Fluorometric and Colorimetric Dual-Readout Immunoassay Based on

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

Fluorometric and Colorimetric Dual-Readout Immunoassay Based on an Alkaline Phosphatase-Triggered Reaction Jiahui Zhao, Shuang Wang, Shasha Lu, Guoyong Liu, Jian Sun, and Xiurong Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01553 • Publication Date (Web): 24 May 2019 Downloaded from http://pubs.acs.org on May 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Fluorometric and Colorimetric Dual-Readout Immunoassay Based on an Alkaline Phosphatase-Triggered Reaction Jiahui Zhao†,§, Shuang Wang†,‡, Shasha Lu†,‡, Guoyong Liu†,‡, 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: Alkaline phosphatase (ALP) usually acts as a signal transmitter in enzyme-linked immunosorbent assay (ELISA), therefore, developing an attractive ALP activity assay, especially, using a preferable substrate would help improve the efficiency and convenience of ELISA in the practical applications. Herein, we have first prepared an original and creative substrate, named m-hydroxyphenyl phosphate sodium salt (m-HPP), with desirable dephosphorylation site for ALP. On the basis of the ALP-catalyzed hydrolysis of m-HPP to resorcinol and its subsequent specific nucleophilic reaction with dopamine, we have exploited a fluorometric and colorimetric dual-readout ALP activity assay and ALP-based ELISA system. Under the employed experimental conditions, highly sensitive and specific assay of ALP and cardiac troponin I (cTnI) have been accomplished in a straightforward way. Furthermore, the commendable sensing performance of our proposed ELISA in the determination of the cTnI level in diluted human serum unambiguously illustrates great potentiality in the early diagnosis of acute myocardial infarction.

As an important zinc-containing dimeric enzyme, alkaline phosphatase (ALP, EC 3.1.3.1) mediates important functions in phosphate metabolism in various biological organisms from bacteria to mammalian.1,2 It is responsible for cleavage of phosphate group in a variety of small biological molecules containing phosphomonoester bonds, and it has the optimum catalytic efficiency with broad substrate specificity in alkaline conditions.3,4 ALP level has been considered to be connected to several diseases, such as bone diseases, liver dysfunction, ovarian and breast cancer, and it has been regarded as an important biomarker in clinic diagnosis.3,5 Recently, great efforts have been devoted on developing efficient methods for ALP sensing due to the close relationships between ALP level and disease states,6,7 as well as its extensive application as a signal reporter in the enzyme-linked immunosorbent assay (ELISA). Based on the avidity and specificity of the antigen-antibody recognition, ELISA usually takes advantage of the highly efficient biocatalytic property of the labeled enzyme (typically ALP).8-11 Each labeling enzyme could catalyze the numerous substrates to generate a measurable spectroscopic signal, which in turn indicates the concentration of the target antigen with a signal amplification effect.8 Therefore, the development of reliable methods for the sensitive detection of ALP activity is extremely valuable for the development of clinical diagnosis and biomedical research. Among various sensing strategies, spectral assays, especially fluorometric and colorimetric assays are

highlighted for their easy implementation and real-time monitoring capability. ALP has broad substrate specificity and ability in dephosphorylation of phosphoryl compounds, and many colorimetric or fluorometric ALP assays have been established with routine p-nitrophenyl phosphate (pNPP), pyrophosphate ion (PPi), L-ascorbic acid 2-phosphate (AA2P) and 4-methylumbellyferyl phosphate (MUP) as the substrates.12,13 Especially, pNPP as one of the conventional and commercial chromogenic substrates is extensively utilized in monitoring ALP activity through colorimetric readout. Various fluorescent nanomaterials, including carbon dots, conjugated polymers, silicon nanoparticles, and gold nanoclusters have been prepared and employed to monitor ALP activity, based on the different interaction of the phosphoryl substrate and enzymatic product with the nanomaterials.14-20 However, these probes generally involved complex preparation process and complicated sensing mechanisms, accompanied with low sensitivity and poor reproducibility. In this regard, few studies paid attention to the development of more preferable and straightforward substrates for ALP. Although fluorometric assay of ALP activity has improved the detection sensitivity than the colorimetric one, the fluorometric assay could be susceptible to different factors including fluctuations of the light source, photobleaching,15 and self-quenching defects of high concentration of fluorophore. Besides, the single-modal readout signal is susceptible to the external interference and is not beneficial to realize the precise,

