Signal Amplification and Detection of Small Molecules via the

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Signal Amplification and Detection of Small Molecules via the Activation of Streptavidin and Biotin Recognition Yu-Hsuan Chen, Wan-Ching Chien, Di-Chi Lee, and Kui-Thong Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03144 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 28, 2019

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Analytical Chemistry

Signal Amplification and Detection of Small Molecules via the Activation of Streptavidin and Biotin Recognition Yu-Hsuan Chen,† Wan-Ching Chien,† Di-Chi Lee† 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) ‡

ABSTRACT: Molecular recognition (e.g. antigen-antibody, DNA-DNA and streptavidin-biotin) is a generic, yet highly versatile and powerful strategy employed in enzyme-catalyzed signal amplification process. However, this approach is not applicable to metals, anions and small reactive species (e.g. O2‒ and F‒) as these molecules are too small to bind effectively to the macromolecules. In this paper, we demonstrate an enzyme-catalyzed signal amplification approach based on the controlled binding between streptavidin and target activated affinity-switchable biotin (ASB) probes, for the detection of O2‒ and F‒, using electrochemical and fluorescent detection techniques. The underlying rationale behind this design is that while the ASB probe would not bind with streptavidin-enzyme conjugate due to its low binding affinity with streptavidin, in the presence of the target analyte, the ASB probe on the immobilized surface will be activated to form biotin, which can then bind with the enzyme-tagged streptavidin to initiate signal amplification process. This versatile approach can also be applied in the imaging of endogenously secreted O2‒ along the plasma membrane of living cells using streptavidin conjugated with multiple fluorescent dye reporters. We believe that this ASB probe strategy will be useful for a wide range of applications, such as in basic biological research and medical diagnosis, where highly specific signal enhancement is required.

INTRODUCTION Most of the signal amplification strategies follow some basic principles. In all cases, the interaction of a target recognition probe with an analyte initiates a process that changes the properties of multiple reporters through multivalency or results in the formation of a large amount of reporter molecules through catalysis.1-3 Although many elegant approaches, such as allosteric regulation,4-7 steric effect,8,9 supramolecular aggregates,10-13 and split-protein systems14,15 have been reported to convey the target recognition event to the signal amplification transducer, molecular recognition (e.g. antigenantibody, DNA-DNA, sugar-lectin, and streptavidin-biotin) remains the most versatile, general and powerful approach in analytical methods to initiate signal amplification process. The importance of molecular recognitions in signal amplified detection has been very well-illustrated by numerous state-of-the-arts techniques, such as western blots, ELISA, and immunocytochemistry, for the detection of a low abundance of proteins using antibodies conjugated with different reporter tags, e.g. enzymes and fluorescent dyes.16-18 In these methods, target-recognition and signal-amplification are separated by extensive washing steps. As all the steps can be carried out independently, this stepwise protocol holds certain advantages, such as applications in diverse detection techniques. Therefore, various highly sensitive techniques, such as fluorescence, chemiluminescence and electrochemical methods, can be applied on demand, to detect the amplified reporter molecules in a clean buffer without interference from the sample matrix. Although a powerful and valuable approach, signalamplification based on molecular recognitions are generally not applicable for metals, anions and small reactive species

(e.g. O2‒, H2O2, ONOO‒ and etc.) as these molecules are generally too small to bind effectively to the macromolecules. To this end, many other methods have been used such as selfimmolative probes,19-22 DNAzymes23-25 and allosterically controlled synthetic catalysts,26,27 to achieve the desired signal amplified detection. However, all of these approaches suffer from some limitations, such as being able to detect only a particular class of target analytes, requiring long synthetic steps to obtain the chemical probes and are not directly applicable in living cells. Thus, the development of a new approach for small molecules detection that can harness the powerful molecular recognition strategy to generate specific amplified signals would definitely be beneficial to many aspects of chemistry and biology. In this paper, we introduce a generic signal amplification approach based on the controlled binding between streptavidin and target activated affinity-switchable biotin (ASB) probes, for the detection of small reactive molecules using diverse detection techniques, such as electrochemical, fluorescence and live cell imaging (Figure 1a). The streptavidin−biotin binding has been used extensively in bioanalytical research, particularly in signal amplification.28-30 The binding of streptavidin with biotin is the strongest known noncovalent interaction (Kd = 10−14 M) and, once formed, is unaffected by extremes of pH, temperature, and other denaturing agents. However, it is also reported that chemical modification of biotin at its N’-1 urea nitrogen position can decrease significantly its binding affinity with streptavidin (Kd ≈ 10−5 M). Although controlling the binding affinity of biotin with streptavidin can be a powerful approach in many applications, most of biotin probes reported previously were applied in protein sitespecific immobilization

