Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its

Dec 20, 2017 - NaOH and fetal bovine serum were purchased from SangonBiotech Co. ... Volumes of 160 μL of phosphate buffer (200 mM, pH 7.0), 160 μL ...
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Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its Application to Fluorescence Immunoassay Jian Sun, Jiahui Zhao, Lei Wang, Hongwei Li, Fan Yang, and Xiurong Yang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00830 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its Application to Fluorescence Immunoassay Jian Sun†,§, Jiahui Zhao†,‡,§, Lei Wang†, Hongwei Liǁ, Fan Yang†, 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 ǁ State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, Jilin 130012, China *Fax: +86 431 85689278. E-mail: [email protected] KEYWORDS: p-phenylenediamine, fluorescein, fluorescent sensor, inner filter effect, fluorescent ELISA, alpha-fetoprotein ABSTRACT: Being an important model peroxidase, horseradish peroxidase (HRP) has been thoroughly understood, and the detection of HRP is not only directly related to peroxidase-triggered catalytic process, but also linked to the development of HRP-based enzyme-linked immunosorbent assay (ELISA). Herein, we have reported an unconventional fluorescent sensor for convenient assay of HRP activity based on the HRP-catalyzed specific conversion of p-phenylenediamine (PPD) into chromogenic PPDox with H2O2 as the oxidizing agent, accompanied with the fluorescence quenching effect on fluorescein. By combining UV−vis absorption spectrum, isothermal titration calorimetry and fluorescence lifetime analysis, we have confirmed the inner filter effect as a main quenching mechanism in our proposed fluorescent assay. According to the intrinsic sensitivity of fluorescent sensor and high selectivity, our PPD/fluorescein-based sensing system can be utilized to real-timely monitor the HRP activity in real biological samples. Furthermore, the unambiguous response mechanism and excellent sensing performance encourage us to extend such HRP assay into the HRP-based fluorescent ELISA, which has a broad prospect of application in fluorescent diagnosis of hepatocellular carcinoma (HCC) by sensing alpha-fetoprotein, the well-known serologic HCC marker.

Being composed of a subset of oxidoreductases, peroxidases (EC 1.11.1.X) have been found in multiple isoforms in a large variety of biological organisms and well characterized in physiological and pathological processes for several decades or even a century.1 In general, peroxidases are known as biocatalysts to regulate various oxidative reactions of organic and inorganic substrate molecules by combining with hydrogen peroxide (H2O2) as the oxidizing agent. As a typical peroxidase isolated from the horseradish roots (a perennial plant belongs to the Brassicaceae family), horseradish peroxidase (HRP, EC 1.11.1.7) is one of the most understood and applied enzymes in biomedical, electrochemical and environmental fields.2-4 Owing to the identified three-dimensional structure, commercial availability in pure form, and high thermal resistance, HRP is usually regarded as the first option for the research and application of peroxidases. It is frequently utilized in enzyme-based sensing, enzyme-linked immunosorbent assay (ELISA),5-9 and industrial bio-catalysis.10-11 Moreover, numerous efforts have been alternatively dedicated to the enzyme immobilization, enzyme modification, and even enzyme mimic, in order to obtain the ideal peroxidase efficiencies.12-13 Therefore, it is still necessary and important to develop the convenient assay for HRP activity in this regard. By means of the unambiguous catalytic mechanism, numerous proposals for detecting HRP activity have been developed including spectrophotometric, chemiluminescent and electroanalytical methods, during which several desirable substrates

