Article pubs.acs.org/ac
Steric-Dependent Label-Free and Washing-Free Enzyme Amplified Protein Detection with Dual-Functional Synthetic Probes Chia-Wen Wang,† Wan-Ting Yu,† Hsiu-Ping Lai,† Bing-Yuan Lee,† Ruo-Cing Gao,† and Kui-Thong Tan*,†,‡ †
Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC) Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan (ROC)
‡
S Supporting Information *
ABSTRACT: Enzyme-catalyzed signal amplification with an antibody−enzyme conjugate is commonly employed in many bioanalytical methods to increase assay sensitivity. However, covalent labeling of the enzyme to the antibody, laborious operating procedures, and extensive washing steps are necessary for protein recognition and signal amplification. Herein, we describe a novel label-free and washing-free enzyme-amplified protein detection method by using dual-functional synthetic molecules to impose steric effects upon protein binding. In our approach, protein recognition and signal amplification are modulated by a simple dual-functional synthetic probe which consists of a protein ligand and an inhibitor. In the absence of the target protein, the inhibitor from the dual-functional probe would inhibit the enzyme activity. In contrast, binding of the target protein to the ligand perturbs this enzyme−inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme, thereby activating the enzyme to initiate signal amplification. With this strategy, the fluorescence signal can be amplified to as high as 70-fold. The generality and versatility of this strategy are demonstrated by the rapid, selective, and sensitive detection of four different proteins, avidin, O6-methylguanine DNA methyltransferase (MGMT), SNAP-tag, and lactoferrin, with four different probes.
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tion methods, most studies have focused on the detection of nucleic acids.7−9 In contrast, label-free and washing-free enzyme-amplified protein detection remains scarcely reported. Unlike nucleic acids, which have extremely well-defined complementary strands and robust binding affinity, the complex and diverse protein structures have made the development of a general label-free and washing-free enzyme amplified protein detection method more challenging. Currently, most of the strategies rely on allosteric enzymes,10−13 DNAzymes,14,15 or split-enzymes,16−18 which require either enzyme conformational change or reassembly of the fragmented enzymes upon the target protein binding to activate the enzymes for signal amplification. However, these often require careful design of polypeptide sequences and involve extensive mutations, which have only been applied to a few target proteins. Recently, a strategy based on the electrostatic interaction between the anionic β-galactosidase and positively charged nanoparticles was developed to regulate the enzymatic activity for label-free and washing-free enzyme amplified protein detection.19
nzyme-catalyzed signal amplification is a common technique in many bioanalytical methods (e.g., enzyme linked immunosorbent assay (ELISA) and Western blot) to increase assay sensitivity for the detection of ultralow concentrations of biomolecules.1 The advantages of enzyme amplification are high substrate specificity and rapid catalysis to generate optical2 or electrical signals.3,4 In most cases, antibody−enzyme conjugates are used for the specific analyte recognition and signal amplification.5,6 Although undoubtedly valuable and powerful, this method suffers from several inherent problems. For instance, in the essential step where the enzyme is covalently labeled to antibodies, site-specific and quantitative labeling without the loss of antibody specificity remains difficult to achieve. Furthermore, besides the high cost and difficulty in preparing large quantities of antibody−enzyme conjugates, laborious operating procedures and extensive washing steps are mandatory. Thus, the development of a new enzyme-catalyzed signal amplification method which is label-free and washing-free would be highly desirable for the rapid and selective detection of biologically and medically important analytes. Although substantial efforts have been invested in developing label-free and washing-free enzyme-catalyzed signal amplifica© XXXX American Chemical Society
Received: November 25, 2014 Accepted: March 26, 2015
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Figure 1. (a) Schematic illustration of the steric dependent label-free and washing-free enzyme-amplified protein detection strategy. Binding of the target protein to the dual-functional synthetic molecule increases the steric hindrance between the enzyme HCAII and the target protein, thereby perturbing the inhibition affinity of sulfonamide to HCAII and thus increasing HCAII activity for substrate hydrolysis and signal amplification. (b) Chemical structures of four different probes for the detection of avidin (BTSA), MGMT (BGSA-1), SNAP-tag (BGSA-2), and lactoferrin (BASA).
