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An Enzyme Cascade-Triggered Fluorogenic and Chromogenic Re-action Applied in Enzyme Activity Assay and Immunoassay Jiahui Zhao, Shuang Wang, Shasha Lu, Xingfu Bao, Jian Sun, and Xiurong Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01845 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Analytical Chemistry

An Enzyme Cascade-Triggered Fluorogenic and Chromogenic Reaction Applied in Enzyme Activity Assay and Immunoassay Jiahui Zhao†,§, Shuang Wang†,‡, Shasha Lu†,‡, Xingfu Bao†, Jian Sun*,†, and Xiurong Yang*,† †State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China §University of Chinese Academy of Sciences, Beijing 100049, China ‡ University of Science and Technology of China, Hefei, Anhui 230026, China *Fax: +86 431 85689278. E-mail: [email protected], [email protected] ABSTRACT: An enzyme cascade-triggered reaction with novel signal generation mechanism is beneficial for the development and insight of the enzyme cascade, which is extensively used for signal transduction in potential applications. Inspired by the fluorogenic and chromogenic reaction between dopamine and resorcinol, and the specific catalytic properties of alkaline phosphatase (ALP) and tyrosinase, we designed and synthesized an unconventional substrate of ALP, named p-aminoethyl-phenyl phosphate disodium salt (PAPP). As expected, the ALP and tyrosinase-incubated PAPP solution exhibited pale yellow with intense blue fluorescence upon addition of resorcinol, owing to the ALP-catalyzed transformation of PAPP into an intermediate tyramine, and the tyrosinasecatalyzed hydroxylation of tyramine to dopamine, as well as the specific reaction between dopamine and resorcinol. Therefore, an enzyme cascade system has been developed herein based on the ALP and tyrosinase coupled enzymes-triggered fluorogenic and chromogenic reaction. According to the direct relationship between the activity of ALP/tyrosinase and absorbance/fluorescence intensity of the resultant solution, the proposed enzyme cascade-triggered reaction was utilized for assaying ALP and tyrosinase activity with fluorometric and colorimetric dual read-out signals. Furthermore, such enzyme cascade catalysis process was integrated into the ALP-based cascade enzyme linked immunosorbent assay with dual readout signals, resulting in the sensitive detection of cardiac troponin I in diluted serum.

As a classical molecular biological technique, enzyme cascade sequentially perform multiple enzymatic reactions, and is extensively used for signal transduction in potential applications in biotechnology.1-4 Recently, ongoing efforts have been devoted for the exploitation of enzyme cascade mechanism and behaviors on several platforms,5-10 and there are growing interests in the integration of several biocatalytic transformations in a multi-enzyme cascade system. However, the insight in existing enzyme cascade systems are almost characterized and quantized through the specific colorimetric signals of the enzymatic product,11-14 in which horseradish peroxidase (HRP)-triggered chromogenic reaction is dominant. In current systems, the substrate/product pairs and signal generation modes are relatively scanty, and thus the development of exquisite enzyme cascade-triggered reaction with novel signal generation mechanism is appealing but challenging. Despite the intrinsic advantage of fluorometry, the insight in enzyme cascade system has been confined to colorimetry due to the insufficient exploration in facile fluorogenic substrate or product for proper and applicable system. As an important hydrolytic enzyme in phosphate metabolism, alkaline phosphatase (ALP, EC 3.1.3.1) can catalyze the hydrolysis and transphosphorylation of a wide variety of phosphate esters in proteins, nucleic acids, and other small molecules.15-17 Thus, ALP has been considered as a significant biomarker in laboratory research and clinic diagnosis of pros-

