Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To

Jul 27, 2016 - Despite the promising improvements made recently on fluorescence probes for the detection of enzymes and reactive small molecules, two ...
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Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity Yan-Syun Zeng,† Ruo-Cing Gao,† Ting-Wei Wu,† Chien Cho,† and Kui-Thong Tan*,†,‡ †

Department of Chemistry, and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Sec. 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC S Supporting Information *

ABSTRACT: Despite the promising improvements made recently on fluorescence probes for the detection of enzymes and reactive small molecules, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag protein and three environment-sensitive fluorescence probes to overcome these two problems. The probes were constructed by following the current probe design for enzymes and reactive small molecules but with an additional benzylguanine moiety for selective SNAP-tag conjugation. The SNAP-tag conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma, whereas analyte concentrations were overestimated up to 700-fold when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically, as our probes displayed 390-fold fluorescence enhancement upon SNAP-tag conjugation, in stark contrast to the weak fluorescence of the free probes in aqueous solutions. Compared with the conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcame many key issues such as dye leakage, long preparation steps, inconsistent dye−host ratios, difficulty in constructing in situ in a complex medium, and limited application to detect only small metabolites.

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INTRODUCTION

most of the cases, the real signals generated by the target are masked by nonspecific fluorescence signal. Depending on the type of fluorescent dyes and macromolecules, fluorescence caused by dye−macromolecule interaction can be very complex, and in most of the cases, it results in fluorescence increase and spectra shift. Many dyes such as fluorescein, rhodamine, cyanine, and ethidium bromide can display fluorescence enhancement upon binding with proteins and DNA in plasma and intracellular environments.20−22 Among them, environment-sensitive fluorophores exhibit the most significant fluorescence increase upon binding with macromolecules. Environment-sensitive dyes are sensitive to their environments due to a longer lifetime and large dipole moments at the excited state.23,24 Typically, they display weaker emission in polar solvents but stronger emission in nonpolar

Analyte-responsive fluorescence probes are important tools in basic biology research and medical diagnosis because they allow for the rapid and sensitive detection of target molecules.1−3 Currently, many fluorescence probes have been developed for the detection of proteins,4−7 reactive small molecules,8−10 metal ions,11−13 etc.14−16 Although most fluorescent probes show excellent performance in clean buffers, they are easily foiled by nonspecific signals in complex contaminant-ridden “real” samples, predominantly due to the nonspecific binding of fluorescent dyes with other macromolecules.17−19 This problem is most severe when the probes are applied in samples containing high protein levels, such as blood plasma and intracellular environments. As the detection of analyte with fluorescence activation probes is normally performed in homogeneous solutions for rapid and convenient analysis, the elimination of nonspecific binding by extensive washing (such as in heterogeneous assays) is not feasible. Unfortunately, in © XXXX American Chemical Society