methanol) were from Sigma-Aldrich and TCI and used without further treatment and distillation. Besides HCAII and SNAPtag proteins, which were purified in our laboraotry, all other proteins used in the selectivity test were purchased from SigmaAldrich. Thin layer chromatography (TLC) was performed on TLC-aluminum sheets (Silica gel 60 F254, Merck). Flash column chromatography was performed with silica gel (230− 400 mesh, Merck). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance II 400 with chemical shifts (δ) reported in ppm relative to the solvent residual signals of acetone-d6 (2.04 ppm), CD3OD (3.30 ppm), CDCl3 (7.24 ppm), dimethylsulfoxide-d6 (DMSO-d6; 2.49 ppm), and coupling constants reported in Hz. HPLC analysis and purification were performed with an analytical column (EC 150/4.6 Nucleosil 300-5 C18, Macherey-Nagel) and a semipreparative column (VP 150/21 Nucleosil 300-5 C18, Machrey-Nagel). Fluorescence spectra were recorded using TECAN Infinite M200Pro. High resolution mass spectra (HRMS) were recorded on HPLC/MS-MS (Varian 901FTMS). HCAII and SNAP-Tag Protein Expression and Purification. HCAII vector pET51b_hCAII was transformed and expressed with N-terminal His-tag in E. coli strain BL21. Bacterial cultures in LB medium were grown at 37 °C to an OD 600 of 1.0. Expression of the HCAII protein was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The bacteria were grown for an additional 16 h at 18 °C and harvested by centrifugation. They were lysed by sonication, and the insoluble protein and cell debris were removed by centrifugation. The protein was purified with Ni-NTA (Qiagen) according to the instructions of the suppliers. The purified protein was snap frozen in liquid nitrogen and stored in N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer (50 mM HEPES, 50 mM NaCl, pH = 7) at −78 °C until further use. The protein concentration was determined using a Thermo Scientific Pierce BCA Protein Assay Kit. SNAP-tag protein expression and purification is similar to the protocol of HCAII. See Figure S16, Supporting Information, for the purity of the proteins after His-tag purification.
However, given the nonspecific nature of electrostatic interactions between the nanoparticles and the enzyme, the challenge in this setup is the issue of protein selectivity. Herein, we describe a novel approach to develop label-free and washing-free enzyme-amplified protein detection by using synthetic molecules to generate steric effects upon protein binding to modulate enzyme activity for signal amplification. Although steric effects have been known to influence the binding of a receptor to a ligand,20−22 the effects have never been applied in enzyme-catalyzed protein detection. In our approach, protein recognition and enzyme activity regulation are achieved by a dual-functional synthetic probe which consists of a protein ligand for target protein recognition and an inhibitor to regulate enzyme activity. In the absence of the target protein, the inhibitor from the dual-functional probe would bind to the enzyme to inhibit the enzyme activity. Binding of the target protein to the ligand perturbs the enzyme−inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme, therefore activating the enzyme to initiate signal amplification (Figure 1a). In this design, the distance between the ligand and the inhibitor is crucial as a shorter linker would maximize the steric effects to perturb enzyme−inhibitor interaction during protein recognition. Human carbonic anhydrase II (HCAII) was chosen as the signal enzyme for its esterase function to hydrolyze nonfluorescent fluorescein diacetate (FLDA) to generate strongly fluorescent fluorescein (FL).23 In addition, the wide range of sulfonamide inhibitors, which inhibit HCAII esterase activity, provides us with the means to control the HCAII activity.24 As a proof-of-principle, four synthetic probes were prepared for the specific and rapid detection of four different proteins, avidin, O6-methylguanine DNA methyltransferase (MGMT), SNAP-tag, and lactoferrin (Figure 1b).