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

reliable and highly sensitive bioassay. Thus, it is of great value to develop an integrated dual readout assay for ALP sensing, taking both sensitivity and linear range into account. Recently, a specific and biocompatible reaction between resorcinol and dopamine has been described and identified in detail, and the resultant cyclization product (named azamonardine) displays yellow color and bright blue fluorescence with high quantum yield at high-alkaline pH.21 Inspired by this, we originally designed and synthesized m-hydroxyphenyl phosphate sodium salt (m-HPP, Scheme 1) in this work through the introduction of a phosphate group to one of phenolic hydroxyl groups in resorcinol. In the absence of ALP, m-HPP and dopamine could not initiate any fluorogenic and/or chromogenic reaction to produce visible and detectable characteristic spectral signals. The phosphate group in m-HPP could inhibit the chemical reactivity of nucleophilic attack. As expected, the anticipant ALP-catalyzed hydrolysis of phosphomonoester bonds could transform m-HPP into resorcinol anion under basic condition. Subsequently, the resorcinol anion initiates a nucleophilic attack to dopamine and cyclization with dopamine to produce azamonardine fluorophore, accompanied with distinct fluorometric and colorimetric signals. Furthermore, such ALP-triggered reaction and accessible reaction conditions encourage us to develop an original fluorometric and colorimetric ALP activity assay and ALP-based ELISA system with m-HPP as the enzyme substrate (Scheme 1). Our proposed assay provides new insight into the ALP assays and the signal generation mechanism, served as a useful supplement or potential alternative for existing enzymatic biosensors.

Scheme 1. Schematic representation of the ALP-enabled chromogenic and fluorogenic reactions with m-HPP as the substrate.

EXPERIMENTAL SECTION Materials and Characterization. p-nitrophenyl phosphate (pNPP), 4-methylumbellyferyl phosphate (MUP) and sodium vanadate (Na3VO4) were purchased from Sigma-Aldrich. m-hydroxyphenyl phosphate sodium salt (m-HPP) was synthesized with the help of SANBANG Chemical (Changchun, China) based on the synthetic procedure in Scheme S1 in Supporting Information, and the purity (> 95%) of m-HPP was confirmed by 1HNMR and mass characterization (Figure S1-2). ALP (EC 3.1.3.1, from bovine intestinal mucosa), glucose oxidase (GOX), horseradish peroxidase (HRP), bovine serum albumin (BSA), human serum albumin (HSA), Immunoglobulin G

(IgG), trypsin and lysozyme were bought from Sigma-Aldrich. Cardiac troponin I (cTnI), its paired antibodies, ALP-conjugated secondary antibody, alpha-fetoprotein (AFP), and carcinoembryonic antigen (CEA) were bought from Abcam (Cambridge, MA). Dopamine, resorcinol, ammonium hydroxide, and magnesium chloride were purchased from Aladdin Industrial Corporation (Shanghai, China). Clinical human serum samples were provided by the Second Hospital of Jilin University (Changchun, China). Absorption spectra were acquired on a CARY 500 UV−Vis−NIR Varian spectrophotometer (CA, USA). Fluorescence spectra were acquired on a Hitachi F-4600 spectrofluorometer (Tokyo, Japan). The mass spectra were obtained from the electrospray ionization (ESI)-Ion Trap (IT) LTQ Mass Spectrometer (Thermo, US). Dual-readout Sensing Assay for ALP Activity. Different concentrations of ALP solutions varied from 0 to 20 mU/mL were firstly injected into the mixture solutions containing 100 μM m-HPP, 200 μM Mg2+ and 50 μM dopamine in 20 mM NH3·H2O-NH4Cl buffer (pH 10.0). The absorption and fluorescence spectra were monitored after incubation at 37°C for 2 h. The selectivity of the proposed ALP sensing system was evaluated by using other non-specific enzymes and proteins as control. Dual-readout Sandwich ELISA System for cTnI. The proposed cTnI assay was performed with m-HPP as the substrate for ALP. First, 100 μL of monoclonal mouse anti-cTnI antibody (10 μg/mL) in coating buffer were injected into the wells of the ELISA plate, then the plate was covered and incubated at 4°C overnight. The coating solution was removed and the wells were washed for three times with ELISA washing buffer, and 200 μL of 2% BSA solution was added into each well and incubated for 1 h at 37°C to to block the nonspecific adsorption sites. After removal and washing steps, 100 μL of cTnI standards with various concentrations ranging from 0 to 8.0 ng/mL were injected into the wells, followed by incubation at 37°C for 1 h. After another removal and washing step, 100 μL polyclonal rabbit anti-cTnI antibody (2 μg/mL) was injected and incubated at 37°C for 1 h. Last, 100 μL of ALP-conjugated second antibody (0.5 μg/mL ) was added and incubated after removal and washing steps. The ALP-conjugated second antibody was removed and the plate was washed for five times. Afterward, the mixture solutions of 300 μL containing 100 μM m-HPP, 200 μM Mg2+ and 50 μM dopamine in 20 mM NH3·H2O-NH4Cl buffer (pH 10.0) were added into each well of above plates. The fluorescence and absorption spectra were monitored after incubation at 37°C for 2 h. Determination of cTnI Concentration in Diluted Serum. The detailed sandwich ELISA procedures were similar to that for the cTnI standard just by the replacement of cTnI standard solutions by 5% normal human serum containing different cTnI level.