Received: June 7, 2016 Revised: July 19, 2016

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DOI: 10.1021/acs.bioconjchem.6b00290 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry organic solvents and viscous environments. Representative fluorophores include Nile red, NBD, and dansyl, which are also effective probes for protein polarity. Many of these environment-sensitive dyes have high binding affinity with plasma proteins, e.g., albumin, and produce large fluorescence increase upon binding.25−27 Due to these two fundamental problems, it is extremely difficult for probes based on environment-sensitive dyes to provide accurate and reliable information about analyte concentrations in complex samples. Although many previous nanosensor strategies have attempted to prevent nonspecific dye−proteins interaction by incorporating fluorescent dyes into polymers, liposomes, and nanoparticles,28,29 these strategies met many problems such as dye leakage, long preparation steps, inconsistent dye−host ratios, difficulty in constructing in situ in complex medium, and limitation to sensing small metabolites, which have hampered their useful applications in real samples. Furthermore, the dye loading density must be optimized to avoid self-quenching which is typically observed for most organic dyes at high concentrations or upon aggregation. The key issues behind all these problems is that nanoparticles and polymers do not have specific reaction or binding domains that can interact rapidly, specifically, and stoichiometrically with functionalized fluorescent probes. To date, nonspecific signals generated in protein rich samples still remain the most difficult problem to be solved for fluorescent probes. Herein, we describe a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAPtag protein and three environment-sensitive fluorescence probes to eliminate nonspecific fluorescence and increase detection sensitivity (Figure 1a). Although SNAP-tag and many other self-labeling proteins have been used to conjugate with fluorescent probes for analyte detection, all of them employed these proteins as localization tags for living cells experiments.13,30−32 In our FPEPC approach, SNAP-tag acts as a protein shield to block out nonspecific interactions and enhance fluorescent signals for the detection in blood plasma. The construct of the probes are based on the common fluorescence probes design for enzymes and reactive small molecules,33 but with an additional O6-benzylguanine (BG) moiety introduced on the probes for selective SNAP-tag conjugation. The SNAP-tag protein is well-known for its quantitative, rapid, and specific covalent reaction with BG derivatives even in highly complex environments.34 Upon the reaction of BG moiety with SNAP-tag, the probe is transferred to SNAP-tag and covalently encapsulated inside the protein interior to cut off its contact with other proteins. As the SNAPtag interior is hydrophobic,35 this strategy also enables fluorescence amplification of the environment-sensitive probe after it is activated by the target analyte. In this approach, fluorescence probes based on three different environment-sensitive dyes, 4-sulfamonyl-7-aminobenzoxadiazole (SBD),36,37 naphthalimide (NAPH),38 and αcyanocinnamic acid (CCA),39 were constructed to demonstrate the protein-shield principle (Figure 1b). These dyes exhibit weak fluorescence in aqueous solution and are susceptible to large nonspecific fluorescence increase upon nonspecific binding with proteins in complex samples. As a proof-ofprinciple, these fluorescence probes were constructed for the quantitative determination of two biologically important targets in blood plasma, nitroreductase (NTRase) and hydrogen sulfide (H2S). NTRase is highly expressed in hypoxic tumors,40,41 while H2S is an important molecule in cell

Figure 1. (a) Schematic illustration of our FPEPC strategy based on SNAP-tag protein for fluorescence amplification and nonspecific signal suppression of fluorescence probes. Two schemes are shown for the detection of analytes of different molecular sizes. Scheme i is used for the detection of enzymes, while scheme ii is employed for the analysis of small reactive molecules. (b) Chemical structures of environmentsensitive fluorescence probes for the demonstration of SNAP-tag based protein-shield strategy. BGSBD-NO2 is for NTRase, and BGSBD-N3, BGNAPH-N3, and BGCCA-N3 are for H2S. BGSBD, BGNAPH, and BGCCA are products of the corresponding reactions.

signaling pathways.42,43 Currently, H2S concentrations in human blood plasma remain very controversial. Some papers reported that H2S concentration in human blood plasma is between 30 and 300 μM, while others stated that H2S concentration should be in subnanomolar range.44 The fluorescence specific detection of NTRase and H2S by our FPEPC approach is simple and rapid, as separation and washing to remove nonspecific binding are not required.

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RESULTS AND DISCUSSIONS Fluorescence Amplified Detection of NTRase with Environment-Sensitive Fluorescence Probe BGSBD-NO2 and SNAP-Tag. To show that the protein encapsulation approach can be used for fluorescence amplification and nonspecific signal elimination in complex samples, we first constructed fluorescence probe BGSBD-NO2 for the quantitative analysis of NTRase in blood plasma (Figure 1b). The probe which is based on an environment-sensitive SBD fluorophore, contains a BG moiety for SNAP-tag conjugation and a nitro moiety for recognition and sensing NTRase. Like many fluorescence probe designs based on push−pull fluorophores, the introduction of an electron-withdrawing carbamate functional group onto SBD sets BGSBD-NO2 to be in its fluorescence inactive “OFF” state. NTRase reduces the nitro group to amine which concomitantly triggers the removal of the carbamate group to activate the probe to form fluorescence “ON” BGSBD (Figure S1 in Supporting Information).45 B

DOI: 10.1021/acs.bioconjchem.6b00290 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry For the analysis of NTRase, BGSBD-NO2 was first reacted with NTRase. Subsequently, SNAP-tag was added for fluorescence amplification and nonspecific signal suppression. In its fluorescence “OFF” form, BGSBD-NO2 exhibited extremely weak fluorescence in aqueous buffer (Figure 2a).