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EXPERIMENTAL SECTION Materials and Apparatus. Chemicals and peptide coupling reagents were purchased from Sigma-Aldrich, Alfa Aesar, TCI, and Advanced Chemtech and used without further purification. Solvents (DMF, DCM, hexane, ethyl acetate, and B
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Figure 2. Label-free and washing-free detection of avidin with BTSA. (a) Time course of fluorescence intensity in the presence and absence of avidin. The inset shows the images of the solution in a microcentrifuge tube before (left) and after (right) the addition of avidin under excitation with a UV lamp (365 nm). Detection condition: 2 μM BTSA, 1 μM HCAII, and 2 μM avidin were incubated in pH 7 HEPES buffer at 37 °C for 10 min. Measurements began immediately after the addition of 1 μM FLDA in a 96-well microtiter plate (total volume = 100 μL, 1% DMSO and 1% ACN). λex = 470 nm, λem = 520 nm. (b) Time course of fluorescence intensity in the presence of different avidin concentrations. (c) Protein detection with BTSA probe in the presence of different proteins at 2 μM concentration. Relative intensity (F/F0) was calculated after 60 min of fluorescent amplification. Error bars were calculated from three independent measurements.
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Label-Free and Washing-Free Enzyme-Catalyzed Protein Detection. One μM HCAII and the corresponding probe were incubated with the target proteins for different periods (avidin: 10 min; lactoferrin: 60 min; MGMT: 90 min; SNAP-tag: 60 min) in HEPES buffer (50 mM HEPES, 50 mM NaCl, pH = 7.0) at 37 °C. Measurements began immediately after the addition of 1 μM FLDA in a 96-well microtiter plate (total volume = 100 μL, 1% DMSO and 1% acetonitrile (ACN)). The fluorescence increase was monitored every 5 min over 60 min at 520 nm using TECAN Infinite M200Pro. The excitation wavelength is 470 nm. For the calculation of limit of detection (LOD), we used the following equation: LOD = mean fluorescent signal of blank (without target protein) + three times the standard deviation corresponding to the blank controls (N = 15).
RESULTS AND DISCUSSION To test our design for the label-free and washing-free enzymeamplified protein detection, BTSA, which consists of a biotin for the binding with avidin and a sulfonamide inhibitor for HCAII, was prepared. The binding of avidin with biotin is wellestablished and has been used in many bioanalytical methods.25 For avidin detection, BTSA was incubated together with avidin and HCAII for 10 min at 37 °C followed by the addition of FLDA to initiate enzyme catalysis to generate a fluorescence signal. In the absence of avidin, the activity of HCAII was completely inhibited by BTSA and only very weak background fluorescence was observed. In contrast, a gradual increase of fluorescent signal was observed when avidin was added (Figure 2a). After 60 min of enzymatic amplification, the emission spectra showed that the signal was enhanced by around 24-fold as compared to the control experiment without avidin (Figure S1, Supporting Information). The amplification was inhibited completely when 50 μM free biotin was preincubated with C
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Figure 3. Detection of MGMT and SNAP-tag with BGSA-1 and BGSA-2. (a) Time course of the fluorescence intensity in the presence and absence of MGMT. Incubation condition: 2 μM BGSA-1, 1 μM HCAII, and 2 μM MGMT were incubated at 37 °C for 90 min in pH 7 HEPES buffer. (b) Time course of the fluorescence intensity in the presence and absence of SNAP-tag protein. Incubation condition: 1 μM BGSA-2, 1 μM HCAII, and 1 μM SNAP-tag were incubated at 37 °C for 60 min in pH 7 HEPES buffer.