tatic cancer, bone disease, hepatic dysfunction, diabetes and so on.18-22 Moreover, ALP is extensively adopted as a marker enzyme in the enzyme linked immunosorbent assay (ELISA) to produce detectable signals because of its high catalytic activity, mild reaction conditions, broad substrate specificity, good stability, and easy conjugation to antibodies.23-26 Although nature has already evolved a large number of substrates for ALP,11,27-30 there are still many potential substrates with desirable regioselective dephosphorylation site.31 To our knowledge, there is no report about enzyme cascade system that involved participation of ALP accompanied with fluorometric and colorimetric dual read-out signals, which is evidential probative for the cascade catalysis of the cascaded enzyme, as well as significant for both mechanism research and tailored applications in ELISA. In light of this, we set out to develop an alternative and more generic ALP-participated enzyme cascade system. In addition, tyrosinase is a widely distributed copper-containing oxidase in plants and animal tissues, and it plays crucial roles in many physiological processes.32,33 Tyrosinase has been anticipated to serve as the biomarker of skin diseases,34 and usually perform the specific hydroxylation of monophenolamine substrates to catecholamines. With tyramine as the model substrate, we have found that the tyrosinase-incubated tyramine solution exhibits pale yellow color and intense blue fluorescence in the presence of resorcinol under alkaline condition in our previous work.35

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Inspired by such well-established tyrosinase-enabled fluorogenic and chromogenic reactions, we have designed and synthesized the named p-aminoethyl-phenyl phosphate disodium salt herein (PAPP, Scheme 1). We hypothesized that the ALP-catalyzed cleavage of the phosphate group in PAPP could induce the transformation of PAPP into an intermediate tyramine, which is demonstrated as the substrate of tyrosinase to be hydroxylated to dopamine. On the basis of the cascaded enzymatic reaction of ALP and tyrosinase, PAPP served as an unconventional substrate for the insight into the cascade catalysis of ALP and tyrosinase through optical spectrum strategy, a conceptual enzyme cascade reaction could be triggered accordingly. In this prospective experiment, the ALP and tyrosinase-incubated PAPP solution exhibits pale yellow color and intense blue fluorescence, upon addition of resorcinol in the Na2CO3 solution. The absorbance and fluorescence intensity are directly related to the concentrations of ALP and tyrosinase, thereby realizing the fluorometric and colorimetric dual read-out assay of ALP or tyrosinase in one system. As far as we are aware, there are no reports about enzyme cascade systems incorporated ALP and tyrosinase, and such enzyme cascade-triggered fluorogenic and chromogenic reactions display promising prospects and preponderance in the extensive biosensing applications and the enzyme cascade mechanism research due to the outstanding dual-readout mode. Furthermore, the convincing ALP-participated cascade catalysis process was integrated into the cascade ELISA (ALP as the marker enzyme and tyrosinase as the signal-out reporter), resulting in sensitive detection of target antigen. To our best knowledge, this is the first cascade ELISA with dual read-out modes. The response could point out a novel strategy for extensive insight into the multi-enzyme biocatalysis research, as well as tailored applications in biosensing.

Scheme 1. Chemical structures of PAPP, tyramine, dopamine, resorcinol, and the azamonardine product.

EXPERIMENTAL SECTION Chemicals and Materials. p-aminoethyl-phenyl phosphate disodium salt (PAPP) were synthesized according to the synthetic procedure described in Scheme S1 by SANBANG Chemical (Changchun, China) with purity > 95% comfimed by 1HNMR characterization displayed in Figure S1. Sodium carbonate, resorcinol, dopamine, diethanolamine (DEA), kojic acid, sodium vanadate (Na3VO4), magnesium chloride and other inorganic salts were purchased from Aladdin Industrial Corporation (Shanghai, China). ALP (EC 3.1.3.1) from bovine intestinal mucosa, tyrosinase from mushroom (EC 1.14.18.1), EcoR I, bovine serum albumin (BSA), human serum albumin (HSA), cyclooxygenase (COX), acetylcholinesterase (AChE), glucose oxidase (GOX), lysozyme, and trypsin were

