures 2a, 4a, and 5c,d,h,i were taken using the Maestro 2 In Vivo imaging system (CRI Inc., Woburn, MA; excitation filter: 616-661 nm, emission filter: 675 nm long pass). The detection was set to capture images automatically at 10 nm increments from 670 to 900 nm. The resulting TIFF spectral image cube was analyzed in the vendor’s software. The quantitative fluorescence intensity was analyzed by AxioVs 40 V 4.8.1.0 (Carlzeiss Imaging Solutions).

’ RESULTS AND DISCUSSION IFP was expressed and purified from Escherichia coli cells (Supporting Information, Figure S1). Analysis of IFP using electrospray mass spectrometry (Figure 1c, see details in Figure S2 and S3, Supporting Information) confirmed that, upon addition of 1.0 mol equiv of Hg(II), an increase in mass ∼200 Da that corresponds to the addition of one mercury atom was observed. The 200 Da mass increase can be observed when mercury was added to 4 mol equiv. The addition of Hg(II) did not change the secondary structure of IFP, as shown in Figure S4, Supporting Information. Whereas addition of BV to IFP resulted in the increase in mass of 580 Da, addition of BV to Hg(II)modified IFP no longer led to detectable BV-IFP complexes. When BV and Hg(II) were coadministered to IFP, only the Hg(II)-modified mass signals were detected. To confirm the covalent addition of both BV and Hg(II) were to the same C24 cysteine thiol, we expressed a mutant IFP that harbors the C24A mutation. As expected, no binding of either BV or Hg(II) toward IFP was detected (Figure 1c). Taken together, we conclude that Hg(II) specifically binds to C24 of IFP, which irreversibly inhibits formation of the fluorescent IFP-BV adduct. We then tested the detection scheme in Figure 1b and evaluated the selectivity of the IFP-based fluorescent probe toward Hg(II) in the presence of other metal ions. Various metal ions, including alkaline ions (K(I), Ca(II), and Mg(II)), transition-metal ions (Mn(II), Pd(II), Cr(III), Ce(III), Zn(II), Fe(II), Ni(II), Cd(II), Co(II), Cu(II), and Hg(II)), and the common heavy-metal contaminant Pb(II) were added into the same amount of IFP solution (pH = 7.4, Sorensen’s phosphate buffer, 0.1 M), followed with the addition of BV. As is shown in Figure 2a, in the absence of BV, no infrared fluorescence can be detected (Supporting Information), while bright emission was acquired with the addition of 6.0 μM BV for 10 min. The concentration and incubation time of the assay were then optimized at a saturated level of photoluminescence (PL) intensity (Figures S5, S6, and S7, Supporting Information). As hypothesized, no infrared emission can be found in the presence of Hg(II) compared to samples containing other metal ions. It is reported9 that the fluorescence of IFP is stable over a wide pH range from 5 to 9. We, therefore, quantitatively assessed IFP detection of 2325

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Figure 2. Detection selectivity of the IFP sensor. (a) Selectivity assay imaged using the Maestro 2 imaging system: each well (diameter: 3 mm) contains 7 μL of sample of 3.2 μM various metal ions together with 3.0 μM IFP and 6.0 μM BV (excitation filter: 616-661 nm, emission filter: 675 nm long pass). Blank: PBS only. (b) The normalized fluorescence intensity via fluorescence spectrometer for 3.2 μM various metal ion samples with 3.0 μM IFP and 6.0 μM BV at different pH values of 5.2, 7.4, and 8.8.

Figure 3. Detection sensitivity of the IFP sensor. (a) Fluorescence response of 3.0 μM IFP to the addition of Hg(II) at 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 1000, 1200, 1400, 1600, 2000, 2400, 3200, and 4000 nM and followed the addition with 6.0 μM BV. Excitation wavelength: 675 nm. (b) Stern-Volmer emission intensity “turn-off” plots for samples with different addition orders of BV and Hg(II). The fluorescence intensity was monitored at 705 nm.

Hg(II)-containing samples in buffer solutions at different pH values of 5.2, 7.4, and 8.8 by fluorescence spectrometry. As shown in Figure 2b, inhibition of IFP fluorescence by Hg(II) can be observed over a broad pH range. To assess the sensitivity of the IFP-BV complex toward Hg(II), the emission spectra of IFP-BV in the presence of different amounts of Hg(II) was performed at pH 7.4. Figure 3a shows a drastic “turn-off” effect on the fluorescence intensity of IFP-BV combination upon increasing the concentration of Hg(II) and subsequently adding BV. Up to >98%, attenuation of fluorescence was observed following the addition of Hg(II) at 3.2 μM, which is consistent with the 1:1 stoichiometry. To further quantitatively assess the sensitivity, the Stern-Volmer equation was applied as: PL0/PL = 1 þ Ksv[Hg], where PL0 is the steadystate emission intensity of IFP-BV in the absence of Hg(II) and PL is the emission intensities monitored at 705 nm in the presence of Hg(II). The Stern-Volmer constant (Ksv) provides a direct characterization of the “turn-off” efficiency of the complex. Interestingly, Figure 3b shows that two linear response