avidin. This indicates that fluorescence amplification is specific and controlled by the recognition of avidin with a biotin moiety from BTSA. Due to the high brightness of fluorescein and efficient signal amplification, the bright emission in the presence of avidin can be easily detected by the naked eye with a hand-held UV lamp (Figure 2a, inset). With increasing concentration of avidin, a higher fluorescence signal was obtained due to the increase in catalytically active free HCAII (Figure 2b). The fluorescence response is concentration dependent and linear in the range from 0.1 to 0.6 μM, and the limit of detection (LOD) for BTSA to detect avidin was estimated to be around 140 nM from three times the standard deviation corresponding to the blank controls (N = 15) without avidin (Figure S2, Supporting Information). To validate that fluorescence amplification is steric dependent and due to the close proximity of HCAII and avidin, another dualfunctional probe, which consists of a longer linker in between the biotin and sulfonamide moieties, was synthesized. A lower fluorescent amplification ratio of about 12-fold was obtained with the longer linker dual-functional molecule (Figure S3, Supporting Information). This is consistent with our proposition that fluorescence amplification is due to the steric effects generated between the two bulky biomolecules and that a shorter linker will lead to an increase in the steric hindrance to weaken the HCAII−sulfonamide interaction. Furthermore, we found that the affinity of the inhibitor to the enzyme also plays a key factor, as the probe with a weaker meta-sulfonamide inhibitor for HCAII gave insignificant fluorescence enhancement (Figure S3, Supporting Information). We proceeded to study the selectivity of our protein detection method by incubating 11 other nontargeted proteins with BTSA. As shown in Figure 2c, BTSA shows exceptional selectivity toward its target protein avidin. In all cases, a dramatic fluorescence increase was observed only when avidin is present. Besides HSA and alcohol dehydrogenase, which displayed a marginal fluorescence increase due to their weak esterase activity to hydrolyze FLDA (about 2-fold), all the other nontarget proteins did not trigger significant fluorescence amplification. To demonstrate the modular feature of our protein detection strategy, we exchanged the biotin with O6-benzylguanine to create BGSA-1 for the detection of O6-methylguanine DNA methyltransferase (MGMT). The MGMT protein repairs naturally occurring mutagenic DNA lesion O6-methylguanine
back to guanine in living cells.26,27 In chemotherapies that are based on alkylating agents such as bischlorethylnitrosourea (BCNU) and Temozolomide, MGMT plays a significant role in the development of drug resistance and is considered a crucial biomarker for individual susceptibility to alkylating agents.28,29 MGMT activity can be inhibited irreversibly by O6benzylguanine (BG) which acts by transferring the benzyl group to the protein at the cysteine side chain (Figure S4, Supporting Information).30 Currently, methods to detect MGMT activity include radioisotope substrates and laborious multistep fluorescent assays.31−33 In the absence of MGMT, BGSA-1 is a potent inhibitor against HCAII to prevent FLDA hydrolysis (Figure 3a and Figure S5, Supporting Information). In the presence of MGMT, the protein transfers the benzylsulfonamide moiety from the BGSA-1 probe to its cysteine side chain and the inhibition of sulfonamide to HCAII is perturbed by the steric effects due to the close proximity of HCAII and MGMT, resulting in a gradual increase of fluorescence signal (Figure 3a, black line). The fluorescence was amplified by about 12-fold as compared to the control measurement without MGMT. When the inhibitor O6-benzylguanine (BG, 50 μM) was incubated together with MGMT and BGSA-1, the fluorescence enhancement was reduced dramatically due to the occupation of the MGMT active site by BG, demonstrating the specificity of the interaction between BGSA-1 and MGMT (Figure 3a, blue line). The detection of MGMT with BGSA-1 displayed a LOD of about 173 nM with a linear range from 0.1 to 1 μM (Figure S6, Supporting Information). To validate that the detection of MGMT is also steric dependent, probe BGSA-2, which has a long benzyl linker between the guanine and sulfonamide moieties, was synthesized and applied for MGMT detection. As expected, BGSA-2 exhibited only slight fluorescence amplification (Figure 3a, gray line). Interestingly, when BGSA-2 was used for the detection of SNAP-tag protein,34 a self-labeling protein derived from MGMT that uses the same BG substrate and follows the same reaction mechanism as MGMT, a rapid and tremendous fluorescence enhancement of around 70-fold was obtained after 60 min of HCAII amplification (Figure 3b). Similar to MGMT detection, SNAP-tag detection by BGSA-2 can also be inhibited by 50 μM BG inhibitor. As MGMT undergoes conformational change and partial unfolding upon alkyl transfer, we postulate D
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Figure 4. (a) Label-free and washing-free enzyme-catalyzed glycoprotein detection with BASA probe. Incubation condition: 4 μM BASA, 1 μM HCAII, and 4 μM glycoproteins were incubated at 37 °C for 60 min in pH 7 HEPES buffer. SBA = soyabean agglutinin. (b) Detection of MGMT with BGSA-1 in 10% urine. Incubation condition: 2 μM BGSA-1, 1 μM HCAII, and 2 μM MGMT were incubated at 37 °C for 90 min in 10% urine mixed with HEPES buffer.