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endogenously secreted O2‒ along the plasma membrane of living cells.

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Figure 1. (a) Selective molecular recognition of streptavidin with biotin (“ON”) and ASB probe (“OFF”) which has chemical modification at N’-1 urea nitrogen of biotin. (b) Chemical structures of ASB probes for the detection of fluoride (ASB-F) and superoxide (ASB-SO). ASB-SO(Neg) is a superoxide non-responsive probe. by photo- or electrochemical stimulation to retain protein activities.31-33 We believe that the activation of biotin and streptavidin recognition by biomolecules can be a big advantage in bioanalysis to obtain highly specific and sensitive signals. Previously, we have demonstrated the first example of target-activated affinity-switchable biotin (ASB) probe for the controlled binding of biotin and streptavidin to detect secreted peroxynitrite (ONOO−) from macrophages by using streptavidin-Cy5 dye as a fluorescent reporter.34,35 In this paper, we expanded the scope of this ASB probe approach to the enzyme-catalyzed signal amplified detection. To demonstrate our strategy, we synthesized ASB-F and ASB-SO probes, which can be used for detecting fluoride (F‒) and superoxide (O2‒) molecules by using electrochemical and fluorescent detection techniques (Figure 1b). In the absence of the target analyte, the surface immobilized ASB probes would not be able to bind with the streptavidin-enzyme conjugate due to the low binding affinity of streptavidin with ASB probes. As a result, the streptavidin conjugate can be washed away effectively. In the presence of the target analyte to trigger the ASB probes activation, the enzyme-tagged streptavidin would bind to the activated biotin. As a result, enzymatic catalysis can be initiated to produce a large amount of reporter molecules that can be detected through either electrochemical or fluorescent techniques, akin to protein detection assays such as ELISA and western blot. Besides the enzymatic-catalysis approach, we also show that this general method can be used for imaging