RESULTS AND DISCUSSION ALP-Triggered Chromogenic and Fluorogenic Reaction. The specific and biocompatible cyclization reaction between resorcinol and dopamine has already


Page 2 of 8

Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry been reported,21 the ambient reaction conditions and admirable spectral property of cyclization product indicate great potential of such reaction in extensive biosensing applications. m-hydroxyphenyl phosphate sodium salt (m-HPP) was synthesized according to the synthetic procedure presented in Scheme S1, based on the introduction of one phosphoester bond to the phenolic hydroxyl group of resorcinol. The 1HNMR and mass spectral characterization of m-HPP are presented in Figure S1 and S2, respectively. As shown in Figure 1A, the characteristic absorption peaks of resorcinol, m-HPP, dopamine were all center around 280 nm approximately. As a verification of precondition, resorcinol was directly injected into dopamine solution under alkaline condition (20 mM NH3·H2O buffer, pH = 10.0), the absorption spectrum of the product solution arose a new absorption peak centered at 420 nm and blue fluorescence emission maximumed at 460 nm emerged, which could be preliminarily attributed to the characteristic peak of azamonardine. The reaction shows admirable spectral “turn on” signals, accompanied with attractive yellow color and fluorescence emission at the same time. The product solution was submitted to ESI-Mass spectrum analysis, and the intense ion peak at m/z=258.1 could be approximately attributed to [azamonardine-H]- (Figure S3). We have further purified the product by virtue of semi-preparative chromatography, and the azamonardine fraction was submitted to EI-Mass spectrum analysis to verify the production of azamonardine. The ion peaks in Figure S4 were all attributed to the quasi-molecular ion peaks of azamonardine, further indicating the generation of azamonardine. Prior to the introduction of ALP, m-HPP solution was injected into the 50 μM dopamine and incubated in the NH3·H2O buffer (20 mM, pH = 10.0) containing Mg2+, and neither new typical absorption band nor identifiable fluorescence could be recognized in the absorption spectrum and fluorescence emission spectrum of the mixture solution, respectively(Figure 1). Besides, no molecular ion peak of azamonardine could be identified in both negative ion mode and positive ion mode of ESI-mass spectra of the mixture solution (Figure S5). In this regard, we consider that m-HPP and dopamine could not initiate such fluorogenic and chromogenic reactions to produce azamonardine without ALP incubation. However, if the dopamine was injected into the m-HPP solution in the presence of ALP, the typical absorption band centered at 420 nm emerged, and intense fluorescence emission maximized at 460 nm could be observed as excited at 415 nm (Figure 1). ESI-mass spectrum analysis of the ALP incubated mixture solution further confirmed the generation of azamonardine (Figure S6, m/z=258.1). Thus, it is suggested that ALP-catalyzed hydrolysis of m-HPP has effectively triggered the fluorogenic and chromogenic reaction from scratch. Since the pH values have a remarkable influence on both ALP-catalyzed enzymatic process and subsequent reaction efficiency, the influence of pH values on the specific reaction between resorcinol and dopamine was firstly investigated in 20 mM NH3·H2O buffer. As shown in Figure S7, both the absorbance values at 420 nm (A420) and