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purchased from Sigma-Aldrich. Lyophilized cardiac troponin I (cTnI) standard, mouse anti-cTnI antibody, rabbit anti-cTnI antibody, ALP-conjugated secondary antibody and other control antigen were brought form Abcam (Cambridge, MA). The sample/antibody diluent buffer and wash buffer for ELISA were purchased from Sangon Biotechnology Co. Ltd (Shanghai, China). The tetramethylbenzidine-horseradish peroxidase (TMB-HRP) based cTnI ELISA kit was purchased from RayBiotech (Norcross, GA, USA). Clinical human serum samples were kindly supplied by the Second Hospital of Jilin University (Changchun, China). Ultrapure water (18.2 MΩ cm) from a Millipore system was used in all aqueous solution. Apparatus and Characterization. Fluorescence excitation and emission spectra of all samples were recorded on a Hitachi F-4600 spectrofluorometer (Tokyo, Japan). Absorption spectra were obtained with a CARY 500 UV−Vis−NIR Varian spectrophotometer (CA, USA). The mass spectrometry analysis was performed on the electrospray ionization (ESI)Ion Trap (IT) LTQ Mass Spectrometer (Thermo, US) in the negative ion mode. Dual-readout Assay of ALP and Tyrosinase Activity. A fluorescent and colorimetric dual-readout assay of ALP activity was performed as follows. The ALP solutions with different activities ranging from 0 to 100 mU mL-1 were firstly added into mixture solution containing 200 µM PAPP and 1 U mL-1 tyrosinase in 10 mM DEA buffer (pH 8.0) containing 200 µM Mg2+. After incubation at 37°C for 1 h, resorcinol solution (200 µM) and sodium carbonate solution (50 mM) were successively injected into the above mixture solutions. The fluorescence and absorption spectra measurements were carried out after vibration for 20 min at room temperature. The selectivity of the proposed sensing system for ALP detection was evaluated by using other control enzymes and biological cations in the absence and presence of ALP, respectively. The detailed procedures of the proposed dual-readout assay of tyrosinase activity were similiar to that for ALP just by the replacement of ALP with tyrosinase with different activities ranging from 0 to 4 U mL-1. The selectivity for tyrosinase detection was evaluated by using other proteins/enzymes and biological cations in the absence and presence of tyrosinase, respectively. Dual-readout Cascade Immunoassay for Cardiac Troponin I. Our proposed assay was performed as follows. First, 100 µL of mouse anti-cTnI antibody (10 µg mL-1) were added into the wells. Then the wells were covered and incubated overnight at 4°C. The coating solutions were removed and the wells were washed for three times. Then, 200 µL of 1% BSA solution was added into each well and incubated at 37°C for 1 h with gentle shaking. After removal and washing steps, 100 µL of cTnI standards with various concentrations ranging from 0 to 80 ng mL-1 were injected into the wells, followed by incubation at 37°C for 1 h. Then the cTnI standard solutions were removed and the wells were washed for four times, followed by injection of 100 µL rabbit anti-cTnI antibody (2 µg mL-1) and incubation at 37°C for 1 h. After removal and washing steps, 100 µL of ALP-conjugated second antibody was added and incubated for 1 h, followed by removal steps and washing for five times. Afterwards, 15 µL of DEA buffer (200 mM, pH 8.0), 30 µL of PAPP (2 mM), 30 µL of Mg2+ (2 mM), 30 µL of tyrosinase (10 U mL-1) and 150 µL of water was added. After incubation at 37°C for 60 min, resorcinol solution (200 µM) and sodium