that this conformational change may expose the MGMT bound sulfonamide moiety to the protein surface and result in less steric hindrance between MGMT and HCAII protein.35,36 As such, MGMT demonstrates greater sensitivity to the linker length of the probe as compared to the SNAP-tag protein. For SNAP-tag detection, further extensions of the linker length between benzylguanine and sulfonamide moieties led to the dramatic decrease of fluorescence enhancement to about 5-fold (Figure S7, Supporting Information). The detection of SNAPtag with BGSA-2 displayed a LOD of about 12 nM (Figure S8, Supporting Information). Both BGSA-1 and BGSA-2 exhibited high protein selectivity as a dramatic fluorescence enhancement was observed only when MGMT and SNAP-tag were present (Figure S9, Supporting Information). While both ligands, biotin and O6-benzylguanine, display very strong binding to their target proteins, we also tested the applicability of our approach to detect proteins which have weaker binding affinities with synthetic ligands. To this end, we incorporated a boronic acid moiety to the sulfonamide inhibitor to create BASA for the detection of glycoproteins. Boronic acids can interact reversibly with cis-diol biomolecules such as sugars or carbohydrate residues of glycoproteins to form boronate ester compounds with weak dissociation constants ranging from 10−1 to 10−3 M.37,38 We first tested BASA against a collection of six different glycoproteins, lactoferrin, avidin, fetuin, soybean agglutinin (SBA), transferrin, and IgG. Surprisingly, fluorescence amplification was observed only when BASA and HCAII were incubated with lactoferrin which is a tear-specific biomarker for the Sjörgen syndrome,39 while the other 5 glycoproteins exhibited almost no fluorescence enhancement after 60 min of enzymatic amplification (Figure 4a). Fluorescent amplification was suppressed when 50 μM phenylboronic acid was added to the mixture of BASA, lactoferrin, and HCAII which validated the specific interaction of boronic acid moiety in BASA with the carbohydrate residues of lactoferrin (Figure S10, Supporting Information). The high selectivity for BASA to detect lactoferrin is remarkable and demonstrates a novel approach to use boronic acid as a ligand to selectively recognize a specific glycoprotein. Furthermore, we have also studied the possible interference of cis-diol compounds by incubating six different carbohydrates (fructose, glucose, galactose, sucrose, mannose, and hyaluronic acid) and
ascorbic acid. At 5 mM concentration, only fructose gave a 2.5fold fluorescence increase, while the other analytes did not show fluorescence enhancement (Figure S11a, Supporting Information). Increasing fructose and glucose concentrations to 50 mM gave similar fluorescence turn-on ratios (Figure S11b, Supporting Information). As small cis-diol molecules are not large enough to exert significant steric effects, these results also strongly support our signal amplification mechanism for protein detection. The detection limit for lactoferrin using our BASA probe was estimated to be about 350 nM with a linear range from 0.3 to 4 μM (Figure S12, Supporting Information). Currently, the concentration of lactoferrin is analyzed mostly by antibody-based detection methods.40,41 Finally, we demonstrated our protein detection method for the potential to detect the target protein