fluorescence intensity at 460 nm (I460) of the resultant solution display significant increase with pH values from 7.5 to 10.0 and slow rise as pH 10.5 to 12.5. Thus, the reaction could be desirably triggered under pH value of 10.0, and it is compatible with ALP functioning. The reaction time was also investigated, and the results in Figure S8 showed that the absorbance and fluorescence of the product solution almost reached the maximum in 10 min. Thus, the specific fluorogenic and chromogenic reaction between resorcinol and dopamine could complete in a very short time, and it is very benefit for the subsequent ALP sensing application. By using m-HPP as the substrate, the effect of the ALP-incubation pH was studied, and the results were shown in Figure S9. As the incubation pH varied from 7.5 to 12.5, the A420 and I460 maximized between pH 10~10.5. Thus, the ALP-catalyzed dephosphorylation of m-HPP and the subsequent reaction could be performed under similar condition to realize the signal activated detection of ALP. Furthermore, the ALP incubation time was evaluated with 100 μM m-HPP as substrate in 20 mM NH3·H2O (pH = 10.0) containing 200 μM Mg2+ and different levels of ALP. The results in Figure S10 illustrated that A420 increased promptly in the first 40 min and the increase gradually leveled off during 40-120 min; I460 enhanced gradually and almost reached its maximum as the incubation time extended to 20 min. It’s worth noting that the A420 and I460 values of the sensing system showed prompt increase upon the injection of ALP, indicating that the reaction between resorcinol and dopamine could take place in situ immediately after the ALP-catalyzed hydrolysis.

Figure 1. (A) Absorption spectra and (B) fluorescence emission spectra of product solutions. The inset shows the pictures under natural light and 365 nm UV light, respectively.



Colorimetric and Fluorometric Sensing ALP activity. The ALP-triggered fluorogenic and chromogenic reaction encouraged us to develop the ALP activity assay with synthetic m-HPP as the substrate. Under the above optimal conditions, the sensing performance of our proposed assay was evaluated by analyzing various ALP activities. As expected, the typical absorption and fluorescence spectra of the ALP-incubated solutions display ALP concentration-dependent enhancement, as the ALP activities ranging from 0 to 20 mU/mL (Figure 2). A420 and I460 of the product solution enhanced promptly with the increase of ALP activities. In particular, a linear relationship between absorbance and ALP activities can be fitted and described as A420 = 0.0455 + 0.0646 CALP (R2 = 0.978) as the ALP activity ranging from 0.05 to 4 mU/mL. The linear relationship of fluorometric readout can be described as I460 = 7.13 + 713.18 CALP (R2 = 0.992) as ALP activity increasing from 0.02 to 2 mU/mL. Compared with most reported ALP sensing systems (Table S1), our proposed assay has a lower limit of quantitation and convincing dual readout signals, which has great potential in further practical applications. To further verify the superiority of our proposed assay, other commercial chromogenic and fluorescent substrates of ALP were employed to assay ALP activity under similar condition. pNPP as a common chromogenic substrate for ALP is frequently utilized in the standard colorimetric assay of ALP activity, which can be converted to p-nitrophenol (pNP) together with an absorption spectrum red-shift (from 312 nm to 405 nm). On the other hand, MUP as a commercially fluorescent substrate can be dephosphorylated by ALP to produce 4-methylumbelliferone (MU, i.e. 7-hydroxy-4-methylcoumarin), accompanied with blue fluorescence emission.22,23 The colorimetric and fluorometric assay of ALP were carried out with the same concentration of pNPP and MUP (100 μM) as substrate,