conjugation of SNAP-tag to BGSBD. In contrast, the SNAP-tag conjugate BGSBD-NO2 displayed very weak fluorescence which indicates that the SNAP-tag hydrophobic interior only amplifies the fluorescence-active dye. The result is consistent with the fluorescence spectra measurement of the two probes in different solvents in which BGSBD displays strong fluorescence in ACN, DMSO, and MeOH, while BGSBD-NO2 shows only weak emission in those hydrophobic solvents (Figure S2). To obtain high fluorescence amplification, it is essential that SNAPtag is added to the sample only after BGSBD-NO2 has been reacted with NTRase (Figure S3). When BGSBD-NO2 is initially conjugated with SNAP-tag, the probe is buried inside the protein interior which hinders the reaction of the nitro moiety with the NTRase. The reduction of BGSBD-NO2 by NTRase to form BGSBD was confirmed by HPLC trace studies (Figure S4). When NTRase was added in increasing concentrations, the fluorescence which is amplified by SNAPtag shows a linear and concentration dependent enhancement (Figure 2b and inset). With 10 μM BGSBD-NO2 and 12.5 μM SNAP-tag, the limit of detection (LOD) to detect NTRase was estimated to be as low as 1 ng/mL. When BGSBD-NO2 was not labeled to SNAP-tag, the LODs for the detection of NTRase were estimated to be around 32 ng/mL. SNAP-Tag Encapsulation To Eliminate Nonspecific Fluorescence Signal in Blood Plasma. After successfully demonstrating that SNAP-tag can amplify the weak fluorescence of SBD dye, we proceeded to investigate whether the crowded protein interior can be used to block the nonspecific binding of SBD dye and eliminate nonspecific signals. We first studied the fluorescence intensity of BGSBD-NO2 and BGSBD in the absence and presence of SNAP-tag in 25% of fetal bovine serum (FBS) and human blood plasma. The samples contain about 13 200 μg/mL (25% FBS) and 21 500 μg/mL (25% plasma) total proteins as determined by BCA assay. Under such high protein concentrations, BGSBD-NO2 did not show any significant fluorescence increase as compared to the fluorescence intensity in Tris buffer (Figure 3a). In contrast, the product BGSBD exhibited dramatic fluorescence increase due to nonspecific binding in FBS and plasma. These results are consistent with the push−pull characteristics of BGSBD-NO2, whereby the introduction of an electron-withdrawing carbamate group switches the dye to its fluorescently inactive form. For fluorescence-inactive probes, nonspecific binding alone cannot turn on the fluorescence. Subsequently, we studied how the SNAP-tag encapsulation can eliminate the nonspecific fluorescence signals. The fluorescence spectra of SNAP-tag conjugated BGSBD-NO2 and BGSBD were recorded in the 25% FBS and blood plasma (Figure 3a). Weak fluorescence was also observed for SNAP-tag conjugated BGSBD-NO2. However, in contrast to the free BGSBD which shows significant nonspecific signal in FBS and plasma, SNAP-tag conjugated BGSBD exhibited no change in fluorescence intensity in Tris buffer, FBS, and human blood plasma. Even in 90% FBS and plasma, SNAP-tag conjugated BGSBD displayed only slight fluorescence decrease (about 20%), which can be attributed to the self-absorption quenching by the high concentration of proteins (Figure S5a). Furthermore, the ability of SNAP-tag to suppress nonspecific binding of fluorescence probes was also studied in high concentrations of human serum albumin (HSA) and bovine serum albumin (BSA). In 250 μM HSA and BSA mixtures, free BGSBD exhibited large fluorescence increase while the SNAPtag conjugated BGSBD showed no change in fluorescence