EXPERIMENTAL SECTION Materials and Instruments. All solvents (hexane, ethyl acetate, CH3CN, CH2Cl2, DMSO, DMF, and MeOH) were from Sigma-Aldrich and used without distillation or further treatment. D-Biotin, 2,6-Difluorophenol, Trifluoromethanesulfonic anhydride, Glutathione, 4-Hydroxybenzaldehyde, Hydroquinone, Sodium hydrosulfide, Hexamethylenetetramine, tert-butyl Hydroperoxide solution, Disodium sulfide, Potassium superoxide, Ammonium iron(II) sulfate hexahydrate, PMA, SIN-1, TEMPO and Superoxide dismutase (from bovine erythrocytes) were purchased from Sigma-Aldrich. FeTMPyP was from Calbiotech, SA-Cy5 and SA-ALP were from Jackson Immuno Research Inc. A 50 mM tris-buffered saline (TBS) was used for the preparation of L-ascorbic acid 2-phosphate (Sigma, U.S.A) solution and SA-ALP solutions. Maleimide activated plates (black, 96-well) were purchased from Thermo Fisher Scientific. Screen-printed carbon electrodes (SPCE, surface area 0.071cm2) were obtained from Zensor R&D (Taichung, Taiwan). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were carried out with CHI621D electrochemical workstation (CH Instrument, Austin, TX, USA). Modification of Au-NP Coated ASB-F Electrodes. AuNP electrode was prepared by electrochemical deposition. The deposition of Au-NP film was carried out by immersing the screen-printed carbon electrodes (Zensor R&D, Taichung, Taiwan) into the solution containing 100 mg/L AuCl4 and 2 M H2SO4 at a working potential of －0.6 V (vs. Ag/AgCl) under hydrodynamic conditions (500 rpm) for 240 seconds. During the process of deposition, the nitrogen was led-in the solution to reduce the oxygen content. After rinsing by DI-water and dried with nitrogen, the Au-NP electrode was coated with 20 µL of 0.2 mM ASB-F probe in isopropanol at room temperature for 12 hours. Electrochemical Detection of Fluoride with ASB-F Modified Electrodes. Tetra-n-butylammonium fluoride (TBAF) and sodium fluoride were dissolved in ddH 2O to make up 1 mM and 200 mM stock solution, respectively. 20 L of the fluoride sample was added on the ASB-F electrode and then placed in a dried screw-thread vial at room temperature for 60 minutes. The electrode was rinsed by DI-water and dried with nitrogen. 500 ng SA-ALP was added onto the electrode and incubated at room temperature for 30 minutes. The electrode was rinsed with DI-water to remove the unbound SA-ALP. To amplify the signal, 5 mM AAP was added to the electrode and the electrochemical measurement was initiated after incubating for 60 minutes at room temperature. Fluorescent Detection of Superoxide with ASB-SO Modified Microtiter Plates. 1 mM Cysteamine solution in ddH2O (100 L) was added to a black 96-well maleimide activated plate (Thermo Fisher Scientific, USA) and the plate was incubated at room temperature overnight. After washing three times with deionized water, the plate was incubated with 50 M ASB-SO (100 L in ddH2O) for 30 minutes at room temperature. After removing the unreacted probe, KO2 was added and the plate was incubated at room temperature for 1 hour. After washing three times with deionized water, the plate was


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Analytical Chemistry (a)

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Figure 2. (a) Schematic illustration of the enzyme-catalyzed electrochemical signal amplified sensing of fluoride ion using ASB-F electrode. (b) DPV spectra of ASB-F electrodes treated with or without 500 μM TBAF for 30 minutes. Detection condition: after removing the unreacted TBAF, 0.5 g SAALP was added to the electrode and incubated for 30 minutes. After washing, the spectra were recorded after incubating for 60 minutes with 5 mM AAP using scan rate of 30 mV/s and pulse amplitude of 50 mV. (c) Time course of faradaic currents for the ASB-F electrodes treated with or without 500 M TBAF. incubated with 200 ng SA-ALP in tris-buffer for 30 minutes at room temperature. The plate was blocked by a tris-buffer solution containing 2 % BSA and 0.5 % Tween 20. To amplify the fluorescent signals, 1 mM 4-MUP was added and the fluorescent intensity was recorded using a TECAN microplate reader (Infinite M200 Pro). Imaging of Superoxide on the Cell Surface of RAW264.7 Macrophages. RAW264.7 cells were cultured in DMEM medium supplemented with 1% penicillin-streptomycin and 10% FBS. 1x105 cells were seeded in 8-well chamber slides (Thermo-Nunc) and cultured at 37 °C with 5% CO2 overnight. After washing the cells three times with DMEM, 0.1 M Hoechst 34580 was added to stain the nuclei. To label the cells, 10 M ASB-SO in HBSS buffer (1.0 % ACN, v/v) was added and incubated at 37 °C for 30 minutes. To image exogenous O2‒, corresponding concentrations of KO2 were added to the cells and incubated in DMEM for 1 hour. For the imaging of secreted O2‒, the cells were treated with the activators for the indicated period in DMEM medium. SA-Cy5 (1 g) was added and incubated with the cells for 10 minutes to label the biotin probe. After removing the excess SA-Cy5, Laser Scanning Confocal Microscope (LSM 700, Zeiss, Germany) was used to image the cells. The Cy5 channel was taken by using 639 nm laser and LP640 emission filter. The nuclei were imaged by using 405 nm laser and SP490 emission filter. RESULTS AND DISCUSSION ASB-F Electrodes in Enzyme-Catalyzed Electrochemical Signal Amplified Sensing of Fluoride Ion. To illustrate our ASB probe approach in the enzyme-catalyzed signal amplified