in biological fluids. When MGMT protein and analytical mixture were spiked in HEPES buffer containing 10% urine, significant fluorescence enhancement was obtained (Figure 4b) against a slightly higher fluorescent background. The fluorescence amplification was concentration dependent, and the LOD was calculated to be around 1.3 μM (Figure S13, Supporting Information). For the detection of avidin and SNAP-tag proteins in urine with our dual-functional probes, the LODs were determined to be as low as 700 and 32 nM, respectively (Figure S14, Supporting Information). The high LOD obtained is likely due to the presence of HSA in the urine sample which can also hydrolyze FLDA and, thus, interferes with the signal amplification process. For protein detection involving covalent bond formation, such as SNAP and MGMT proteins, this problem can be overcome by first incubating the probe in the sample followed by denaturing the sample mixture at 95 °C for 5 min to deactivate the esterase. Finally, HCAII and FLDA can be added to initiate signal amplification (Figure S15, Supporting Information). In this case, the esterase interference from the biological samples can be suppressed and the denatured protein probe complex is still large enough to exert a steric effect to produce signal amplification. These results indicate the potential usefulness of this steric-dependent enzyme amplification method for protein detection in complex biological samples. E
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(11) Banala, S.; Arts, R.; Aper, S. J.; Merkx, M. Org. Biomol. Chem. 2013, 11, 7642−7649. (12) Brennan, C. A.; Christianson, K.; La Fleur, M. A.; Mandecki, W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5783−5787. (13) Goodey, N. M.; Benkovic, S. J. Nat. Chem. Biol. 2008, 4, 474− 482. (14) Huang, Y.; Chen, J.; Zhao, S.; Shi, M.; Chen, Z. F.; Liang, H. Anal. Chem. 2013, 85, 4423−4430. (15) Teller, C.; Shimron, S.; Willner, I. Anal. Chem. 2009, 81, 9114− 9119. (16) Shekhawat, S. S.; Porter, J. R.; Sriprasad, A.; Ghosh, I. J. Am. Chem. Soc. 2009, 131, 15284−15290. (17) Stains, C. I.; Furman, J. L.; Porter, J. R.; Rajagopal, S.; Li, Y.; Wyatt, R. T.; Ghosh, I. ACS Chem. Biol. 2010, 5, 943−952. (18) Shekhawat, S. S.; Ghosh, I. Curr. Opin. Chem. Biol. 2011, 15, 789−797. (19) Miranda, O. R.; Chen, H. T.; You, C. C.; Mortenson, D. E.; Yang, X. C.; Bunz, U. H.; Rotello, V. M. J. Am. Chem. Soc. 2010, 132, 5285−5289. (20) Hlavacek, W. S.; Posner, R. G.; Perelson, A. S. Biophys. J. 1999, 76, 3031−3043. (21) Monine, M. I.; Posner, R. G.; Savage, P. B.; Faeder, J. R.; Hlavacek, W. S. Biophys. J. 2010, 98, 48−56. (22) Wall, S. T.; Saha, K.; Ashton, R. S.; Kam, K. R.; Schaffer, D. V.; Healy, K. E. Bioconjugate Chem. 2008, 19, 806−812. (23) Shih, P. M.; Liu, T. K.; Tan, K. T. Chem. Commun. 2013, 49, 6212−6214. (24) Krishnamurthy, V. M.; Kaufman, G. K.; Urbach, A. R.; Gitlin, I.; Gudiksen, K. L.; Weibel, D. B.; Whitesides, G. M. Chem. Rev. 2008, 108, 946−1051. (25) Dundas, C. M.; Demonte, D.; Park, S. Appl. Microbiol. Biotechnol. 2013, 97, 9343−9353. (26) Pegg, A. E.; Byers, T. L. FASEB J. 1992, 6, 2302−2310. (27) Pegg, A. E.; Dolan, M. E.; Moschel, R. C. Prog. Nucleic Acid Res. Mol. Biol. 1995, 51, 167−223. (28) Hegi, M. E.; Diserens, A. C.; Gorlia, T.; Hamou, M. F.; de Tribolet, N.; Weller, M.; Kros, J. M.; Hainfellner, J. A.; Mason, W.; Mariani, L.; Bromberg, J. E.; Hau, P.; Mirimanoff, R. O.; Cairncross, J. G.; Janzer, R. C.; Stupp, R. N. Engl. J. Med. 2005, 352, 997−1003. (29) Brent, T. P.; Houghton, P. J.; Houghton, J. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 2985−2989. (30) Gerson, S. L. J. Clin. Oncol. 2002, 20, 2388−2399. (31) Ishiguro, K.; Shyam, K.; Penketh, P. G.; Sartorelli, A. C. Anal. Biochem. 2008, 383, 44−51. (32) Damoiseaux, R.; Keppler, A.; Johnsson, K. ChemBioChem 2001, 2, 285−287. (33) Wu, R. S.; Hurst-Calderone, S.; Kohn, K. W. Cancer Res. 1987, 47, 6229−6235. (34) Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9955−9959. (35) Mollwitz, B.; Brunk, E.; Schmitt, S.; Pojer, F.; Bannwarth, M.; Schiltz, M.; Rothlisberger, U.; Johnsson, K. Biochemistry 2012, 51, 986−994. (36) Wibley, J. E.; Pegg, A. E.; Moody, P. C. Nucleic Acids Res. 2000, 28, 393−401. (37) Wu, X.; Li, Z.; Chen, X. X.; Fossey, J. S.; James, T. D.; Jiang, Y. B. Chem. Soc. Rev. 2013, 42, 8032−8048. (38) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291−5300. (39) Ohashi, Y.; Ishida, R.; Kojima, T.; Goto, E.; Matsumoto, Y.; Watanabe, K.; Ishida, N.; Nakata, K.; Takeuchi, T.; Tsubota, K. Am. J. Ophthalmol. 2003, 136, 291−299. (40) Karns, K.; Herr, A. E. Anal. Chem. 2011, 83, 8115−8122. (41) Tuccari, G.; Barresi, G.; Arena, F.; Inferrera, C. Arch. Pathol. Lab. Med. 1989, 113, 912−915.
CONCLUSIONS In conclusion, on the basis of the concept that steric hindrance can perturb the binding of a receptor to a ligand, a label-free and washing-free enzyme-amplified protein detection method was developed by using dual-functional probes to control the enzyme activity. As compared to the traditional immunoassays, which require enzyme labeling to the antibody, laborious operating procedures, and extensive washing during protein detection and signal amplification, our method presents the first general approach by using a simple molecular probe to overcome the protein labeling problem and provide a simple operation procedure for rapid and selective protein detection. The syntheses of the probes are easy and straightforward as most of them involved only a one step reaction. The protein detection method is modular and highly versatile as demonstrated by the selective detection of avidin, MGMT, SNAP-tag, and lactoferrin with four different probes. Furthermore, it is also possible to change the amplification unit by simply replacing HCAII with another enzyme and incorporating a corresponding inhibitor of the enzyme to the ligand. We believe that, with further optimization to increase sensitivity and selectivity, this steric-dependent enzyme amplification strategy will be useful for a wide range of applications, such as in medical diagnosis where high signal enhancement and straightforward detection methods are required.
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ASSOCIATED CONTENT
S Supporting Information *
Complete experimental methods and figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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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 We are grateful to the National Science Council (Grant Nos. 102-2113-M007-004-MY2 and 101-2113-M009-006-MY2) and Ministry of Education (“Aim for the Top University Plan”; Grant No. 102N2011E1), Taiwan (ROC), for financial support.
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