of HRP have been described and verified.14-16 Especially in electrochemical assays, a series of benzene derivatives such as phenol, o-phenylenediamine (OPD), 1,4-diaminobenzene (pphenylenediamine, PPD) and 3,3’,5,5’-tetramethylbenzidine (TMB) have been successfully employed as substrates.15-17 On the other hand, current routine methods for measuring HRP activity in both laboratory research and industrial/clinic application involve colorimetric techniques based on the conversion of chromogenic substrates into colored products. In this regard, the chromogenic substrates usually include 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (namely ABTS), OPD, TMB and so on.17-18 In contrast, it is very short of fluorescent substrate or fluorimetric method for sensing the HRP activity, although fluorescence analysis has many intrinsic advantages, such as the high sensitivity, fast response time and practical feasibility in biological system.19-20 In our opinion, such deficiency might be ascribed to the fact that most fluorophores could interact with the essential peroxides (usually is hydrogen peroxide, H2O2) and the resultant fluorescence changes hindered the potential fluorescent detection.20-22 As a vigorous benzene derivative, PPD is prone to be oxidized by oxygen, ferricyanide, or hydrogen peroxide in mild alkaline aqueous solution.23-25 As shown in Figure 1A, the structure of major oxidation product has been regarded as 2,5diamino-NN’-bis-(p-aminophenyl)-l,4-benzoquinone di-imine (i.e. PPDox or Bandrowski’s base) by Bandrowski for more than one hundred years and the detailed formation mechanism

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has been gradually ascertained from 1960s.23 In 2000, Jiao et al. found that PPD could be oxidized into PPDox by H2O2 in weak acid or neutral solution in the case of HRP as catalyst.15 Based on the fact that the PPDox molecule exhibits good electrochemical activity, an assay for HRP activity and resultant HRP-mediated voltammetric immunoassay can be developed by using PPD as an electrochemical substrate. In fact, there are very great differences in the colors and absorption spectra between the PPD and PPDox aqueous solutions. However, such HRP-triggered discriminability has scarcely been utilized in the development of optical sensors for measuring HRP activity and other relative analytes.26 Inspired by the aforementioned HRP-catalyzed specific oxidation of PPD to PPDox by H2O2, we develop a colorimetric strategy for recording the HRP activity by using commercially available PPD as chromogenic substrate in neutral aqueous solution. Furthermore, a conventional fluorophore, fluorescein, has been introduced into the sensing system and HRPtriggered formation of PPDox would lead to fluorescence quenching of fluorescein. In view of the sufficient spectral overlap, the confirmed non-interaction between fluorescein and PPDox and the negligible change of lifetime of fluorescein, an inner filter effect (IFE)-based fluorescent assay for HRP activity has been proposed and identified for the first time. In consideration that HRP is one of most typically used enzymes in the routine colorimetric ELISA, we have further extended the enzyme assay into fluorescent immunoassay with HRP as a signal-output enzyme.27 For the proof-of-concept, we have taken advantage of such fluorescent ELISA in the detection of a well-known serologic marker of hepatocellular carcinoma (HCC), alpha-fetoprotein (AFP).28-30 The excellent sensing performance and distinguishable results of the normal adults and HCC patients clearly indicate that the simple substrateand fluorophore-based fluorescent ELISA exhibits promising prospect in fluorescent diagnosis.

EXPERIMENTAL SECTION Chemicals and Materials. PPD was purchased from TCI Chemical Industries (Tokyo, Japan). H2O2 (35%), NaH2PO4·2H2O, and Na2HPO4·12H2O were purchased from Beijing Chemical Reagent Co. (Beijing, China). Fluorescein sodium salt, peroxidase from horseradish (HRP, EC 1.11.1.7), alkaline phosphatase, bovine serum albumin, EcoR I, glucose oxidase, human serum albumin, lysozyme, trypsin, tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). NaOH and fetal bovine serum were purchased from SangonBiotech Co. Ltd. (Shanghai, China). Human alphafetoprotein (AFP) ELISA Kit (including the plate coated with anti-human AFP, lyophilized AFP protein standard, biotinylated AFP detection antibody, HRP-conjugated streptavidin, ELISA colorimetric TMB reagent, ELISA stop solution, sample diluent buffer and wash buffer) were purchased from RayBiotech (Norcross, GA, USA). Clinical human serum samples were gifted from the Second Hospital of Jilin University (China). Ultrapure water (18.2 MΩ cm) was used in all aqueous solution. The phosphate buffer was prepared by dissolving Na2HPO4·12H2O and NaH2PO4·2H2O into the ultrapure water and adjusting the pH to 7.0. Apparatus and Characterization. Absorption and fluorescence spectra of all samples were obtained from a CARY 500 UV−vis−NIR Varian spectrophotometer (CA, USA) and a Hitachi F-4600 spectrofluorometer (Tokyo, Japan). Isothermal