Figure 3. Sensitivity and selectivity test of ASB-F electrodes in signal amplified detection of fluoride. (a) DPV spectra of ASB-F electrodes treated with different concentrations of TBAF. (b) Plots of the peak current with increasing TBAF concentrations. The inset shows that the current increases linearly up to a TBAF concentration of 1 M. (c) Selectivity test of ASB-F electrodes in the presence of different analytes. Except TBAF which was in 50 M concentration, all the other tested analytes were in 1 mM concentration. detection of fluoride (F‒), we constructed ASB-F, which consists of a fluoride responsive TBDMS (tert-butyldimethylsilyl) moiety and a lipoic acid (Figure 2a). In this study, lipoic acid was employed as a linker for the attachment of ASB-F to the gold-deposited carbon electrode. The reaction of F‒ with the TBDMS moiety triggers the cleavage of the carbamate group, thereby activating the probe for binding with streptavidinalkaline phosphatase (SA-ALP).36,37 After removing the unbound SA-ALP by washing, enzyme-catalyzed electrochemical signal amplification can be initiated by the addition of ascorbic acid phosphate (AAP). The alkaline phosphatase hydrolyzes AAP to become ascorbic acid (AA) which is an excellent electrochemical signal reporter. Using differential pulse voltammetry (DPV) to investigate the resultant ASB-F electrode which has been treated with TBAF (tetrabutylammonium fluoride), SA-ALP and AAP, we observed a readily measurable faradaic current at 0.42 V (vs Ag/AgCl) in Tris buffer (Figure 2b). The oxidation of AA at 0.42 V was confirmed by cyclic voltammetry (CV) measurement which shows only one irreversible redox peak (Figure S1). On the other hand, the ASB-F electrode was not able to bind with SA-ALP in the absence of F‒ source and a very weak background current was observed. The specific cleavage of TBDMS and carbamate groups from ASB-F probe in the presence of fluoride ion to form biotin was confirmed by



HPLC and TLC analyses (Figure S2). Similar to most enzymecatalyzed signal amplification techniques, the electrochemical signal of the ASB-F electrode can be enhanced by extending the incubation time with AAP. After 120 minutes of amplification, the DPV spectra showed that the electrochemical signal was increased by about 6-fold as compared to the control experiment without F‒ treatment (Figure 2c and Figure S3). To expand the scope of the ASB-F electrode, we also showed that the electrode can be applied in detecting inorganic sodium fluoride (NaF) which is known to have lower reactivity with TBDMS group (Figure S4). Therefore, a higher NaF concentration must be added to generate significant faradaic current from the ASB-F electrode.38 These results suggest that like most of the enzyme-catalyzed signal amplification methods, the activation of ASB probe for the binding with SA-ALP is critical to obtain high amplified signals. Sensitivity, Selectivity and Precision of ASB-F Electrode in Signal Amplified Detection of Fluoride. With increasing TBAF concentration, a higher electrochemical current was obtained due to the increase in free biotin for binding with SAALP (Figure 3a, 3b and S5). The electrochemical response is concentration dependent and the limit of detection (LOD) for the ASB-F electrode to detect TBAF was calculated to be approximately 0.3 nM (N = 10). For NaF detection, the LOD was calculated to be around 5 mM (Figure S4). Next, we proceeded to investigate the detection selectivity of our approach by incubating the ASB-F electrodes with other non-target analytes (Figure 3b). ASB-F electrode shows exceptional selectivity towards TBAF (50 M), KF (1 mM) and NaF (1 mM). However, all the other non-target analytes at 1 mM concentration did not exhibit any electrochemical response. Finally, to investigate the precision of the measurement, 20 ASB-F electrodes were prepared separately to measure the electrochemical current generated under positive (500 M TBAF) or negative (without TBAF) conditions (Figure S6). The relative standard deviation (RSD) is 5.0% (positive) and 10.7% (negative), respectively, indicating relatively good precision and reproducibility. Enzyme-Catalyzed Fluorescent Signal Amplified Detection of Superoxide on Microtiter Plates using ASB-SO Probe. To illustrate the modular feature of our ASB probe approach, we exchanged the TBDMS moiety with difluorophenyl trifluoromethanesulfonate (Tf) group to obtain ASBSO for the detection of superoxide (O2‒) through fluorescence amplification. Previous results have shown that the Tf group can react specifically with superoxide to form phenols.39,40 Thus, nucleophilic attack of the small but strong electronwithdrawing Tf group by superoxide can activate the sulfonate ester to yield a free phenol, which concomitantly triggers the cleavage of the carbamate group to form biotin for binding with streptavidin. To immobilize the probe, a maleimide functionalized 96-well plate was coated with cysteamine which was then reacted with the probe through the OSu chemistry (Figure 4a). Reaction of O2‒ with the ASB-SO probe activates the biotin for binding with SA-ALP. After removing the excess SA-ALP, enzyme-catalyze signal amplification can be initiated by the addition of 4-methylumbelliferyl phosphate (4MUP) which can be hydrolyzed by ALP enzyme to afford highly fluorescent 4-methylumbelliferyl (4-MU). Figure 4b shows the fluorescence spectra of the ASB-SO plate after 60 minutes of incubation with 1 mM 4-MUP. A strong and characteristic 4-MU emission spectra can be obtai-

