Fluorescence Immunoassay Based on the Alkaline Phosphatase

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Fluorescent Immunoassay Based on the Alkaline Phosphatase-Triggered in Situ Fluorogenic Reaction of o-Phenylenediamine and Ascorbic Acid Dan Zhao, Juan Li, Chuanyun Peng, Shuyun Zhu, Jian Sun, and Xiurong Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05203 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Analytical Chemistry

Fluorescent Immunoassay Based on the Alkaline PhosphataseTriggered In Situ Fluorogenic Reaction of o-Phenylenediamine and Ascorbic Acid Dan Zhao,†‡ Juan Li,† Chuanyun Peng,† Shuyun Zhu,§ Jian Sun,*,‡ and Xiurong Yang‡ † School

of Environmental Engineering and Chemistry, Luoyang Institute of Science and Technology, Luoyang, Henan 471023, China ‡State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130012, China. §College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu City, Shandong Province, 273165, China. *Fax: +86 431 85689278. E-mail: [email protected] ABSTRACT: Inspired by the special reducing capability of ascorbic acid (AA), ascorbic acid 2-phosphate (AA2P) has been extensively utilized as a substrate in current alkaline phosphatase (ALP) activity assays owing to the ALP-triggered transformation of AA2P into AA. However, such assays usually require AA-related complicated and laborious synthesis and/or signal generation procedures. Herein, we report an interesting in situ fluorogenic interaction between o-phenylenediamine (OPD) and AA, which inspires us to put forward a novel and simple AA2P/OPD-participated fluorescence turn-on ALP activity assay for the first time, and then the corresponding ALP-based fluorescent enzyme-linked immunosorbent assay (ELISA) has also been developed by means of the conventional ELISA platforms. According to the convenient and facile detection process with clear response mechanism, our fluorogenic reaction-based assay exhibits good sensitivity, selectivity and excellent sensing performance, which ensures fluorescent ELISA to potentially apply in the clinic diagnosis by employing a well-studied biomarker of hepatocellular carcinoma, alpha-fetoprotein (AFP) as the model analyte. Such original ELISA via in situ formation of fluorophore from scratch gives a new sight to develop other potential immunoassay platforms in early clinic diagnosis by controlling the target antigens in the near future.

As the preferred strategy of immunoassay, enzyme-linked immune-sorbent assay (ELISA) simultaneously makes use of extremely high catalytic ability of labeled enzymes and high specific antibody-antigen binding affinity.1,2 Recently, the development of immunoassay techniques has witnessed the prosperity of ELISA on account of its low cost, extraordinary specificity and high-throughput analysis. And thus ELISA has been widely applied in foods quality control, clinical diagnosis, environmental monitoring, as well as general laboratory research.3-6 Alkaline phosphatase (ALP, EC 3.1.3.1) is one of the most widely used labeling tracer in ELISA to generate detectable signals due to its high catalytic activity, high turnover number, good stability, broad substrate specificity, and easy conjugation to antibodies.7-8 More impressively, ALP is an important hydrolytic enzyme in the phosphate metabolism and its abnormal level in serum is closely related to various diseases, including breast and prostatic cancer, bone disease, liver dysfunction, and diabetes.9-11 Thus, the exploration of novel protocols for detecting ALP activity with simple procedures, low cost and high sensitivity is of vital importance in measuring the ALP levels in clinical samples and designing ALP-triggered fluorescence immunoassays. On the basis of the unambiguous catalytic mechanism, various proposals have been established to detect the ALP activity, including electrochemistry, fluorimetry, colorimetry and surface enhanced Raman spectroscopy.12-16 Among these methods mentioned above, optical approaches, especially

fluorimetry featuring with reliability, high sensitivity, convenience, fast response, and accessible instrument requirement, are well-suited for high-throughput analysis and real-time detection. Usually, the fluorimetric methods for ALP detection are realized by comparing the fluorescence responses of the enzyme substrate and ALP-triggered hydrolysis product.17-19 For instance, many fluorometric sensors have been designed by using the fluorescence quenching ability of the ALP-resulting product and the discrimination of pyrophosphate and phosphate by Cu2+.20-22 Unfortunately, such turn-off modes are always associated with low signal outputs, formidable background, and relatively low sensitivity and selectivity By contrast, the fluorescent switch-on assays have attracted overwhelming attention due to the superior properties such as less false signals, excellent selectivity and enhanced sensitivity. In this regard, ascorbic acid 2-phosphate (AA2P) is one of the most frequently used specific substrates in the turnon ALP activity assays, which could be hydrolyzed and transformed into ascorbic acid (AA) in the presence of ALP.23,24 In comparison with AA2P, the enzymatic product AA exhibits more considerable reducing capability and can be dehydrogenized more easily under alkaline condition.25-27 By means of the ALP-catalyzed hydrolysis of AA2P and the reducing ability of AA, a series of booming fluorimetric ALP assays have been also established. Actually, various fluorescent indicators, including organic dyes, coordination polymers and functional nanomaterials such as polydopamine