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titration calorimetry (ITC) experiments were performed at 25 °C using a GE Healthcare MicroCal ITC200 calorimeter (MA, USA). Fluorescence lifetime measurements were carried out in the time-correlated single-photon counting unit of a Horiba-Jobin-Yvon Fluorolog-3 spectrofluorometer (NJ, USA) with 455 nm NanoLEDs excitation source. PPD-Based Colorimetric Response to HRP Activity. Volumes of 160 µL of phosphate buffer (200 mM, pH 7.0), 160 µL of PPD (10 mM), 160 µL of H2O2 (10 mM), and 720 µL of water were injected into a 2.0 mL microcentrifugal tube. And then 400 µL of freshly prepared HRP aqueous solutions with different concentrations ranging from 0.1 to 50 ng/mL were added into the tube, respectively. After a quick mixing (approximately 10 sec), the absorption spectra of the mixture solution were consecutively monitored at 1 min intervals at room temperature. Fluorescent Assay for HRP Activity. A fluorescent HRP activity assay was performed using the following procedures. Volumes of 160 µL of phosphate buffer (200 mM, pH 7.0), 160 µL of PPD (10 mM), 160 µL of H2O2 (10 mM), 160 µL of fluorescein (100 µM) and 560 µL of water were injected into a 2.0 mL microcentrifugal tube. And then 400 µL of freshly prepared HRP aqueous solutions with different concentrations ranging from 0.01 to 40 ng/mL were added into the tube, respectively. After a quick mixing, the fluorescence spectra of the mixture solution were consecutively monitored at 1 min intervals at room temperature. The proposed fluorescent assay for HRP activity in diluted fetal bovine serum sample (FBS, 1%) was carried out under the same conditions. Fluorescence Immunoassay for the Human AFP. Our proposed assay was performed on the commercial Human AFP ELISA Kit by using PPD as substrate instead of TMB, and the assay procedure was as follows. First, 100 µL of AFP standards with various concentrations ranging from 0.05 to 50 ng/mL were added into the wells coated with anti-human AFP. Then the wells were covered and incubated overnight at 4°C. After incubation, the removal of AFP solutions and washing steps were executed sequentially. Then 100 µL of 80-fold diluted biotinylated detection antibody was added into each well, followed by incubation at room temperature for 1 h with gentle shaking. After removal and washing again, 100 µL of 100fold diluted HRP-conjugated streptavidin was added, followed by incubation for 45 min with gentle shaking. After removal and washing steps, subsequently, the 30 µL of phosphate buffer (200 mM, pH 7.0), 30 µL of PPD (10 mM), 30 µL of H2O2 (10 mM), 30 µL of fluorescein (100 µM) and 180 µL of water was added. The fluorescence spectra measurements were carried out after gentle shaking for 20 min at room temperature. Fluorescence Immunoassay for AFP in the Real Human Serum Samples. Serum samples from three normal adults and three patients with HCC were kindly provided by the Second Hospital of Jilin University. The detailed procedures of the fluorescence ELISA referred to that for the model protein by just adding the diluted human serum samples instead of the AFP standard solutions.

RESULTS AND DISCUSSION HRP-Catalyzed Oxidation of PPD by H2O2. As shown in Figure 1B, the aqueous solution of sole PPD in 20 mM phosphate buffer at pH 7.0 has remained colorless all the time under the incubation at room temperature for 30 min, where the representative UV−vis spectrum exhibits strong absorption