respectively. The results in Figure S11 indicated that the analytical sensitivity for ALP with m-HPP as the substrate was superior to that of pNPP (linear range of 0.2 to 20 mU/mL), and comparable with that of MUP (linear range of 0.02 to 2 mU/mL). We have further investigated the molar extinction coefficient and quantum yield of the produced chromophore and fluorophore to reveal the essence of the sensitivity and superiority of the proposed assay. The molar extinction coefficient (ε) of azamonardine (ε420) and pNP (ε405) were measured and calculated to be ε420 = 218000 L/(mol cm) and ε405 = 19160 L/(mol cm) in 20 mM NH3·H2O-NH4Cl buffer (pH 10.0), respectively. As shown in Figure S12, the quantum yield (Φ) of azamonardine and MU were evaluated based on the investigation into the relationship between the fluorescence intensity and absorbance of azamonardine and MU, respectively. The quantum yield of azamonardine was approximately one-third of the quantum yield of MU, indicated from the slopes of the fitted line in Figure S12. In consideration of that MU is a typical coumarin derivative and commercially available fluorescent dye, our product azamonardine exhibits a desirable fluorescence quantum yield. Moreover, both the absorbance and fluorescence of azamonardine showed a bit bathochromic-shift than that of pNP and MU, and especially ε420 of azamonardine was more than 10 times higher than ε405 of pNP, indicating the potential superiority of m-HPP based ALP sensing by colorimetric readout and naked eye recognition. It is noteworthy that the azamonardine possesses satisfactory molar extinction coefficient and quantum yield, which is beneficial to the improvement of the assay performance. The proposed m-HPP-based fluorescent readout mode displays higher sensitivity than the colorimetric mode with a detection limit (LOD) of 0.0023 mU/mL based on 3σ/slope, and the colorimetric readout mode exhibits wider linear range with a LOD of 0.081 mU/mL.

Figure 2. (A) Absorption spectra and (B) fluorescence emission spectra with different ALP concentrations. (C) A420 and (D) I460 versus various concentrations of ALP. (E) the photographs under natural light and 365 nm UV light. (F) I460 of the proposed


Page 4 of 8

Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry sensing system in the presence of ALP (1 mU/mL) or several other proteins (10 ng/mL). All measurements were performed with 100 μM m-HPP and 50 μM dopamine in NH3·H2O buffer (20 mM, pH 10.0) containing 200 μM Mg2+.

More significantly, the colorimetric and fluorometric modes in the spectral analysis are complementary, and the combination of both readouts could provide more convincing results and avoid interference to some extent. Besides of pNPP- and MUP- based ALP sensing system, other ALP sensing systems with PPi, AA2P, or ATP as substrates usually need the help of other nanomaterials to produce detectable signals. The preparation process was usually time-consuming and environmentally unfriendly with complicated sensing mechanism (Table S1). The facile and straightforward sensing process and elaborate dual readouts of our proposed assay have more advantages and potentials in versatile biosensing applications. In addition, our proposed ALP assay system also shows high selectivity against several proteins including GOX, BSA, HRP, IgG, and lysozyme, and none of these proteins could induce distinct fluorescence signals under the same conditions (Figure 2F). Inhibitor Screening for ALP. Generally, the inhibitors of enzymes have a significantly negative influence on the enzymatic reaction, and the effect of inhibitor provides indirect evidence in demonstrating the enzymatic reaction. Na3VO4, as a typical inhibitor for ALP, has been usually adopted to evaluate the inhibition efficiency on ALP activity.18,24-25 For the proposed m-HPP-based ALP activity assay, various concentrations of Na3VO4 solution were preincubated with ALP for 10 min before being injected into m-HPP solution. The results in Figure 3 indicate that the fluorescence intensities of the system gradually decrease with increased Na3VO4 concentrations, indicating the ALP activity has been effectually inhibited by Na3VO4. Furthermore, the relationship between fluorescence intensities and the logarithms of the Na3VO4 concentrations can be well fitted into the sigmoidal curve. The IC50 was evaluated to be approximately 74.13 μM, which is comparable with those of previous ALP inhibitor screening.12 Furthermore, the colorimetric mode of our developed assay can also be used in the inhibitor screening for ALP under similar conditions (data not shown).

Figure 3. (A) Fluorescence emission spectra with different concentrations of Na3VO4 in the presence of 2 mU/mL ALP. (B) Sigmoidal fitting of I460 of the sensing system versus the logarithm of the Na3VO4 concentrations. Dual-readout Sandwich ELISA for cTnI Determination. As a specialized myocardial regulatory protein, cardiac troponin I (cTnI) has been regarded as an important biomarker of acute myocardial infarction (AMI),