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nanoparticles, semiconductor quantum dots (QDs), carbon dots, gold nanoclusters, and so on,28-32 have been reported to assess the ALP activity with turn-on fluorescence response. However, the synthesis, modification and purification of these organic probes or nanomaterials are typically tedious and time-consuming. Meanwhile, high costly or toxic constituents and complicated signal generation procedures may severely impede their practical applications. Undoubtedly, it is still challenging to explore more rapid and convenient assays for detecting ALP activity, especially taking advantage of direct simple reactions between the enzymatic product AA and certain commercially available reagents. Herein, we have discovered a simple and interesting phenomenon that the mixture solution of o-phenylenediamine (OPD) and AA exhibited intense blue fluorescence under alkaline conditions with a featured emission peak centered at about 425 nm. More impressively, we have further verified that no fluorescent product can be obtained if using AA2P instead of AA or using p-phenylenediamine (PPD) and mphenylenediamine (MPD) instead of OPD. In virtue of the ALP-triggered hydrolysis of AA2P into AA and such specific fluorogenic reaction between OPD and AA, we have successfully designed a convenient ALP assay by using AA2P as substrate. As a proof of concept, ALP-triggered fluorescence ELISA using α-fetoprotein (AFP) as a model antigen target has been further constructed. The extent of the fluorescence enhancement was directly associated with the AA level released from ALP reaction (i.e., the amount of ALPsecondary antibody conjugates), and was thus correlated with the AFP concentration indirectly. In addition, this fluorescence ELISA was also used to detect patient serum samples for investigating the efficiency and robustness. Such facile and specific reaction between OPD and AA allows the appearance of strong fluorescence from scratch, endowing the presented ALP assay and ALP-enabled ELISA method with special selectivity and sensitivity. EXPERIMENTAL SECTION Materials and Apparatus. Sodium L-ascorbate, ophenylenediamine (OPD), tris(hydroxymethyl)aminomethane (Tris), sodium vanadate (Na3VO4), and L-ascorbic acid 2phosphatetrisodium salt (AA2P) were obtained from Aladdin Industrial Corporation (Shanghai, China). p-Phenylenediamine (PPD) and m-phenylenediamine (MPD) were bought from Beijing Chemical Reagent Corporation (China). Alkaline phosphatase from bovine intestinal mucosa (ALP), lysozyme, casein, trypsin, papain, human serum albumin (HSA), glucose oxidase (GOx), acetylcholinesterase (AChE), endonuclease I (ECoR I), human immunoglobulin G (IgG), bovine serum albumin (BSA), and pepsin were purchased from SigmaAldrich. ALP-conjugated goat antirabbit secondary antibody (ALP-ab2) was obtained from Abcam (Cambridge, MA). Alphafetoprotein (AFP), rabbit anti-AFP (Ab1) and mouse monoclonal antibody were supplied by ProSpec (Ness Ziona, Israel). Unless otherwise stated, chemicals in all the experiments were analytical reagent grade. High-purity water (18.2 MΩ) was used throughout the work. Fluorescence spectra and ultraviolet-visible (UV-vis) spectra measurements were carried out with an F-4600 fluorescence spectrofluorometer (Tokyo) and a Cary 500 UVvis-NIR spectrophotometer (Varian), respectively. Enzymatic assay for ALP Activity. The interaction between OPD and AA was performed by adding different amounts of AA stock solution into freshly prepared OPD