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ACS Sensors peak around 305 nm and no obvious absorption in visible region. Under the similar conditions, the individual addition of either H2O2 or HRP into the PPD solution can hardly induce any interference on the absorption spectrum. In consideration of the featureless absorption of H2O2 and HRP, it looks like the sole H2O2 could not oxidize the PPD in neutral solution (phosphate buffer at pH 7.0), although it has been demonstrated that PPD is tend to be oxidized in alkaline aqueous solutions.25, 28, 31 On the other hand, the neutral PPD solution exhibits a marvelous absorbance enhancement and an apparent absorption peak centered at about 500 nm in the simultaneous presence of H2O2 and HRP, where HRP can catalyze the reaction of its active and colorless substrate PPD into the colored PPDox with H2O2. In this regard, several studies have ascertained that the structure of major oxidation product of PPD by H2O2 is 2,5diamino-NN’-bis-(p-aminophenyl)-l,4-benzoquinone di-imine (PPDox) or named as Bandrowski’s base (Figure 1A). As shown in Figure S1, Bandrowski’s base could exist in its neutral form, or in its protonated form according to the pH value, where the reported pKa value of monoprotonated Bandrowski’s base is about 7.4. Meanwhile, the absorption maximum in visible region of free base and protonated species are around 460 nm and 530−540 nm, respectively. The absorption peak in our experiment is centered at 500 nm, which indicates both free and protonated bases are coexisting components in the HRP-catalyzed oxidation of PPD with H2O2 at pH 7.0.18, 23 PPD-Based Colorimetric Response to HRP Activity. In fact, the phenomenon that HRP can specifically catalyze an in situ H2O2-based chromogenic reaction of PPD gives us opportunity to develop a colorimetric response to HRP activity. Usually, it is acknowledged that the neutral pH value is beneficial to the HRP activity and stability.32 Thus, the different concentrations of HRP are assessed in a real-time colorimetric response by employing PPD (1 mM) solution in 20 mM phosphate buffer at pH 7.0 containing H2O2 (1 mM). The mixture solutions have been monitored by UV−vis absorbance spectroscopy after the addition of different concentrations of HRP standards (0 – 50 ng/mL) at room temperature. Figure S2 describes that the typical absorbance spectra were recorded each minute and the increased concentration of HRP could give rise to a higher increasing rate of absorption. Almost all the absorption spectra have an obvious absorption peak centered at about 500 nm, where the absorbance values are directly related to the HRP concentration and incubation time. Therefore as shown in Figure 2A, we have summarized the time-dependent absorbance at 500 nm from the absorption spectra and the absorbance enhancements are nearly proportional to the increase in HRP concentration at a certain time. Such colorimetric response to HRP is sensitive, where the 0.1 ng/mL of HRP could also clearly result in an apparent gradual increase as a function of time (Figure 2B). To obtain the relationship between the absorption response and HRP concentration, the absorbance values at 500 nm at a reaction time of 15 min are plotted as a function of concentrations of HRP (Figure 2C). The inset graph shows that the concentration-response curve is quasi-linear from 0.1 – 14 ng/mL for HRP and the fitted linear data could be expressed as A500 = 0.0167 – 0.102 CHRP (ng/mL), R2= 0.998. Besides, the color change of the solutions induced by 1 ng/mL of HRP concentrations can be easily identified by naked eyes (Figure 2D). Furthermore, the aforementioned quasi-linear relationship and sensitive response between the absorbance values and concentrations of HRP would be

beneficial to the subsequent development of fluorescent HRP sensing.

Figure 1. (A) Schematic representation of HRP-catalyzed oxidation of PPD into PPDox by H2O2 (B) Absorbance spectra of PPD (1), PPD + H2O2 (2), PPD + HRP (3), H2O2 + HRP (4) and PPD + H2O2 + HRP (5) in 20 mM phosphate buffer at pH 7.0.

Figure 2. (A) Time-dependent variation of absorbance values at 500 nm recorded each 1 min during the real-time response system with different concentrations of HRP standards (0 – 50 ng/mL). (B) Corresponding expanded region of graph A with HRP activity from 0 to 1 ng/mL. (C) Plots of absorbance values at 500 nm for 15 min as a function of HRP activities (0 – 50 ng/mL). Inset: expanded linear region. (D) Photographs under natural daylight for 15 min of the response system toward HRP with various activities.