Figure 2. Fluorescence amplified detection of NTRase with BGSBDNO2 and SNAP-tag. (a) Fluorescence spectra of BGSBD-NO2 in the absence and presence of NTRase and SNAP-tag. The inset shows the image of the solution in a cuvette before (left) and after SNAP-tag addition for fluorescence amplification (right) under a 405 nm laser. The fluorescence spectra of BGSBD-NO2 with or without NTRase and SNAP-tag conjugated BGSBD-NO2 were magnified by 5 times. Detection conditions were the following. A microcentrifuge tube containing 10 μM BGSBD-NO2 was incubated with 500 μM NADH and 1.5 μg/mL NTRase in pH 7.4 Tris buffer at 37 °C for 1 h. 12.5 μM SNAP-tag was then added and incubated for another 0.5 h. λex = 440 nm. The fluorescence turn-on ratio was calculated from the relative fluorescence intensity of BGSBD-NO2 at λem = 516 nm. (b) Fluorescence spectra of 10 μM BGSBD-NO2 with increasing concentrations of NTRase and 500 μM NADH in Tris buffer. The fluorescence was amplified with 12.5 μM SNAP-tag. The inset (y = 4737x + 157, R2 = 0.995) shows that the fluorescence response was linear in the range of 0−2 μg/mL NTRase with the LOD of about 1 ng/mL NTRase.

Upon enzymatic reaction with 1.5 μg/mL NTRase in Tris buffer for 1 h at 37 °C, the fluorescence of BGSBD remained very weak (ε = 13 000 cm−1 M−1, ϕ = 0.0008) and only showed a slightly higher intensity than BGSBD-NO2. The addition of SNAP-tag protein to the reaction mixture enhanced the fluorescence dramatically with a high turn-on ratio of 390fold (ϕ = 0.1430) as compared with BGSBD-NO2. The fluorescence enhancement by SNAP-tag was so significant that it can be observed easily with the naked eye (Figure 2a, inset). The dramatic fluorescence increase was solely due to the C

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Figure 3. (a) Fluorescence intensity of 10 μM BGSBD-NO2, BGSBD, SNAP-tag conjugated BGSBD-NO2, and BGSBD in Tris buffer, 25% FBS, and 25% human blood plasma. Analysis conditions were the following. In the absence or presence of 12.5 μM SNAP-tag, 10 μM probes were added to Tris buffer, 25% plasma, and FBS samples (v/v, diluted with Tris buffer). Fluorescence spectra were recorded after the mixtures were incubated at 37 °C for 30 min. Error bars were calculated from three independent measurements. Shown are fluorescence spectra of 1.0 μg/mL NTRase in Tris buffer and 10% plasma as analyzed by (b) 10 μM BGSBD-NO2 and 12.5 μM SNAP-tag and (c) 10 μM free BGSBD-NO2. The insets in the figure (b, y = 4080x + 2, R2 = 0.996) and (c, y = 159x − 14, R2 = 0.975) show the fluorescence intensity (red triangles) of 1 μg/mL NTRase in 10% plasma and their interpolation/extrapolation to the calibration curve created in Tris buffer in the presence or absence of SNAP-tag protein. In all the measurements, the fluorescence intensity was obtained by subtracting the FBS and plasma background fluorescence from the original spectra.

intensity as compared to the measurement performed in Tris buffer (Figure S5b). Molecular modeling of SNAP-tag-BGSBD complex shows that the probe is surrounded by amino acid residues of SNAP-tag which hinder the nonspecific interaction of probe with other proteins (Figure S6). Together, these results indicate that the BG reaction domain of SNAP-tag is sufficiently crowded to block out nonspecific protein binding which in turn led to the elimination of nonspecific fluorescence. The labeling efficiency of SNAP-tag to the probes in FBS and blood plasma was also studied, and it was found that SNAP-tag can achieve quantitative conjugation with the probes in the samples (Figure S7). Due to the rapid, specific and stoichiometric reaction of SNAP-tag and BG-probe, an excess of 0.25 equiv of SNAP-tag protein to probe is enough to encapsulate all the probes. Finally, we studied the potential of this FPEPC strategy to quantify NTRase in complex mediums by performing a recovery experiment. When 1 μg/mL NTRase was spiked to 10% blood plasma (ratio of plasma proteins to NTRase is about 8000 fold, w/w), we observed similar fluorescence spectra as when the measurements were performed in Tris buffer (Figure 3b). Interpolating the fluorescence intensity obtained in plasma samples to the calibration curve constructed in Tris buffer gave percent recovery of 84%, 87%, and 88% for three different samples (Figure 3b, inset). The slightly lower recovery ratio obtained is most likely due to matrix interference that resulted