aqueous solutions. The fluorescence spectra were recorded after incubation at 37°C for 120 min. Typically, 100 μL of ALP with various activities was mixed with the substrate of 100 μL 25 mM of AA2P in pH 9.0 TrisHCl buffer solution (640 μL, 50 mM) containing 60 μL of 0.5 mM MgCl2. Following that, the above mixtures were reacted at 37°C for 50 min. Afterwards, 100 μL of OPD (10 mM) was introduced to the reaction system and the fluorescence intensities were monitored after vibration at 37°C for 120 min. To examine the specificity of our proposed strategy toward ALP activity, some competing proteins and enzymes were investigated instead of ALP under identical conditions. Fluorescence ELISA for AFP Detection Using the AA2POPD system. Our proposed assay toward target AFP by coupling with ALP-enabled fluorogenic reaction was performed as follows. First, each well of a 96-well plate was coated with 50 μL of mouse monoclonal antibody (1:200) at 4°C overnight. During this time, antibody can be firmly immobilized on the plate via the physical adsorption between hydrophobic groups of antibody and polystyrene plate.2,6 After removing the coating solutions and washing away the unbound antibody, 1% BSA blocking solution was introduced into each well and incubated for 60 min to prevent nonspecific adsorption, followed by washing again. Afterwards, 50 μL of AFP with the desired final concentrations (0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 20, 30, 40, 60, 80, 100, 120 and 150 ng/mL) were introduced and kept reacting at 37°C for 60 min to form the antigen-antibody complex. After washing as before, 50 μL of Ab1 (1:500) was injected and reacted at 37°C for 60 min to allow the formation the sandwiched immunocomplex. Subsequently, the plate was rinsed thoroughly with wash buffer followed by the addition of 50 μL of dilutions of ALPab2 (1:3000) and incubated for another 60 min at 37°C. Then, the wells were kept at 37°C for 50 min in Tris-HCl buffer containing 25 μL of 100 mM AA2P and 30 μL 0.25 mM of MgCl2. After that, 25 μL of OPD (10 mM) was injected into the wells and left to stand for 120 min at 37°C before the fluorescence spectra measurements. Detection of AFP in Clinical samples. The clinical samples were obtained from six volunteers including healthy people and patients with hepatocellular carcinoma (HCC) at the Second Hospital of Jilin University. Before detection, the patient serum samples were diluted 20 times. Subsequent procedures were carried out following the AFP assay just by replacing the AFP standards with serum samples. RESULTS AND DISCUSSION Fluorogenic Reaction between OPD and AA. Being a common phosphated molecule, AA2P is frequently utilized as the substrate of ALP, and the chemical or physical properties between AA2P and the corresponding enzymatic product AA could be distinguished by many studies.25-27 As a vigorous, simple and cheap aromatic 1,2-diamines, the colorless and non-emissive OPD is one of the most widely used organic molecular indicator and involved in various chromogenic and fluorogenic reactions.33-35 In this work, it is surprised that the mixture solution of OPD and AA displayed an apparent emission peak at about 425 nm when excited at 360 nm, generating a spectacularly intense blue emission under the ultraviolet light (Figure 1A). By contrast, the incubation of AA2P with OPD under alkaline environment could not induce any featured fluorescence emission (Figure S1). Additionally, the aqueous solution of sole AA2P, AA or individual OPD presented an ideal non-fluorescent state. This result further


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Analytical Chemistry revealed that the specific reaction between OPD and AA could lead to the fluorescence enhancement from scratch, and the high discrimination ability of OPD to distinguish AA and AA2P is the necessary prerequisite to the following ALP activity assay. In addition, we have speculated that the generation of the fluorophore might derive from the keto-form of AA and 1,2diamines structure of OPD. To make the mechanism more explicit, the reactions of various aromatic diamines including OPD, MPD and PPD with AA were investigated. It is demonstrated that MPD and PPD are unable to invoke similar fluorogenic reactions and thus no typical fluorescence can be obtained (Figure S2). In fact, there are several investigations described in detail that a category of biocompatible reactions between aromatic/heteroaromatic 1,2-diamines and αdiketones derivative would take place to yield quinoxaline core skeleton.36-38 In this case, once AA was introduced to the OPD solution under alkaline environment, it was readily dehydrogenized and the keto-form of AA reacted with OPD to form an N-hetaeocyclic fluorophore. To further demonstrate the reaction between OPD and AA, UV-Vis spectra of different solutions were recorded. As shown in Figure S3, the aqueous solution of sole OPD lacked a characteristic peak while AA displayed an intense absorption at around 250 nm. After incubation with OPD, the original absorption of AA at 250 nm decreased and a new obvious absorption peak around 350 nm appeared, which was consistent with the characteristic absorption of the resultant quinoxaline of the afore-mentioned reactions in former literatures.36,39,40 By contrast, under the same conditions, the individual addition of either MPD or PPD into the AA solution can hardly generate apparent absorption peak around 350 nm. In addition, the structure of the major product derived from OPD and AA is 3-(1,2dihydroxyethyl)furo[3,4-b]quinoxalin-1(3H)-one (Figure 1B) and the detailed formation mechanism has been gradually ascertained.40 These results demonstrated the pivotal role of the OPD and AA in the formation of quinoxaline derivative and the specific fluorogenic reaction may lay the foundation for ALP activity assay. Otherwise, when the OPD concentration was fixed with 1 mM, the influence of the concentration of sodium ascorbate on the fluorescence intensities of the as-formed fluorophore was also studied. Upon the addition of an increasing concentration, the fluorescence intensities of the resultant quinoxaline gradually enhanced (Figure S4) and the concentrationdependent fluorescence response provided a possibility for implementing versatile bioassays for the subsequent detection of ALP activity. In addition, we compared the results of the sole OPD and the mixture of OPD and AA (Figure S5). During the continuous six days, the results showed negligible changes, demonstrating good reproducibility of our developed method.