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Figure 3. (A) Schematic representation of the fluorescent assay for HRP activity based on the IFE between fluorescein and PPDox. (B) Absorption spectra of PPD (black) and PPDox (red). Fluorescence excitation (blue) and emission (green) spectra of fluorescein. (C) Fluorescence spectra of fluorescein (1), fluorescein mixed with PPD (2), PPD + H2O2 (3), PPD + HRP (4), H2O2 + HRP (5) and PPD + H2O2 + HRP (6) in 20 mM phosphate buffer at pH 7.0. (D) Time-dependent variation of relative fluorescence intensities (I/I0 at 515 nm) recorded each 1 min during the assay with different concentrations of HRP standards (0 – 40 ng/mL). (E) Plots of I/I0 at 515 nm for 15 min as a function of HRP activities (0 – 40 ng/mL). Inset: expanded region. (F) Plots of the corresponding logarithm of I/I0 at 515 nm for 15 min as a function of HRP activities (0 – 40 ng/mL). Inset: expanded linear region.

Establishment of PPD and Fluorescein-Based Sensing System. As one of the most conventional manufactured organic fluorophore, fluorescein has been widely used for fluorescent sensing, imaging and labeling.33-35 It is reported in previous literatures that both the maximum fluorescence excitation and emission of fluorescein in aqueous solution are around 500 nm,36 which are well overlapped with the absorption spectrum of PPDox, providing the potentiality for the fluorescence quenching. We have investigated the effects of the concentrations and pH values on the fluorescence spectrum of fluorescein before the establishment of fluorescein−PPD sensing system for assaying HRP activity. As shown in Figure S3A, the fluorescence emission of fluorescein has a continual bathochromic shift with the concentration increase. While the fluorescence intensity exhibits a gradual enhancement at first and then drastically decrease at high concentrations owing to the inherent self-absorption effect. On the other hand, the lower pH values induce a gradual decrease on the fluorescence intensity and the maximal intensity can be obtained in neutral and alkaline solutions (Figure S3B). Thus, we have chosen 10 µM as the fluorescein concentration in our following experiments and the fluorescein remains the property of fluorescence in the neutral solution (pH 7.0), which is also beneficial to the HRP activity and PPD-based colorimetric response to HRP. Furthermore, we have assessed the possibility of developing fluorescent sensor by introducing fluorescein into PPD-based response system. As shown in Figure 3B, fluorescein in neutral solution exhibits a fluorescence maximum excitation around 490 nm and maximum emission around 515 nm, both of which are well overlapped with the absorption spectrum of PPDox. In contrast, the neutral PPD solution exhibits no obvious absorption in such visible region. Accordingly, the individual addition of PPD into the fluorescein solution can never induce any interference on the fluorescence spectrum (Figure 3C). Similarly, the fluorescence of fluorescein solution could also be remained by introducing the mixture solution of PPD/H2O2,

PPD/HRP, or H2O2/HRP, partly due to that none of these mixture solutions exhibits obvious absorption in the visible region (Figure 1B). At the same time, it is illustrated that neither H2O2 nor HRP has a direct interaction with fluorescein. In the case of the simultaneous addition of PPD, H2O2 and HRP into the fluorescein solution together, however, a significant fluorescence quenching is observed, owing to the formation of PPDox and the resultant apparent absorption peak centered at about 500 nm. In this regard, the corresponding absorption spectra shown in Figure S4 also indicate that the maximum absorption of fluorescein around 490 nm can hardly be changed, unless the presence of PPD, H2O2 and HRP together. As illustrated in Figure 3A, we have used the mixture solution of fluorescein, PPD and H2O2 in phosphate buffer (pH 7.0) as the assay solution. The mixture solution remains pale yellow-green in visible light, and shows intense yellow-green emission under ultraviolet light of 365 nm. After the addition of HRP and incubation for several minutes with gentle shaking, the mixture solution has changed from initial pale yellow-green to reddish-brown, and the bright yellow-green fluorescence has been substantially quenched. Moreover, the degree of the fluorescence quenching is related to the addition of HRP (i.e., the HRP-catalyzed oxidation reaction proceeds), which provides the foundation for the development of PPD and fluorescein-based fluorescent HRP assay. Sensitive and Selective Detection of HRP Activity. Besides fluorescein concentration, the concentrations of PPD and H2O2 also influence the fluorescence behaviour in the detection process. As shown in Figure S5 and S6, we have summarized the time-dependent intensity ratios I/I0 at 515 nm (where I and I0 are the corresponding intensities in the presence and absence of HRP, respectively) and the results demonstrate the higher quenching rate and ratio are observed with an increased concentration of PPD or H2O2. Comprehensive considering the reagents consumption and the fluorescence quenching of resultant assay solution, the activity of HRP has been determined