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Figure 4. (a) Schematic illustration of the enzyme-catalyzed fluorescent signal amplified detection of superoxide with ASB-SO microtiter plate. (b) Fluorescent spectra of ASB-SO plates treated with or without 100 μM KO2 for 60 minutes. Fluorescent spectra of 4-MUP was included for comparison. Detection condition: after removing the unreacted KO2, 200 ng SA-ALP was added to the plates and incubated for 30 minutes. After washing, the spectra were recorded after incubating for 60 minutes with 1 mM 4-MUP. ex = 360 nm. (c) Time course of fluorescence increase for the ASB-F plates treated with or without 100 M KO2. (d) Selectivity test of ASB-SO plates in the presence of different analytes at 100 M concentration. ned for the plate treated with 100 M KO2 (O2‒ source). In contrast, the microtiter plate shows only very weak background fluorescence in the absence of KO2. HPLC analysis confirmed the formation of biotin after the reaction of ASBSO with KO2 (Figure S7). After 120 minutes of enzymatic amplification, the fluorescent signal was increased by about 24-fold as compared to the control experiment without KO2 (Figure 4c). The fluorescence signal was suppressed by the O 2‒ scavengers, superoxide dismutase (SOD) and TEMPO, indicating that the amplification is specific and regulated by the controlled binding of biotin with SA-ALP (Figure S8). It is important to note that the two fluoride atoms on diflurophenyl moiety are critical for O2‒ to react with the Tf group. Weak