Figure 1. (A) Absorption, fluorescence excitation, and emission spectra of mixture solution of OPD with AA. Inset: photographs of the mixture solution under visible (1) and ultraviolet light (2), respectively. (B) Schematic illustration of the fluorogenic reaction between OPD and AA. OPD, 1 mM; AA, 2 mM; pH 9.0 (50 mM Tris-HCl). The absorption spectrum was measured by diluting the mixture solution 10 times.

Figure 2. (A) Schematic illustration of the ALP-triggered enzymatic and fluorogenic reaction. (B) Fluorescence emission spectra of the individual OPD (a), OPD+ALP (b), OPD+AA2P (c), OPD+AA2P+ALP (d) and OPD+Pi (e) in 50 mM Tris-HCl buffer at pH 9.0 containing 30 μM Mg2+. Inset illustrates the corresponding photographs under 365 nm ultraviolet light. OPD, 1 mM; AA2P, 2.5 mM; Pi, 2.5 mM and ALP, 100 mU/mL. λex =360 nm.



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Figure 3. (A) The fluorescence responses of our developed strategy upon addition of various activities of ALP activity. (B) Plots of the fluorescence intensity versus ALP concentration, and (C) the corresponding photographs of this strategy toward different concentrations of ALP under 365 nm ultraviolet light. Inset in B illustrates the linear fitting plots for ALP detection. (D) The fluorescence signals of the method upon addition of the competing components (20 μg/mL) with (red) and without (black) the co-existence of 40 mU/ mL ALP. OPD, 1 mM; AA2P, 2.5 mM and Tris-HCl buffer (pH 9.0, 50 mM). λex =360 nm.

Principle of ALP Activity Assay Based on the Fluorogenic Reaction. Figure 2A schematically illustrates the enzymatic and fluorogenic processes for ALP activity assessing based on the enzymatic product AA-triggered in situ fluorogenic reaction. In this strategy, ALP could trigger the dephosphorylation reaction of the enzyme substrate AA2P, thus yielding AA to react with non-emissive OPD and allowing the subsequent formation of quinoxaline fluorophore. The nearly colorless resultant solution shows an intense blue fluorescence, visible to the naked eye under ultraviolet light at 365 nm. Thus, by means of the ALP-catalyzed dephosphorylation of AA2P and the AA-controllable in situ fluorogenic reaction, superior performance of our fluorescence method for sensing ALP could be expected based on the obvious discriminability between AA2P and AA. Following the design, to further understand the exact mechanism of the developed AA2P/OPD-based protocol for sensing ALP, several control tests were implemented. As shown in Figure 2B, the sole OPD solution (1 mM) presented a non-fluorescent state and the addition of individual AA2P or ALP could not have obvious effect. As expected, only when AA2P and ALP were simultaneously added, substantial enhanced fluorescence readout could be observed due to the generation of AA. This reaction, however, in alkaline conditions yields one more hydrolysate, phosphate ion (Pi), which can also be used to quantify the ALP activity in the recent reports.6,41 In addition, the fluorescence emission spectrum of the mixture of OPD and Pi was almost identical to that of the individual OPD, which illustrated that there was no obvious interaction between OPD and Pi. Besides, the fluorescence color change under ultraviolet light also unequivocally demonstrated the corresponding process (inset image, Figure 2B). Summing up the above, the ALP-triggered liberation of AA from the AA2P substrate contributed to the emergence of fluorescence from scratch. Quantitation Analysis of ALP Activity. To assess the extreme sensing ability of our ALP-enabled fluorogenic