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ACS Sensors in a real-time assay using fluorescein (10 µM) in 20 mM phosphate buffer buffer (pH 7.0) containing PPD (1 mM) and H2O2 (1 mM) as the standard assay solution. After the addition of different concentrations of HRP standards (0 – 40 ng/mL), we can utilize fluorescence emission spectroscopy to monitor the real-time reaction. As shown in Figure S7, the typical fluorescence emission spectra were recorded each 1 min and the increased concentration of HRP could give rise to a higher decline rate of fluorescence intensity. More distinctly, we have summarized the time-dependent intensity ratios I/I0 at 515 nm from the fluorescence spectra, and the results shown in Figure 3D demonstrate the higher quenching rate and ratio are observed with an increased concentration of HRP. Meanwhile, 0.01 ng/mL of HRP clearly results in an apparent gradual decrease in fluorescence intensity as a function of time and could be easily detected. Furthermore, I/I0 values at a reaction time of 15 min are plotted as a function of concentration of HRP (Figure 3E). It is obvious that the sensitivity in the fluorescent assay for HRP is higher compared with the aforementioned colorimetric response (Figure 2). Moreover, the inset graph of Figure 3F shows a quasi-linear relationship between the logarithm of I/I0 and the HRP concentration in the range of 0.01 – 16 ng/mL, and the fitted linear data could be expressed as Log (I/I0) = –0.068 – 0.010 CHRP (ng/mL), R2= 0.991. The selectivity of our proposed assay has been investigated by using other non-specific biomolecules, such as alkaline phosphatase (ALP), bovine serum albumin (BSA), EcoR I, glucose oxidase (GOx), human serum albumin (HSA), lysozyme, trypsin and tyrosinase as controls. The fluorescent results in Figure 4A have illustrated that these control biomolecules (10 µg/mL) had ignorable influence on the fluorescent assay both in the absence and presence of HRP. In addition, our developed fluorescein−PPD based sensing system has been successfully used to assay HRP in the diluted fetal bovine serum (FBS, 1%). As shown in Figure 4B, after adding HRP with different concentrations (0, 0.01, 0.1, 1 and 10 ng/mL, respectively), the real-time fluorescence results obtained in presence of FBS are similar to the corresponding ones of FBS free, providing the potential of our assay to selectively and sensitively monitor HRP concentration in real samples. Investigation into the Quenching Mechanism. Undoubtedly, our proposed fluorescent assay possesses an unequivocal sensing principle, and then we have further used physical−chemical methods to ascertain the mechanism in the fluorescence quenching of fluorescein by as-produced PPDox. Generally speaking, all the static quenching, dynamic quenching, as well as IFE can result in the fluorescence quench phenomenon. Among these different mechanisms, static quenching needs to the intense interaction and formation of a groundstate complex between the fluorophore and quencher, which generally changes the absorption spectra.37 In this case, we have investigated the interaction of PPDox titrated with fluorescein by qualitative isothermal titration calorimetry experiments.38 Thereinto, the PPDox stock solution (1 mM) was freshly prepared by mixing PPD (1 mM), H2O2 (2 mM) and HRP (20 ng/mL) in the phosphate buffer (20 mM, pH 7.0) and incubating for 30 min at room temperature. The calorimeter cell contained 200 µL of PPDox solution (50 µM), and 1.5 µL aliquots of 500 µM fluorescein solution in phosphate buffer were injected at 2 min intervals. As shown in Figure 5A, the heat of dilution has been first determined in control titration by injecting fluorescein solution into phosphate buffer. In the next titration of PPDox with fluorescein, each injection of fluorescein into PPDox solution produces a very small endothermic heat flow, which is similar to the control titration and also