in a slower reaction rate of NTRase and BGSBD-NO2 in plasma than Tris buffer. The slightly lower recovery ratio may be due to a lower enzymatic activity of NTRase in blood plasma as compared to the optimized buffer conditions. In comparison, when SNAP-tag was not added into the plasma samples to suppress nonspecific binding, large differences in fluorescence intensity were obtained between the measurements performed in plasma and in Tris buffer (Figure 3c). In this case, the percent recovery was calculated to be about 1000% (Figure 3c, inset). These results indicate that protein encapsulation is essential in protein rich samples to prevent overestimation of the analyte due to an increase in nonspecific fluorescence. By use of BGSBD-NO2 and SNAP-tag, the fluorescence response of NTRase in blood plasma is linear and concentration dependent with a LOD of about 11 ng/mL (Figure S8). Detection of H2S in Blood Plasma by EnvironmentSensitive Fluorescence Probe BGSBD-N3 and SNAP-Tag Protein. To demonstrate that this FPEPC approach is not limited to the quantitative detection of enzymes, we have also constructed another probe, BGSBD-N3, to detect H2S in blood plasma (Figure 1b). Similar to the probe for NTRase, reduction of the azide moiety on BGSBD-N3 by H2S provides the amine, which decomposes to para-quinone methide, CO2, and BGSBD. In aqueous PBS buffer, both BGSBD-N3 and its product BGSBD showed extremely weak fluorescence (Figure 4a). However, addition of H2S to SNAP-tag conjugated D

DOI: 10.1021/acs.bioconjchem.6b00290 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. (a) Fluorescence spectra of BGSBD-N3 and SNAP-tag conjugated BGSBD-N3 with H2S. The inset (y = 17x + 775, R2 = 0.998) shows that the fluorescence response was linear in the range of 0−250 μM H2S. The fluorescence spectra of free BGSBD-N3 in the absence or presence of H2S were magnified 5 times. (b) Fluorescence intensity of 10 μM BGSBD-N3 and SNAP-tag conjugated BGSBD-N3 in PBS buffer, 50% FBS, and 50% human blood plasma (v/v, diluted with PBS buffer). Error bars were calculated from three independent measurements. Shown are fluorescence spectra of 50 μM H2S in PBS buffer and 10% plasma as analyzed by (c) 10 μM SNAP-tag conjugated BGSBD-N3 and (d) 10 μM free BGSBD-N3. The insets in the figure (c, y = 20x + 1270, R2 = 0.990) and (d, y = 0.09x + 265, R2 = 0.750) show the fluorescence intensity (red triangles) of 50 μM H2S in 10% plasma and their interpolation/extrapolation to the calibration curve created in PBS buffer in the presence or absence of SNAP-tag protein. In all the measurements, the fluorescence intensity was obtained by subtracting the FBS and plasma background fluorescence from the original spectra.

BGSBD-N3 triggered large fluorescence amplification of around 185-fold as compared to the fluorescence of free BGSBD-N3. Although SNAP-tag conjugated BGSBD-N3 exhibits a higher fluorescence intensity, a low LOD of about 3.3 μM H2S can still be detected (Figure 4a, inset). In the absence of SNAP-tag, the LOD for the detection of H2S was estimated to be around 194 μM. Unlike NTRase detection which requires the probe to be reacted first with the enzyme followed by SNAP-tag addition to obtain high fluorescence amplification ratio, H2S detection with either SNAP-tag conjugated BGSBD-N3 or the two-step procedure (where BGSBD-N3 first reacts with H2S followed by SNAP-tag addition) gave similar fluorescence amplification ratio (Figure S9). This is because the smaller H2S molecule can diffuse into the SNAP-tag interior for the reaction with the azide moiety. The advantage of preconjugating the probe to the protein is that it can prevent unspecific reaction of the azide with other thiols in the macromolecules. The H2S reaction rate with BGSBD-N3-SNAP-tag conjugate was found to be about 3 times longer than the free probe containing the same azide moiety reported previously (Figure S10).46 This is because the H2S molecule needs to diffuse into the SNAP-tag interior prior to the reaction with the azide moiety. We also studied the fluorescence spectra of free BGSBD-N3 and its SNAP-tag conjugated form in 50% FBS and blood plasma samples, respectively (Figure 4b). In its free form,