reaction system, the concentration of AA2P and activator (MgCl2), pH, temperature, enzymatic and fluorogenic reaction time have been investigated in detail and optimized. The value of ΔF was explored to evaluate and appraise the performance of the proposed fluorescent sensor, where ΔF represented the difference of fluorescence intensity at 425 nm of the incubation solution in the presence and absence of ALP. As the pH values have significant effect on the catalysis efficiency of enzyme and alkaline environment was beneficial to the ALP-catalyzed enzymatic process and fluorogenic reaction, the influence of the pH values ranging from 7 to 10 was firstly investigated. The results in Figure S6A indicated that the ΔF reached maximum as the pH value located at 9.0, and thus pH 9.0 is the optimal value. Temperature is another crucial factor for enzyme-based reactions. It was found that the maximum fluorescence signal could be achieved when the temperature of the enzymatic reaction was kept at 37°C (Figure S6B). We speculated that a higher temperature will inhibit the enzyme activity, resulting in the thermal deactivation of ALP and thereby impeding the whole reaction. Because the substrate concentration directly determines the liner range and detection limit of ALP, AA2P concentration was assessed and optimized to be 2.5 mM (Figure S6C). More importantly and impressively, it was reported that an appropriate level of Mg2+ could effectively activate the ALP activity and prevent its autolysis.42,43 Our results in Figure S6D showed that higher concentration of Mg2+ could induce a more marvelous enhancement of the fluorescence at 425 nm, but has litter impact on the OPD-AA2P system without ALP. However, the fluorescence intensities had litter change when adding Mg2+ concentration more than 30 μM, and this concentration was used in the following assay experiments. Additionally, by compromising the resultant fluorescence intensity of the fluorescent quinoxaline derivative and time consumption, we chose 50 min as the best time point for the reaction between AA2P and ALP and 120 min for the fluorogenic reaction (Figure S7).


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Analytical Chemistry Under the aforementioned optimized conditions, the analytical performance of our proposed assay by analyzing various activities of ALP was studied and demonstrated in Figure 3. As designed, the fluorescence intensity centered at 425 nm increased gradually with increasing ALP activities, due to the gradual hydrolysis of AA2P and the increase of produced AA concentrations (Figure 3A). Furthermore, when the ALP concentration was 2 mU/mL, an obvious fluorescent color change could be easily read out with the help of a UV lamp. The plot of the fluorescence responses as a function of the ALP concentration with a steady-state at 100 mU/mL was demonstrated in Figure 3B. In particular, there is a quasi-linear relation between the fluorescence values and the ALP activities. In the concentration range of 0.1−30 mU/mL, the liner fitting equation could be expressed as F =6.82 + 2.56 [ALP] (mU/mL), R2=0.993. The limit of detection (LOD) was calculated to be 0.06 mU/mL, which was comparable or superior to other existing methods (Table S1). Moreover, none of these control non-specific proteins/enzymes could bring apparent fluorescence impact as ALP did and interfere in the recognition response of ALP (Figure 3D). However, the detection principle of the proposed method is based on the AA-participated fluorogenic reaction, and thereby the interference study of two analogous molecules of AA including uric acid (UC) and dopamine (DA) were investigated. As shown in Figure S8, neither of them could generate a significant fluorescence response owing to the absence of αdiketones form under alkaline environment. Such results unambiguously confirmed the ideal selectivity of the reaction between OPD and AA. Considering the threshold of total ALP in human serum is approximate 40 mU/mL, the as-established assay could satisfy the need of clinical diagnostics in biological samples. Inspired by the excellent performance of this fluorescence assay for ALP activity, we attempt to challenge the proposed biosensor in diluted human serum (1%) samples. Table S2 demonstrated the experimental results of the serum and ALPspiked serum samples. Using this method, the ALP concentration in the two undiluted serum samples was calculated to be 86 and 135 mU/mL, respectively, which lies in the normal range of 40-190 mU/mL.6 It was noteworthy that our proposed method yielded satisfactory recoveries between 99.4% and 111.7% within the relative standard deviations (RSDs) from 2.45% to 4.87%. The above results demonstrated that the established method had high reliability and accuracy in real clinical samples. In fact, our developed ALP strategy could be extended to inhibitor screening by employing a well-known ALP inhibitor (Na3VO4). As depicted in Figure 4A, by increasing concentrations of Na3VO4 with constant activity of ALP (30 mU/mL), the fluorescence intensities of the sensing system displayed concentration-dependent decrease, demonstrating the conspicuous inhibitory effect of Na3VO4 on the ALP activity. Figure 4B manifested the sigmoidal profile of the fluorescence responses as a function of the logarithm of the concentration of Na3VO4. The IC50 value (concentrations of inhibitor when the ALP activity is inhibited by 50%) was estimated to be 108.6 μM, which was in accordance with the values reported previously.13,16,21-25