originates from the heat of featureless dilution. Therefore, it is demonstrated that the direct interaction and formation of ground-state complex could hardly take place between PPDox and fluorescein. Furthermore, the absorption spectra of fluorescein, PPDox and fluorescein/PPDox mixture are obtained to exclude the possibility of static quenching (Figure S8). On the other hand, both the well-known förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) belong to the dynamic quenching mechanism because such energy/electron transfer just occurs in the excited-state and is expected to substantially change the lifetime of the fluorophore.39-40 Herein, the fluorescein shows a single lifetime (~4.01 ns) in phosphate buffer at pH 7.0, which is agreement with the previously reported value.35-36 Upon the addition of PPDox, the fluorescence lifetime of the fluorescein changed very little, and thus the dynamic quenching could also be excluded in our fluorescein−PPD sensing system (Figure 5B). The IFE, based on the re-absorption of the excitation and/or emission light by the quencher, is an easily neglected but very effective mechanism in fluorescence quenching. As far as we know, IFE has already been employed to develop several novel fluorescent detection systems in the last few years.41-43 Generally, an efficacious IFE needs a spectral overlap as large as possible between the absorption band of the quencher and the excitation and/or emission band of the fluorophore.42 In addition, the IFE could have a large influence on the fluorescence intensity of the fluorophore, where the fluorescence lifetime remained unchanged owing to no excited-state energy/electron transfer involved in such quenching. Meanwhile, there would be not a direct interaction between fluorophore and quencher in the IFE. Thus, comprehensive consideration of the experimental results of the fluorescein in the absence and presence of PPDox, the sufficient spectral overlap (Figure 3B), the confirmed non-interaction between fluorescein and PPDox (Figure 5A) and the negligible change of lifetime (Figure 5B) have demonstrated that the fluorescence quenching mechanism should be attributed to the IFE undoubtedly.

Figure 4. (A) Relative fluorescence intensities (I/I0 at 515 nm) of assay solution in addition to the control proteins or enzymes without (10 µg/mL, black columns) and with (red columns) the coexistence of 10 ng/mL HRP after incubation for 15 min. (B) Timedependent variation of I/I0 at 515 nm recorded each 1 min during the assay with different concentrations of HRP standards (0, 0.01, 0.1, 1, 10 ng/mL) with and without 1% fetal bovine serum.

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Figure 5. (A) Raw ITC data output for the interaction of PPDox (in the cell) titrated with fluorescein (in the syringe) at 25 °C. (B) Fluorescence lifetimes of fluorescein in phosphate buffer at pH 7.0 without and with the coexistence of PPDox.

Figure 6. (A) Schematic representation of the IFE-enabled fluorescent immunoassay based on the conventional sandwich ELISA platform. The fluorescence emission spectra (B) and relative intensities (I/I0 at 515 nm) (C) of the immunoassay toward AFP standards (0 – 50 ng/mL). Inset: expanded linear region.

In fact, the IFE-based assay method not only offers more convenience by simply mixing fluorophore and quencher, but also possesses considerable operability on the basis of the unaffected absorption spectra of fluorophore and quencher. For instance, we have already used fluorescein in phosphate buffer containing PPD and H2O2 as the standard assay solution to fluorescently determine the HRP activity. Besides that, this standard assay solution could also be developed as a colorimetric response to HRP activity, although there is a nearly complete spectral overlap between fluorescein and as-formed PPDox. If subtracting the constant absorption spectra of fluorescein, the typical absorption spectra obtained each 1 min from the standard assay solutions in the presence of HRP are consistent with those obtained from the mixture solutions without fluorescein (Figure S9). Therefore, using our standard assay solution (fluorescein in phosphate buffer containing PPD and H2O2), a potential fluorescent and colorimetric dualreadout assay for sensing HRP has been demonstrated. HRP-Enabled Fluorescence ELISA Strategy. Considering that HRP is one of most extensively and successfully used enzymes in ELISA, our developed enzyme assay has been potentially extended into the HRP-labeled ELISA with the help of commercially available antibodies labeled HRP. As schematically illustrated in Figure 6A, we have taken AFP as a model target antigen to evaluate the analytical performance of the HRP-based fluorescent ELISA for proof-of-concept. If