ranges from 0 to 300 nM and 600 to 2400 nM can be observed. As suggested for some polymer-based quenching systems,13 such nonmonotonic linear regions and the concentration-dependent Ksv can be attributed to the formation of protein aggregates or deformation, which result in amplification of the quenching. However, no precipitates were observed in IFP samples treated with different concentrations of Hg(II) during our measurements. The values of Ksv of 4.3  105 M-1 and 6.4  106 M-1 indicate an efficient fluorescence “turn-off” response can be obtained (Figure 3b). The detection limit was determined to be 50 nM, which can be further decreased to sub-10 nM by correspondingly decreasing the original concentration of IFP and adjusting the concentration of BV (Figure S8, Supporting Information). Moreover, as displayed in Figure 3b, when Hg(II) and BV are added together through a prepared mixture, a similar plot showing identical values of Ksv and sensitivity can be achieved, validating the Hg(II) has preferential binding ability to the active site(s) compared to BV. In contrast, when BV is added first for 20 min and then Hg(II) is added, it shows a 2326

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Figure 4. Intracellular detection using IFP. (a) Escherichia coli-based biofilm expressing IFP formed on the 35 mm agar plate (i) and character “A” written by 10.0 μM BV on biofilms treated with Hg(II) with different concentrations (ii: control; iii: 4.0 μM; iii: 40.0 μM) and then imaged by Maestro 2 imaging system. (b) Confocal microscopic bright field and fluorescence images of HEK-293 cells transfected with pcDNA3 encoding IFP and added with BV (10.0 μM) in absence of Hg(II) or with Hg(II) (32 μM). (c) Quantitative analysis of fluorescence intensity (at 705 nm) of transfected HEK-293 cells treated with different concentrations of Hg(II) and subsequent addition of BV (10.0 μM). PL intensity was measured by a 96-well plate reader and normalized to that of control samples. For all data points, n g 3.

distinctly inferior fluorescence “turn-off” efficiency (Figure 3b). The Ksv over the range of 0-3.2 μM is 3.3  105 M-1, about 20fold lower than those under previously discussed assay conditions. These observations are consistent with the competitive binding between BV and Hg (II) to the IFP active site cysteine (Figure S2, Supporting Information). Encouraged by the high selectivity and sensitivity of IFP-based sensor toward Hg(II), we further extended the detection capabilities of this infrared probe toward bacteria and mammalian cells. E. Coli-based biofilm was obtained by collecting BL21(DE3) colonies transformed with IFP expression plasmid and evenly spreading on fresh media (Figure 4ai). After inducing IFP expression by 0.05 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 2 h, the biofilms were treated with Hg(II) at different concentrations. Five minutes thereafter, the letter “A” was evenly “written” onto the surface of the biofilm using a pipet with 10 μL of BV solution (10.0 μM). The otherwise latent “A” can be decoded through the imaging system with an excitation wavelength of 675 nm (Figure 4aii). The average infrared intensity was clearly attenuated in the presence of Hg(II), as shown in Figure 4aiii,iv, where the concentrations of Hg(II) were 4 and 40 μM, respectively. During the imaging process, treated bacteria appeared to survive and no apparent toxicity was observed through transmission light microscopy. Furthermore, HEK-293 cells were transfected with pcDNA39 encoding IFP via FuGENE for 24 h. After a thorough wash, cells were incubated with Hg(II) in serum-free medium for 5 min and then incubated with BV for 10 min before confocal imaging. Compared with the control sample in the absence of Hg(II), a prominent decrease in the infrared emission was observed for cells treated with 32 μM Hg(II), as shown in Figure 4b. The steady “turn-off” trend of the emission intensity can be quantitatively recorded by a fluorescence plate reader (Figure 4c). With the addition of 32 μM Hg(II), the infrared intensity of the treated cells decreased to 20% of the control sample. The Hg(II) detection sensitivity in

living cells can be affected by other free thiol-containing compounds, such as glutathione (GSH). Figure S9 (Supporting Information) demonstrates that, with the addition of GSH to the IFP solution, the “turn off” efficiency was dramatically inhibited due to the competition binding of GSH to Hg(II). Nevertheless, the detection limit can be further optimized by adjusting the IFP expression level. Collectively, these experiments indicate the genetically encoded IFP probe may be useful for reporting Hg(II) levels in living organisms. We next developed a straightforward, portable IFP-hydrogelbased paper assay to detect Hg(II). Paper strips have been used in chemical/biomedical assays for decades, because they offer a low-cost, lightweight, and disposable platform.14 However, protein-containing paper substrates for analytical and diagnostic applications remain elusive due to difficulty in immobilization and stabilization of protein onto papers. Inspired by our previous study on hydrogel-based protein patterning and encapsulation,15 we proposed the idea of forming IFP-encapsulated hydrogel film on the paper surface through the interaction of fiber and gel structure. To accomplish this, the thermo-reversible hydrogel agarose, of which the gel network is stabilized by bound water molecules,16 was applied. The boiling agarose aqueous solution (0.75%) was gradually cooled down to 37 C and then fully mixed with IFP solution (19/1; v/v). The IFP-agarose solution (Figure 5b) incubated in a 35 C water bath was then transferred to the desired areas of a filter paper, printed with “control” (“C”) and “sample” (“S”) tagged circles to confine a small volume of samples (Supporting Information). IFP hydrogel was then formed upon further cooling to room temperature (Figure 5c). The storage modulus and loss modulus of hydrogel with IFP was retained similarly to the free agarose gel (Figure S10, Supporting Information). When permeated with BV, uniform infrared emission can be detected, indicating the activity of IFP is retained in the encapsulation process. Moreover, compared with IFP in PBS solution, the agarose gel containing the same concentration 2327