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Analytical Chemistry carbonate solution (50 mM) were successively injected into the above mixture solutions. The fluorescence and absorption spectra measurements were carried out after gentle shaking for 20 min at room temperature. Dual-readout Cascade ELISA in the Serological analysis. The detailed procedures of the proposed dualreadout ELISA were similiar to that for the model antigen just by the replacement of cTnI standard solutions with human serum. Before detection, all serum samples were diluted 20 and 50 times before being added to the wells, respectively. RESULTS AND DISCUSSION Enzyme Cascade Triggered Fluorogenic and Chromogenic Reactions. Under alkaline condition, a specific fluorogenic and chromogenic reaction takes place between dopamine and resorcinol, yielding strong fluorescent azamonardine with pale yellow color.35,36 Inspired by the explicit reaction mechanism and obvious spectral signal of such reactions,35 we have designed and synthesized PAPP according to the synthetic procedure described in Scheme S1, and the 1HNMR and mass spectra characterization of PAPP are displayed in Figure S1 and Figure S2, respectively. The anticipated ALP-catalyzed cleavage of the phosphate group in PAPP induces the transformation of PAPP into tyramine, which is known to be potently hydroxylated by the monophenolase activity of tyrosinase. In this prospective experiment, in situ fluorogenic and chromogenic reactions were expected to take place between the enzymatic product and resorcinol to produce azamonardine fluorophore, thereby generating dual-readout signals.35,36 In order to verify our assumption, we first investigated the absorption spectra of PAPP, dopamine and resorcinol, and the characteristic absorption peaks of all of them centered around 270 nm (Figure S3). In the absence of ALP and tyrosinase, neither PAPP nor tyramine could generate distinct fluorogenic and chromogenic signals with resorcinol in Na2CO3 solution (Figure S4). Furthermore, compared with the produced azamonardine fluorophore (ion peaks at m/z=258.09 in ESImass spectrum) in the resultant solution of dopamine reacting with resorcinol36 (Figure S5A), there was no homologous ion peaks in the resultant solution of PAPP or tyramine blending with resorcinol in Na2CO3 solution (Figure S5B and S5C). Figure 1A schematically presents the cascaded enzymatic dephosphorylation and hydroxylation process of PAPP and the subsequent in situ fluorogenic and chromogenic reactions upon the addition of resorcinol. In the absence of either enzyme, the absorption spectra of the resultant mixture in Figure 1B display the simple summation of the characteristic peak of PAPP and resorcinol, and there was no new typical absorption peak and negligible fluorescence, as well as the absence of the quasi-molecular ion peak of azamonardine fluorophore in ESI-mass spectrometry analysis (Figure S6A and S6B). As expected, only in the coexistence of ALP and tyrosinase, a typically intense absorption band centered at 420 nm appeared after resorcinol was added into the preincubated PAPP solution (blue line in Figure 1B), and bright blue fluorescence was observed under 365 nm UV light (the inset in Figure 1C). Besides, the ion peaks at m/z=258.12 in the resultant solution attributed to the quasi-molecular ion peak of azamonardine fluorophore was observed as well (red line in Figure S6C), which further confirmed the produce of azamonardine fluorophore by the enzyme cascade catalysis. In this scheme, the sequential dephosphorylation and

hydroxylation process of PAPP (dopamine as the product) is the prerequisite condition for the nucleophilic attack of resocinol monoanion to dopamine and/or corresponding quinone under alkaline conditions, and the generation of azamonardine fluorophore.36 Thus, PAPP served as a substrate for the extensive insights into the cascade catalysis of ALP and tyrosinase through optical spectrum strategy, a conceptual enzyme cascade reaction was triggered accordingly. As the reaction time between dopamine and resorcinol shortened considerably, and azamonardine became anionic with intense absorption band and strong fluorescence emission under strong alkaline conditions35,36, the subsequent fluorogenic and chromogenic reaction was performed in 50 mM Na2CO3 buffer solution (pH = 11).

Figure 1. (A) Schematic representation of the ALP and tyrosinase cascade-triggered fluorogenic and chromogenic reactions with PAPP as the substrate. (B) Absorption spectra of the product solution in the absence of tyrosinase (black line), ALP (red line), and coexistence of ALP and tyrosinase (blue line) with PAPP as the substrate, and dopamine reacting with resorcinol (pink line), respectively. The inset shows the corresponding solution under natural light. (C) Absorption and fluorescence excitation and emission spectra of the ALP-tyrosinase incubated PAPP reacting with resorcinol. The inset shows the resultant solution under natural light and 365 nm UV light, respectively.

Particularly, the experimental condition optimizations including the enzymatic incubation pH, PAPP and resorinol concentrations were implemented to investigate their influence on the ALP-tyrosinase cascade enabled fluorogenic reaction. As the pH values have appreciable impact on enzyme catalysis efficiency and different enzymes has various optimum pH values, we first investigated the influence of the pH values on the ALP-tyrosinase cascade catalysis in 10 mM DEA buffer containing Mg2+ with different pH ranging from 7 to 10. The results in Figure S7 indicate that the fluorescence intensity at 460 nm reaches maximum as the pH value located at 8.0, revealing the optimal pH value is 8.0 in this cascaded enzymatic catalysis process, which is a compromise between the optimum pH value for each enzyme. In addition, at a constant concentration of ALP and tyrosinase, various concentrations of PAPP solutions were incubated with the enzymes at pH value of 8.0, and the fluorescence intensities increased gradually as PAPP concentrations ranging from 0 to 1000 µM. The PAPP concentration was ultimately optimized to 200 µM, due to the slow growth in fluorescence intensities after that (Figure S8B). At PAPP concentration of 200 µM, different concentrations of resorcinol solutions were added to produce fluorescence emission in the Na2CO3 buffer solution, 200 µM resorcinol was selected due to the negligible change