there is an increased concentration of AFP as antigen in ELISA system, incremental amounts of biotinylated detection antibody and HRP-conjugated streptavidin would be immobilized specifically and proportionately. At the same time, the present activity of HRP (i.e., the amount of HRP-streptavidin conjugates) can be determined by monitoring the fluorescence intensity of the fluorescein−PPD sensing system, during which a target antigen (AFP)-dependent varied process of fluorescence intensity could be acquired. The control experiments demonstrate that the isolated existence of AFP standards, biotinylated antibody, HRP-conjugated streptavidin do not affect the fluorescence response of fluorescein and PPDox-based detection system (data not shown). As shown in Figure 6B, the fluorescence emission intensities of the resultant solution exhibit sustaining decrease with the concentrations of AFP increasing from 0 to 50 ng/mL. Furthermore, I/I0 at 515 nm (where I and I0 are the corresponding intensities in the presence and absence of AFP, respectively) are plotted as a function of concentrations of AFP (Figure 6C). The inset graph shows a quasi-linear relationship between I/I0 and the logarithm of AFP concentration from 0.05 – 10 ng/mL, and then the fitted linear data could be expressed as I/I0 = 0.646 – 0.304 Log (CHRP) (ng/mL), R2= 0.985, where 0.05 ng/mL AFP can be easily detected. This value is lower than those in previously reported fluorescence immunoassay for AFP29,44,45, and more significantly, has already satisfied the need for the clinical detection. As an important serologic marker of hepatocellular carcinoma (HCC), abnormally elevated serum AFP level (typically >400 ng/mL) is usually observed in the serum of patients with HCC in contrast to the relative low level for normal adults (lower than or usually close to 5 ng/mL).28-29 Our fluorescein and PPDox-based fluorescence immunoassay was applied to detect the level of AFP in the clinical blood samples (containing serum samples of three normal adults and three patients with HCC). The results in Table S1 have been compared with the TMB-based standard colorimetric ELISA for serologic analysis of AFP in three normal adults and three patients with HCC, which has demonstrated that our developed fluorescence ELISA possesses great potential to be an alternative to the conventional ones in the serologic and clinical diagnosis.

CONCLUSION In summary, a well-defined unconventional fluorescent sensor for convenient assay of HRP has been rationally proposed on the basis of the IFE of PPDox, the HRP-mediated oxidation product of PPD, on the fluorescence of fluorescein. By means of the commercially available HRP-based colorimetric ELISA platform, our developed HRP sensor with clear response principle has been successfully extended into the fluorescent ELISA for the target antigen detection and serologic analysis. Besides of the intrinsic superiority of fluorescence analysis, in our opinion, such fluorescent sensor and ELISA have two following merits: (1) The commercially available PPD, fluorescein and H2O2 make the assay considerably simple, convenient and time-saving, (2) The unambiguous and efficacious IFE of PPDox on the fluorescence of fluorescein is beneficial to develop several other potential PPDox- and/or fluorescein-related sensors in the near future. Furthermore, we envision that such simple substrate- and fluorophore-based novel fluorescent assay could become an attractive and convincing sensing system in extensive bioassays and clinical diagnosis.

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ACS Sensors Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Protonation and deprotonation processes of Bandrowski’s base, fluorescence spectra of fluorescein at different concentrations and different pH values, absorption spectra of fluorescein with other related reagents, influence of PPD and H2O2 concentrations on the detection process, detailed absorption and fluorescence emission spectra of the sensing system in the absence and presence of HRP, the analytical results for AFP in the human serum samples (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Fax: +86 431 85689278

Author Contributions §

J.S. and J.Z. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial supports by the National Key Research and Development Program of China (2016YFA0201301), the National Natural Science Foundation of China (Grant Nos. 21435005, 21627808, 21605139), and Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019).

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