BGSBD-N3 displayed a 6-fold unspecific fluorescence increase in the complex samples, which can be attributed to the unspecific reaction of azide moiety with various proteins in the samples. In contrast, SNAP-tag conjugated BGSBD-N 3 exhibited no change in fluorescence intensity in aqueous buffer, FBS, and human blood plasma. When 50 μM H2S was spiked to three 10% plasma samples and quantified using SNAP-tag conjugated BGSBD-N3 probe, we obtained highly specific fluorescence signals with the recovery ratios of 96%, 99%, and 94%, respectively, as compared to the standard curve constructed in PBS buffer (Figure 4c). In contrast, the percent recovery for the detection of 50 μM H2S in plasma by the free BGSBD-N3 was about 70 000% (Figure 4d). Fluorescence Amplification and Nonspecific Signal Suppression of NAPH- and CCA-Based Probes by SNAPTag. To further illustrate the generality of this FPEPC strategy, we synthesized two more environment-sensitive fluorescence probes for the detection of H2S. BGNAPH-N3 is based on the naphthalimide dye, while BGCCA-N3 is based on the αcyanocinnamic acid dye. Both dyes also exhibit strong environment-sensitive effects: weak fluorescence in aqueous solution but strong emission in hydrophobic solvents (Figure S11). The design of BGNAPH-N3 and BGCCA-N3 is similar to the previously reported fluorescence probes where the only difference is the additional BG moiety on the probes for the conjugation with SNAP-tag protein.39,46 Both BGNAPH-N3 E

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Figure 5. Fluorescence amplification and nonspecific suppression of NAPH- and CCA-based probes by SNAP-tag protein for H2S detection. (a) Fluorescence response of 500 μM H2S with 10 μM BGNAPH-N3 and SNAP-tag conjugated BGNAPH-N3. λex = 440 nm. The fluorescence turn-on ratio was calculated from the relative fluorescence intensity of BGNAPH-N3 at λem = 530 nm. (b) Fluorescence response of 500 μM H2S with 10 μM BGCCA-N3 and SNAP-tag conjugated BGCCA-N3. λex = 383 nm. The fluorescence turn-on ratio was calculated from the relative fluorescence intensity of BGCCA-N3 at λem = 465 nm. (c) Fluorescence intensity of 10 μM BGNAPH-N3, BGNAPH, SNAP-tag conjugated BGNAPH-N3 and BGNAPH in PBS buffer, 10% FBS, and 10% human blood plasma (v/v, diluted with PBS buffer). Error bars were calculated from three independent measurements. (d) Fluorescence spectra of 25 μM H2S in PBS buffer and 10% plasma as analyzed by 10 μM SNAP-tag conjugated BGNAPH-N3. The inset (y = 117x + 2294, R2 = 0.985) shows the fluorescence intensity (red triangles) of 25 μM H2S in 10% plasma and their interpolation to the calibration curve created in PBS buffer in the presence of SNAP-tag protein. Background fluorescence of 10% plasma was subtracted to obtain the fluorescence spectra.

and BGCCA-N3 showed weak fluorescence in aqueous PBS buffer. Addition of 500 μM H2S led to the fluorescence enhancement of only 7-fold for BGNAPH-N3 and virtually no change in fluorescence for BGCCA-N3 (Figure 5a and Figure 5b). However, when BGNAPH-N3 and BGCCA-N3 were conjugated to SNAP-tag, the fluorescence intensity can be amplified to 31-fold and 8-fold, respectively, as compared to their fluorescence “OFF” forms. The LOD values for the detection of H2S by SNAP-tag conjugated BGNAPH-N3 and BGCCA-N3 are estimated to be around 3.9 μM and 10 μM, respectively (Figure S12). As with the BGSBD-N3 probe, BGNAPH-N3 and its H2S activated fluorescence “ON” product BGNAPH are very susceptible to nonspecific binding. Strong nonspecific fluorescence increase can be observed when the probes were in FBS and plasma (Figure 5c). In contrast, we observed virtually no change in fluorescence intensity when SNAP-tag conjugated BGNAPH-N3 and BGNAPH were added in FBS and plasma samples. When 25 μM H2S was spiked into 10% blood plasma and quantified using SNAP-tag conjugated BGNAPH-N3, we obtained very similar fluorescence spectra as with the detection performed in PBS buffer (Figure 5d). The recovery ratios of 25 μM H2S in three different plasma samples were calculated to be 79%, 80%, and 84%, by interpolating the fluorescence intensity obtained in plasma to the calibration curve determined in PBS buffer (Figure 5d, inset). In comparison, quantitative detection