which is one of the most fatal human diseases worldwide. It is still restrained by inaccurate diagnosis at present, although the early detection of cTnI in patients can reduce the risk of death from heart attacks.26-28 In consideration of ALP served as the labeling enzyme in ELISA, our proposed dual-modal ALP sensing system has considerable potential in cTnI detection by means of the conventional ELISA platform and corresponding antibodies. Thus, we attempted to extend such enzymatic fluorogenic and chromogenic reaction into a novel conceptual dual-readout ELISA strategy. As depicted in Figure 4A, the immunological recognition diagram described the sensitive assay strategy for cTnI with m-HPP as the substrate for ALP. Meanwhile, the Ab, Ab1, and ALP-Ab2 in Figure 4A stand for the capture antibody, detection antibody, and ALP-secondary antibody conjugate, respectively. The results in Figure 4 display the fluorescence and absorption spectra of the product solution are dependent on cTnI concentrations, and A420 and I460 improved as the enhancement of cTnI concentrations. As cTnI concentration increased from 0.125 to 8.0 ng/mL, the satisfactory quasi-linear relationship between A420/I460 and cTnI concentrations could be fitted. The colorimetric linear equation can be described as A420 = 0.01 + 0.0015 CcTnI, R2 = 0.978, and the fluorometric linear equation can be described as I460 = 201.0 + 28.2 CcTnI, R2 = 0.951. With commercial pNPP and MUP as substrate, the cTnI assay was also implemented and the analytical sensitivity was compared with that of m-HPP based assay. The results in Figure S13 indicated the sensitivity of our colorimetric readout mode (LOD = 0.04 ng/mL) was superior to that of the conventional pNPP-based assay (LOD = 0.15 ng/mL). The sensitivity of fluorescent readout mode (LOD = 0.04 ng/mL) was comparable with that of ideal MUP-based assay (LOD = 0.04 ng/mL). In addition, the performance of our proposed dual readout assay is comparative or better than that of most previously reported assays (Table S2). Furthermore, other non-specific proteins, including AFP, CEA, IgG, HSA, and trypsin, were selected to investigate the specificity and selectivity of the proposed sandwich ELISA platform. As shown in Figure S14, compared with the blank control (without any antigen), none of these proteins could cause obvious changes in fluorescence signals, indicating the great potential in cTnI determination in biological samples. Determination of CTnI Concentration in Diluted Human Serum. More significantly, the performance of our dual modal ELISA in diluted serum matrix was investigated, through analyzing diluted human serum containing different levels of cTnI. As indicated in Table S3, the cTnI detection results of both the dual modal response results were consistently well with the results from the commercial TMB-HRP based ELISA kits. It is demonstrated that our dual-readout ELISA provides convincing results and possesses potential in clinical diagnosis.



Figure 4. (A) Schematic Diagram of the dual modal sandwich ELISA. (B) Absorption spectra and (D) fluorescence emission spectra of the sandwich ELISA system in the presence of different cTnI concentrations. (C) A420 and (E) I460 versus different cTnI concentrations. Insert shows the pictures under natural light and 365 nm UV light.

CONCLUSION In summary, with creatively prepared m-HPP as the substrate, an attractive ALP-initiated chromogenic and fluorogenic reaction has been proposed and confirmed under ambient condition. We have first developed a straightforward assay for ALP activity determination and its inhibitor screening, with great convenience and excellent dual readout signals. Choosing cTnI as the target antigen, a conceptual fluorometric and colorimetric dual-readout ELISA has been realized, by virtue of the conventional ALP-based ELISA platform. We envision the facile sensing process and convincing dual-readout mode of our proposed m-HPP-based ALP activity assay and corresponding ELISA system would possess high practicality and superiority in both laboratory research and clinical diagnosis in the near future.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Schematic of synthetic procedure of m-HPP, 1HNMR and mass spectrum of m-HPP, mass spectra of azamonardine and control experiments, detailed data of experiment condition optimization. (PDF)

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

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

We thank the financial supports by the National Key Research and Development Program of China (2016YFA0201301), the National Natural Science Foundation of China (No. 21435005, 21627808, 21605139), Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019), the Youth Innovation Promotion Association, CAS (No. 2018258) and Open Project of State Key Laboratory of Supramolecular Structure and Materials (sklssm2019023).