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Figure 5. Protein-gel-based paper assay. (a) Schematic of the fabrication process. (b) A picture of 3.0 μM IFP in agarose solution at 35 C (scale bar: 1 cm). (c) Agarose solution with IFP after gelation. Left: optical picture of IFP hydrogel in the absence of BV and with the addition of 2 equiv of BV; the arrow indicates the diffusion direction of BV; right: infrared fluorescence image of hydrogel samples as shown in the left. (d) Fluorescence image of 3.0 μM IFP in PBS solution (i) or in agarose gel (ii) incubated with 6.0 μM BV after incubation at 37 C for different durations; the figure below shows quantitative emission intensity. (e) Demonstration of the printed filter paper for the IFP-gel-based assay for detection of Hg(II) (scale bar: 1 cm). (f) SEM image of the boundary of IFP-agarose gel film on the filter paper (scale bar: 50 μm). (g) Comparison of one PBS droplet (2 μL) absorbed by filter paper (left) or agarose film covered filter paper (right). (h) The sensitivity of IFP-gel-based paper assay. Each droplet contained 3 μL of agarose gel with 1.5 μM IFP. Two μL test samples with different conc. Hg(II) were added to the tagged area with IFP-gel droplet. Two μL of 4.0 μM BV was subsequently added as an indicator. Fluorescence images of test papers were parallelly recorded, and the intensity of each dot was analyzed as the inset red curves (scale bar: 1 cm). (i) The sample with a concentration of 20 nM Hg(II) can be detected by continuous additions of the test sample for enrichment. Left: before enrichment, with 2 μL sample ; right: after enrichment, with 12 μL (6  2 μL) sample.

of IFP showed only a slight decrease in emission intensity and maintained consistent stability over 4 days (Figure 5d). The SEM image displaying the boundary of the filter paper fiber and hydrogel film suggests that the hydrogel moiety can be compactly integrated with paper fibers (Figures 5f and S11, Supporting Information). Interestingly, in contrast to native filter paper, the hydrogel coated region presents a relatively “slow” water absorption area (Figure 5g), which may facilitate the interaction between the immobilized protein and added BV or Hg(II). To demonstrate the sensitivity of the paper assay for Hg(II) detection, Hg(II) solutions with various concentrations were added to the “S” area coated with IFP-hydrogel, and BV was then added to both “C” and “S”. Parallel with control samples, with an increase of the addition amount of Hg(II) (original concentrations were at 200, 600, and 1200 nM), a corresponding decrease of emission intensity ratios (sample intensity/control intensity) were clearly observed, as shown in Figure 5h. More importantly, when the water absorbing nature of the paper strip is taken advantage of, samples can be readily enriched via multiple addition/drying steps. As it is shown in Figure 5i, the sample with an original Hg (II) concentration of 20 nM can be detected through continuous enrichment with 6 the sample volume.

detection limit can be determined as sub-50 nM. We also developed a protein-hydrogel-based paper assay to immobilize IFP onto the filter paper strips for detection of Hg(II) ions. This method provides a convenient method to immobilize proteins/ enzymes onto paper strips for detection purposes. Meltinghydrogel droplets with proteins can be directly printed onto paper support for high-throughput fabrication and detection. This strategy may be useful in applications where portability, low cost, low volumes of samples, and sensor robustness are required.

’ ASSOCIATED CONTENT

bS

Supporting Information. Complete experimental details and spectroscopic information. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (Z.G.); [email protected] (Y.T.). Fax: (þ1) 310-206-4107 (Y.T.). Present Addresses ^

’ CONCLUSION In summary, we utilize the combination of IFP and its chromophore BV as an infrared fluorescence probe for Hg(II) detection. The detection wavelength is in the infrared range with the merits of low interference background and high tissue penetration. The probe has a favorable selectivity toward Hg(II) over a broad pH range among other metal ions. The in vitro

Department of Chemical Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, United States.

’ ACKNOWLEDGMENT This work was supported by a David and Lucile Packard Foundation grant to Y.T. Fluorescence imaging was performed 2328

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Analytical Chemistry on a Maestro 2 in vivo imaging system at the Advanced Light Microscopy/Spectroscopy Shared Resource Facility at CNSI. We thank Prof. R. Y. Tsien for pENTR1A and pcDNA3 with IFP1.4, Prof. J. P. Whitelegge and Dr. C. M. Ryan for assistance with the mass spectrometry, Dr. K. Joo for the confocal microscopy, and Ms. Y. Li and Mr. S. Chen for assistance with data analysis.

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