of fluorescence intensity as resorcinol concentration was greater than 200 µM (Figure S8D). Fluorescent and Colorimetric Dual-Readout Assays of ALP and Tyrosinase activity. Upon the addition of resorcinol, the in situ cyclization between dopamine (the product of ALP-tyrosinase preincubated PAPP) and resorcinol takes place, accompanied with remarkable fluorogenic and chromogenic signals. Such enzyme cascade fluorogenic and chromogenic reactions encourage us to develop “turn-on” assay of ALP and tyrosinase activity with outstanding dualreadout mode. Under the above optimal conditions, we have firstly developed a fluorometric and colorimetric dual read-out sensing assay for ALP activity by using PAPP as the substrate. As shown in Figure 2, the fluorescence and absorption spectra display concentration-dependent enhancement with increasing activity of ALP from 0 to 200 mU mL-1. Figure 2B illustrates that the assay system exhibits a good linear relationship between fluorescence intensities at 460 nm (I460) and ALP activities over the range from 0.1 to 40 mU mL-1, where the fitted linear equation can be expressed as Log I460 = 1.917 + 0.597 Log CALP (mU mL-1), R2 = 0.993. Moreover, it displays a wider linear range between the absorption values at 420 nm (A420) and ALP concentrations ranging from 0.2 to 100 mU mL-1, where the fitted linear equation can be expressed as Log A420 = -1.167 + 0.359 Log CALP (mU mL-1), R2 = 0.986, as depicted in Figure 2D. It is noteworthy that the proposed fluorescent readout mode displays higher sensitivity with a detection limit of 0.03 mU mL-1, while the colorimetric readout mode exhibits wider linear range. Our rational and effortless dual readout assay reveals excellent performance in ALP activity sensing compared to most of the previously reported ALP sensing methods in Table S1, which indicates promising prospects in extensive bioassay applications. Moreover, other non-specific proteins and biological cations were selected to investigate the biospecificity of the proposed ALP sensing system. The results in Figure 2E show that none of them could induce ignorable changes in fluorescence signals, indicating great potential of ALP sensing in biological samples.

Figure 2. (A) Fluorescence emission spectra at different ALP concentrations excited at 415 nm. (B) Fluorescence intensities at 460 nm versus various concentrations of ALP. (C) Absorption

spectra at different concentrations of ALP. (D) Absorbance values at 420 nm versus different concentrations of ALP. (E) Fluorescence responses of the proposed system against the control enzymes (2 U mL-1) or proteins (10 µg mL-1) in the absence and presence of ALP (40 mU mL-1). (F) Corresponding photographs under 365 nm UV light (1) and natural light (2). All measurements were performed with 200 µM PAPP solutions as substrate in DEA buffer (10 mM, pH 8.0) containing 1 U mL-1 tyrosinase and 200 µM Mg2+.

Furthermore, the assay of tyrosinase activity was implemented with 20 mU mL-1 ALP and various concentrations of tyrosinase under the same conditions. The results in Figure 3 show that the fluorescence and absorption spectra display concentration-dependent enhancement with increasing tyrosinase activity from 0 to 4 U mL-1. The results in Figure 3B illustrate that I460 correlates linearly well with the tyrosinase activities ranging from 0.01 to 1 U mL-1, and the fitted linear equation could be expressed as Log I460 = 2.80 + 0.958 Log Ctyrosinase (U mL-1), R2 = 0.984. The calculated detection limit is as low as 0.003 U mL-1. In the colorimetric readout mode, A420 correlates linearly well with tyrosinase concentrations ranging from 0.05 to 2 U mL-1, where the fitted linear equation could be expressed as Log A420 = 2.80 + 0.958 Log Ctyrosinase (U mL-1), R2 = 0.984. The proposed dual readout assay reveals analogical promising potential in tyrosinase activity sensing which is comparable with or better than the previously reported tyrosinase sensing methods in Table S2. Moreover, other non-specific proteins and biological cations were selected to investigate the biospecificity of the proposed tyrosinase sensing system. The response results in Figure 3E indicate the good biocompatibility of the proposed assay system.