REFERENCES (1) Coleman, J. E. Annu. Rev. Bioph. Biom. 1992, 21, 441-483. (2) Sharma, U.; Pal, D.; Prasad, R. Indian J. Clin. Biochem. 2014, 29, 269-278. (3) Millan, J. L. Purinerg. Signal 2006, 2, 335-341. (4) Kim, E. E.; Wyckoff, H. W. J. Mol. Biol. 1991, 218, 449-464. (5) Sturgeon, C. Clin. Chem .2002, 48, 1151-1159. (6) Szulc, P.; Seeman, E.; Delmas, P. D. Osteoporos. Int. 2000, 11, 281-294. (7) Schiele, F.; Vincent-Viry, M.; Fournier, B.; Starck, M.; Siest, G. Clin. Chem. Lab. Med. 1998, 36, 871-878. (8) Shen, J. W.; Li, Y. B.; Gu, H. S.; Xia, F.; Zuo, X. L. Chem. Rev. 2014, 114, 7631-7677. (9) Pei, X. M.; Zhang, B.; Tang, J.; Liu, B. Q.; Lai, W. Q.; Tang, D. P. Anal. Chim. Acta 2013, 758, 1-18. (10) de la Rica, R.; Stevens, M. M. Nat. Protoc. 2013, 8, 1759-1764. (11) Gao, Z. Q.; Hou, L.; Xu, M. D.; Tang, D. P. Sci. Rep. 2014, 4. (12) Tan, Y.; Zhang, L.; Man, K. H.; Peltier, R.; Chen, G. C.; Zhang, H. T.; Zhou, L. Y.; Wang, F.; Ho, D.; Yao, S. Q.; Hu, Y.; Sun, H. Y. ACS Appl. Mate. Inter. 2017, 9, 6796-6803. (13) Song, Z. G.; Kwok, R. T. K.; Zhao, E. G.; He, Z. K.; Hong, Y. N.; Lam, J. W. Y.; Liu, B.; Tang, B. Z. ACS Appl. Mate. Inter. 2014, 6, 17245-17254. (14) Qian, Z. S.; Chai, L. J.; Tang, C.; Huang, Y. Y.; Chen, J. R.; Feng, H. Anal. Chem. 2015, 87, 2966-2973. (15) Deng, J. J.; Yu, P.; Wang, Y. X.; Mao, L. Q. Anal. Chem. 2015, 87, 3080-3086. (16) Liu, S. G.; Han, L.; Li, N.; Xiao, N.; Ju, Y. J.; Li, N. B.; Luo, H. Q. J. Mater. Chem. B 2018, 6, 2843-2850. (17) Sun, J.; Hu, T.; Chen, C. X.; Zhao, D.; Yang, F.; Yang, X. R. Anal. Chem. 2016, 88, 9789-9795. (18) Li, G. L.; Fu, H. L.; Chen, X. J.; Gong, P. W.; Chen, G.; Xia, L.; Wang, H.; You, J. M.; Wu, Y. N. Anal. Chem. 2016, 88, 2720-2726. (19) Zhao, J. H.; Wang, S.; Lu, S. S.; Sun, J.; Yang, X. R. Nanoscale 2018, 10, 7163-7170.


Page 6 of 8

Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry (20) Zhang, J. Y.; Lu, X. M.; Lei, Y.; Hou, X. D.; Wu, P. Nanoscale 2017, 9, 15606-15611. (21) Acuna, A. U.; Alvarez-Perez, M.; Liras, M.; Coto, P. B.; Amat-Guerri, F. Phys. Chem. Chem. Phys. 2013, 15, 16704-16712. (22) Jin, M.; Liu, X.; van den Berg, A.; Zhou, G.; Shui, L. Nanotechnology 2016, 27, 335102. (23) Obayashi, Y.; Iino, R.; Noji, H. Analyst 2015, 140, 5065-5073. (24) Ma, F.; Liu, W. J.; Liang, L.; Tang, B.; Zhang, C. Y. Chem. Commun. 2018, 54, 2413-2416. (25) Zhao, J. H.; Wang, S.; Lu, S. S.; Bao, X. F.; Sun, J.; Yang, X. R. Anal. Chem. 2018, 90, 7754–7760. (26) Han, X.; Li, S. H.; Peng, Z. L.; Othman, A. M.; Leblanc, R. Acs Sensors 2016, 1, 106-114. (27) Nandhikonda, P.; Heagy, M. D. J. Am. Chem. Soc. 2011, 133, 14972-14974. (28) Kim, K.; Park, C.; Kwon, D.; Kim, D.; Meyyappan, M.; Jeon, S.; Lee, J. S. Biosens. Bioelectron. 2016, 77, 695-701.

7 ACS Paragon Plus Environment


Page 8 of 8

for TOC only

8 ACS Paragon Plus Environment

Fluorometric and Colorimetric Dual-Readout Immunoassay Based on

Recommend Documents