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Analytical Chemistry fluorescence was obtained for the ASB-SO(Neg) which does not contain any fluoride atoms (Figure S9). The reported experimental pKa values of phenol and ortho-diflurophenol are 9.9 and 7.51, respectively.41 Therefore, we believe that the two fluoride atoms on the ortho-position decrease the pKa of the phenol, resulting in a better leaving group. To further evaluate the applicability of our ASB-SO probe for detecting O2‒, we performed selectivity tests with other reactive species (Figure 4d). Strong fluorescence was observed only in the presence of O2‒, while the addition of H2O2, OH●, GSH, SIN-1 (ONOO‒ source), TBHP, HOCl, DTT, Na2S and etc. to the plate gave either no detectable or weak fluorescence after 60 minutes of signal amplification. For the sensitivity study, the detection limit of O2‒ with ASB-SO microtiter plate was determined to be about 4.0 M with a linear range from 0 to 100 M (Figure S10). Although the sensitivities of these affinity-switchable biotin probes are not as high as compared to some of the reported F‒ and O2‒ detection methods,22,39,40,4248 our approach has a distinct advantage, as the electrochemical and fluorescent signals are obtained in clean buffers, in which interferences arising from the sample matrix can be excluded (Table S1 and S2). Overall, the results from the fluoride and superoxide detection demonstrate the first example of enzyme-catalyzed signal amplified detection of small reactive molecules using the molecular recognition strategy. Imaging of Exogenous Superoxide on Living Cell Surface using ASB-SO Probe. While the sensing of F‒ and O2‒ using ASB probes and SA-ALP demonstrate a new enzymecatalyzed signal amplification approach for small reactive molecules detection, the modular and flexibility of the signal generation strategy would also enable us to obtain significant amplified signals using streptavidin that is conjugated with fluorophore units, e.g. SA-Cy5. In fluorescent immunocytochemistry, SA-dyes are frequently employed for signal amplified detection of low abundance proteins in cells. Thus, we show that the ASB-SO probe can also be used to detect and image secreted O2‒ from living macrophage cells by using SACy5 to generate amplified fluorescence. In living cells, O2‒ arises from the one-electron reduction of molecular oxygen catalyzed by several enzyme complexes, such as NADPH oxidases and xanthine oxidase.49-51 It is a vital cellular signaling molecule involved in many pathological and physiological processes including immune defense, signal transduction, apoptosis and metabolic homeostasis, as O 2‒ is the precursor of most of the reactive oxygen or nitrogen species (ROS or RNS), e.g. H2O2, OH●, and ONOO‒. For instance, O2‒ can be converted to H2O2 by scavenging enzymes called superoxide dismutase (SOD) with distinct isoenzymes located in the mitochondria, cytoplasm, and extracellular compartments. If nitric oxide (NO) is present, O2‒ can react with NO at a diffusion-limited rate to generate ONOO‒. Thus, O2‒ is undoubtedly the most important ROS in cells, whose biosynthesis and fate are intertwined with diverse pathological and physiological processes ASB-SO was immobilized onto the cell surface by using its sulfo-NHS moiety which can be reacted with the amine of the cell membrane proteins (Figure 5a). 10 M ASB-SO was incubated with RAW264.7 macrophages for 30 minutes at 37 °C in HBSS buffer. After removing excess ASB-SO by washing with DMEM medium, the cells were treated with 100 M KO2 and incubated for 60 minutes. 1 g SA–Cy5 was incubated with the cells for 10 minutes and fluorescent images were

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Figure 5. (a) Schematic illustration of the membrane-anchored ASB-SO probe for the imaging of O2‒ at the cell surface. (b) Live cell imaging of the ASB-SO labeled RAW264.7 cells in (i) the absence or (ii) the presence of 100 M KO2. (c) Fluorescent images of the ASB-SO labeled RAW264.7 cells in the presence of 100 M (i) O2‒ (ii) HOCl, (iii) H2O2, (iv) NO, (v) OH●, (vi) ONOO‒, (vii) NO3‒, or (viii) NO2‒. Scale bar: 20 m. taken immediately after removing excess unbound SA–Cy5. Strong fluorescence was observed along the plasma membrane for the cells treated with 100 M KO2, while minimal fluorescence was detected without KO2 (Figure 5b). Notably, the fluorescence was barely detectable inside the cells, demonstrating clearly the applicability of our approach to image secreted O2‒ at the cell surface. It is also important to note that, macrophages labeled with ASB-SO(Neg) did not exhibit distinctive fluorescence along the plasma membrane even after treatment with 500 M KO2 (Figure S11). To evaluate the selectivity of ASB-SO for imaging secreted O2‒ at the cell surface, we performed selectivity tests with other RNS and ROS. Strong fluorescence was obtained for the cells treated with KO2, while the addition of HOCl, H2O2, NO, OH●, ONOO‒, NO2‒, and NO3‒ gave either no detectable or weak fluorescence along the plasma membrane (Figure 5c). This selectivity test on the cell surface is consistent with the results from the ASB-SO plate.