Figure 3. (A) Fluorescence emission spectra at different tyrosinase concentrations excited at 415 nm. (B) Fluorescence intensities at 460 nm versus various tyrosinase concentrations. (C) Absorption spectra at different concentrations of tyrosinase. (D) Absorbance values at 420 nm versus different concentrations of tyrosinase. (E) Fluorescence responses of the proposed system against the control enzymes (2 U mL-1) in the absence and presence of tyrosinase (2 U mL-1). (F) Corresponding photographs under 365 nm UV light (1) and natural light (2). All


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Analytical Chemistry measurements were performed with 200 µM PAPP solutions as substrate in DEA buffer (10 mM, pH 8.0) containing 20 mU mL-1 ALP and 200 µM Mg2+.

Inhibitor Screening for ALP and Tyrosinase. In fact, the inhibitors of each enzyme could significantly influence the enzyme cascade-enabled reaction. Therefore, our proposed fluorometric and colorimetric dual read-out sensing assay has been also used to screen ALP and tyrosinase inhibitors. Typically, Na3VO4 and kojic acid served as the inhibitor for ALP and tyrosinase have been employed in the inhibiting efficiency evaluation.37,38 Accordingly, the proposed system was further extended to inhibitor screening for ALP and tyrosinase with Na3VO4 and kojic acid as a model, respectively. The enzymes were preincubated with corresponding inhibitor for 10 min before being submitted to the substrate solution. As depicted in Figure 4, the fluorescence intensities display concentrationdependent slowing down with Na3VO4 and kojic acid concentrations, respectively, which indicates the enzyme activity has been effectually inhibited by each inhibitor. Furthermore, Figure 4B and 4D show the sigmoidal fitting results of the plots of fluorescence intensities versus the logarithms of the inhibitor concentrations, respectively. The IC50 values (the inhibitor concentrations where the enzyme activity is inhibited by 50%) of Na3VO4 and kojic acid were calculated to be approximately 18.4 µM and 88.0 µM, respectively, which are comparable with those of previously reported ALP and tyrosinase activity assays.28,39,40 Likewise, the above results highlight that the proposed ALP and tyrosinase activity assays show great potential in both ALP and tyrosinase inhibitors screening.

Figure 4. Fluorescence emission spectra at different concentrations of Na3VO4 (A) and kojic acid (C). Sigmoidal fitting of the fluorescence intensity of sensing system in 40 mU mL1 ALP and 2 U mL-1 tyrosinase versus the logarithm of the Na3VO4 (B) and kojic acid (D) concentrations.

Enzyme Cascade-Enabled dual-readout ELISA Strategy for CTnI Detection. As a specialised myocardial regulatory protein, cTnI is considered as the gold standard for the early diagnosis of acute myocardial infarction (AMI).41 Immunoassay has become the most extensively used method for cTnI detection,42 and ELISA has aroused widespread concern due to the enzyme-catalyzed signal amplification effect. Inspired by the extensive applications of ALP in conventional ELISA, the proposed dual read-out system for ALP sensing has great potential in cTnI detection, which encourages us to extend such enzyme cascade catalysis