of H2S in 10% plasma with free BGNAPH-N3 gave a recovery ratio of over 8000% (Figure S13). As for the free BGCCA-N3 probe and its SNAP-tag conjugated form, they gave similar performance as the probes based on SBD and naphthalimide dyes (Figure S14). Together, these results indicate that the SNAP-tag based protein-shield strategy is a very powerful method which not only can specifically amplify weak fluorescence signals of many environmental-sensitive probes but most importantly also is able to suppress the nonspecific signals in high protein level samples.

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CONCLUSIONS Over the years, the development of fluorescence probes for the precise quantification of analyte concentrations in complex samples without high dilution has been a challenging task. One of the reasons is the nonspecific signals caused by the nonspecific binding of the probes with other proteins in the samples. For example, total protein level in human blood is estimated to be around 60−80 g/L, comprising nearly 30 000 distinct proteins,47 many of which, such as serum albumin and globulins, exhibit high binding affinity with many fluorescence dyes. As demonstrated by SNAP-tag protein and three environment-sensitive fluorescence probes for the detection of NTRase and H2S in blood plasma, we have shown that FPEPC strategy can be used for increasing detection sensitivity and eliminating nonspecific signal of environment-sensitive F

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fluorescence probes in samples with high protein content. As compared to other fluorescent probes for NTRase and H2S, our FPEPC method is one the most sensitive probes for the detection of H 2 S and NTRase (Tables S1 and S2). Unfortunately, the method is still not sensitive enough to detect endogenous blood plasma H2S, of which we believe that the concentration should be in the subnanomolar range. Without conjugation to SNAP-tag protein, the quantification results by the free probes led to significant overestimation of NTRase and H2S concentration in blood plasma. This can be effectively rectified by using our protein encapsulation strategy. The ability of SNAP-tag to distinguish and amplify the fluorescently active “ON” products instead of the fluorescently inactive “OFF” probes provides a great advantage to precisely quantify analyte concentration. For example, conjugation of BGSBD-NO2 to SNAP-tag gave only a 3-fold fluorescence increase, while conjugation of SNAP-tag to the fluorescently active product BGSBD displayed fluorescence enhancement of greater than 390-fold. Although fluorescence amplification might not seem to be very critical for many bright fluorescent dyes, e.g., rhodamines and cyanine dyes, the shielding of the dyes in SNAP-tag interior to suppress nonspecific binding that can cause nonspecific signal is a particularly important feature of our method. The protein-shield strategy based on SNAP-tag is highly versatile and modular, as it can be adapted to the current design of fluorescence probes for enzymes and reactive small molecules detection by simply introducing an additional BG moiety on the probes. We believe that this simple proteinshield strategy opens up new opportunities to precisely quantify many important enzymes and reactive small molecules directly in blood samples. Due to the possible proteolytic degradation of SNAP-tag protein in the human body, the application of this method in vivo might require further modification to SNAP-tag (e.g., pegylation) to increase protein stability. With advances in protein delivery and imaging techniques, it might be possible to quantify those analytes in intracellular environments. Currently we are working to expand the scope of this strategy by investigating other proteins that can also be adapted with our protein-shield approach for fluorescence amplification and nonspecific signal suppression of fluorescence probes.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00290.

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Article

Complete experimental methods and additional figures (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Ministry of Science and Technology (Grant 104B0084I5) and Ministry of Education (“Aim for the Top University Plan”, Grant 104N2011E1), Taiwan, ROC, for financial support. G

DOI: 10.1021/acs.bioconjchem.6b00290 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.bioconjchem.6b00290 Bioconjugate Chem. XXXX, XXX, XXX−XXX