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activators, such as phorbol-12-myristate-13-acetate (PMA) and phosphatidylinositol 4,5-bisphosphate (PIP2), can also be employed to stimulate NOX.52,53 Besides the PKC activators, cytokine (e.g. IFN-) and endotoxins (e.g. LPS) can also enhance NOX activity by shifting the localization of NOX from the intracellular compartments to the plasma membrane and by increasing expression level of the protein. Therefore, the ability of ASB-SO probe to detect endogenously secreted O2‒ from RAW264.7 macrophages was studied by using lipopolysaccharides (LPS), phorbol-12-myristate-13-acetate (PMA) and Interferon- (IFN-) as stimulants. When ASB-SO labeled RAW264.7 cells were treated with these three activators, we observed bright fluorescence at the cell surface and minimal background fluorescence in the absence of the activators (Figure 6a and Figure S12). When 1, 5 and 10 g/mL of PMA was added to the ASB-SO labeled macrophages, we observed a gradual increase in fluorescence intensity along the plasma membrane, suggesting that the response of our probe to O2‒ on the cell surface is concentration dependent (Figure S13). To confirm the detection of endogenous O2‒ by our ASB-SO probe, we examined the fluorescence response in the presence of three different O2‒ scavengers, SOD, TEMPO, and FeTMPyP, during PMA stimulation (Figure 6b). Weak fluorescence was observed for the PMAstimulated macrophages treated with these three scavengers. These results indicated that the membrane-anchored ASB-SO probe based on the ASB probe approach can specifically image endogenously O2‒ along the extracellular membrane.

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Figure 6. (a) Imaging of ASB-SO labeled macrophages in the (i) absence or (ii) presence of 10 g/mL PMA, (iii) 0.1 g/mL IFN-, and (iv) 1 g/mL LPS. The cells were incubated for 1 hour with PMA and 12 hours with LPS and IFN-. (b) Imaging of ASB-SO labeled RAW264.7 cells in the (i) absence or (ii) presence of 10 g/mL PMA and (iii) PMA + 40U SOD, (iv) PMA + 500 M FeTMPyP, (v) PMA + 500 M TEMPO. The inhibitors and PMA were added together and incubated with the cells for 60 minutes. Scale bar: 20 m Imaging Endogenously Secreted Superoxide under Different Stimulants. In macrophages, latent NADPH oxidases (NOX) can be activated to assemble at the plasma membrane to produce O2‒ by phosphokinase C (PKC). Thus, many PKC

CONCLUSIONS By regulating the specific interaction of ASB probes with streptavidin, we have shown the first example of enzymecatalyzed signal amplified detection of small reactive molecules using the molecular recognition strategy. As compared to all the existing methods, our ASB approach holds certain advantages. For example, the design is modular, since all the steps (detection and signal amplification) can be carried out independently. This allows for the use of various sensitive detection techniques, such as fluorescence, electrochemical and surface plasmon resonance (SPR) and etc. On demand, to generate specific amplified signals in the clean buffer without any interference from the matrix. This was demonstrated by the enzyme-catalyzed signal amplified detection of F‒ and O2‒ using electrochemical and fluorescent techniques. Furthermore, the ASB approach can also be applied in live cell imaging to sense endogenously secreted O2‒ from macrophages upon treatment with different stimulants. Finally, we believe that this affinity-switchable biotin probe (ASB probe) approach will be useful for a wide range of applications, such as in basic biological research and medical diagnosis, where highly specific signal enhancement is required.

ASSOCIATED CONTENT Supporting Information. For complete experimental methods and figures see Supplementary Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]


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Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We are grateful to the Ministry of Science and Technology (grant no. 107-2113-M-007-003) and Ministry of Education (grant no. 107QR001I5), Taiwan (ROC) for financial support. We also thank Hsin-Ru Wu of the Instrumentation Center of National Tsing Hua University for HRMS measurements.

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Signal Amplification and Detection of Small Molecules via the

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