reactions into the cascade ELISA (tyrosinase as the signal-out reporters). With the help of commercially available ALPlabeled antibody, the proposed cascade ELISA keeps away from the cumbersome and expensive labeling process, thereby avoiding the loss of enzyme activity in the labeling process. Thus, we attempted to develop a novel dual-readout cascade ELISA strategy through ALP-tyrosinase enabled fluorogenic and chromogenic reaction. Here, we take cTnI as a model of disease biomarker, and Figure 5A schematically displays the immunological recognition strategy of the proposed cascade ELISA for the sensitive assay of cTnI. The results in Figure 5B and 5C display the cTnI-concentration dependent fluorescence and absorption spectra, and Figure 5D and 5E elucidates that the fluorescence intensities at 460 nm and absorbance values at 420 nm increased gradually with the cTnI concentrations ranging from 0 to 80 ng mL-1. Particularly, the fluorescence intensities at 460 exhibit good linearity with cTnI concentrations from 0.05 - 4 ng mL-1, and 4 - 80 ng mL-1, and the fitted linear equation could be described as I460 = 201.0 + 28.2 CcTnI (ng mL-1), R2 = 0.951, and I460 = 300.8 + 3.48 CcTnI (ng mL-1), R2 = 0.986, respectively. Meanwhile, the absorbance values at 420 nm display good linearity with cTnI concentrations from 0.2 - 80 ng mL-1, and the fitted linear equation could be described as A420 = 0.09992 + 0.00154 CcTnI (ng mL-1), R2 = 0.978. Thus, the proposed dual-readout cascade ELISA can detect cTnI as low as 0.015 ng mL-1 with a fluorescence spectrometer and 0.06 ng mL-1 with a UV spectrophotometer, respectively, which is comparable with or better than other reported methods (Table S3) and commercial TMB-HRP based ELISA kits for cTnI detection (LOD = 0.038 ng mL-1). More significantly, our proposed cascade ELISA has great potential in extensive applications for determining other target analytes just by the replacement of target antigen and corresponding capture and detection antibody. To verify the application potential in real samples, high specificity toward target antigen is necessary. Herein, other non-specific proteins, including CA-125, IgG, CEA, BSA, HSA, lysozyme and trypsin, were selected to challenge the proposed ELISA platform. The response results in Figure 5G imply that none of them could induce conspicuous changes in fluorescence signals, indicating great specificity and potential of our cascade ELISA system in cTnI sensing in biological samples. Determination of CTnI in Diluted Human Serum Sample. The cTnI concentration in normal people is usually lower than 0.2 ng mL-1. After the outbreak of AMI, the cTnI concentration in human serum would ascend to 50 ng mL-1 within 3 - 6 h, and finally climb to a level around 550 ng mL1 43 , and it would remain elevated for a few days, which supplies enough diagnostic window for detection of cTnI through serological analysis.42,44 The performance of our proposed ELISA was investigated through the addition of certain amounts of cTnI standards in 5% and 2% normal human serum, respectively, and the fluorometric and colorimetric response results were compared with commercial TMB-HRP based ELISA kits for cTnI. As shown in Table S4, the results tally well with that of commercial ELISA kits, indicating that this cascade ELISA possesses great prospect in diagnosis of disease biomarkers.



Figure 5. (A) Schematic representation of the cascade ELISA strategy via tandem enzymatic fluorogenic and chromogenic reactions. The fluorescence emission spectra (B) and absorption spectra (D) of the cascade ELISA system with various concentrations of cTnI. Fluorescence intensities at 460 nm (C) and absorbance values at 420 nm (E) versus various concentrations of cTnI. (F) Corresponding photographs under 365 nm UV light and natural light. (G) Fluorescence responses of the cascade ELISA system against cTnI (40 ng mL-1) or other control enzymes/proteins (100 ng mL-1). Ab, Ab1 and ALP-Ab2 in above schematic diagram represent antibody, detection antibody, antigen and ALP-secondary antibody conjugate, respectively. We thank the financial supports by the National Key Research CONCLUSION and Development Program of China (2016YFA0201301), the A rational enzyme cascade system has been developed based National Natural Science Foundation of China (No. 21435005, 21627808, 21605139), Key Research Program of Frontier Sciencon the ALP and tyrosinase coupled enzymes-triggered es, CAS (QYZDY-SSW-SLH019), and the Youth Innovation fluorogenic and chromogenic reactions. The synthesized Promotion Association, CAS (No. 2018258). PAPP served as a substrate for the insight into the cascade

catalysis of ALP and tyrosinase through optical spectrum strategy. Typically, the dual read-out assay of ALP and tyrosinase activity has been accomplished, as well as the ALPbased cascade ELISA for cTnI detection in diluted serum. We envision that the ALP-tyrosinase cascade system provides a new strategy for extensive insight into the cascade biocatalysis mechanism research, as well as tailored applications in biosensing. The response is conducive for the exploitation and regulation of multi-enzyme cascade behaviors on different platforms with the help of appropriate biotechnology and modification methods.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Schematic representation of synthetic procedure of PAPP, 1 HNMR and mass spectra of PAPP, extra mass and absorption spectra, detailed data of parameter optimization, comparisons of performance of different ALP, tyrosinase and cTnI sensors. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86 431 85262056. Fax: +86 431 85689278. *E-mail: [email protected]. Phone: +86 431 85262063. Fax: +86 431 85689278.

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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