Figure 4. (A) Fluorescent emission spectra of our method after addition of various concentrations of Na3VO4-treated 30 mU/mL ALP. (B) Calibration plots of the corresponding fluorescence intensity versus the logarithm of the Na3VO4 concentration. OPD, 1 mM; AA2P, 2.5 mM and Tris-HCl buffer (pH 9.0, 50 mM). λex =360 nm.

ALP-Triggered Fluorescent ELISA Strategy for AFP Detection. The detection of cancer biomarkers is very important for the early cancer treatment which could potentially improve the cure rate of patients. For instance, it is generally known that AFP is a valuable biomarker for HCC patients by monitoring cancer progression.44,45 Most significantly, the immunoassay has become the most extensively used method for AFP detection, and ELISA has aroused widespread concern due to the enzyme-catalyzed signal amplification effect. Inspired by the successful and extensive application of ALP as a biocatalytic label in ELISA, we extended the developed ALP assay to design a fluorescent sandwich ELISA for proof-of-concept. As shown in Figure 5A, the mouse anti-AFP monoclonal antibody, rabbit anti-AFP, and goat anti-rabbit IgG labeled with ALP were employed as the capture antibody, primary antibody, and secondary antibody, respectively. In addition, we also evaluated the response signals of the antibody and antibody-ALP conjugates toward the as-proposed system. As shown in Figure S9, the results unequivocally imply that neither of them could evoke significant fluorescence change of the as-resulted fluorophore. Therefore, a fluorogenic reaction-based fluorescent immunoassay could be designed and proposed. Following the routine co-immobilization of our used antigens and antibodies onto a 96-well plate, AA2P was injected to generate AA, and then OPD was introduced to give a fluorescent readout signal. In this regard, a higher AFP concentration led to the capturing of more ALP on the 96-well plate, thus generating more AA and subsequently more intense fluorescent readout signal. As expected, the fluorescence intensity progressively increased from the non-fluorescence state with the increasing of AFP concentration (Figure 5B), demonstrating a gradual binding of ALP labeled on antibody. The inset graph showed a quasilinear relationship between the fluorescence intensity and AFP concentrations from 0.5−40 ng/mL, and then the fitted linear data could be expressed as F = 7.65 + 1.17 CAFP (ng/mL), R2=0.997. The LOD was obtained as low as 0.21 ng/mL of AFP, which was comparable or much lower than those previous reports for AFP detection (Table S3).



Figure 5. (A) Fluorescent detection of AFP by coupling the conventional ELISA and ALP-guided fluorogenic reaction. (B) Fluorescence emission spectra at 425 nm in the presence of different concentrations of AFP. (C) Fluorescence intensities of our proposed assay versus the concentrations of AFP. Inset: response linearity of the assay at low AFP concentrations. (D) Selectivity of our ELISA for AFP (80 ng/mL) over other competing components (200 ng/mL). OPD, 1 mM; AA2P, 2.5 mM and Tris-HCl buffer (pH 9.0, 50 mM). λex =360 nm.

To validate the analytical reliability of our designed sensor in complex biological samples, high specificity is the basic requirement to distinguish AFP from other non-specific proteins. The comparative study was performed by measuring the low-concentration target AFP and high-concentration interfering components. As shown in Figure 5D, the potential interfering proteins induced negligible change in the fluorescence signal, and only AFP could evoke a convincing fluorescence response due to the antigen-antibody specificity recognition, manifesting the excellent selectivity of our assay toward AFP detection. Detection of AFP in Diluted Human Serum Samples. Encouraged by the outstanding discrimination capability and sensitivity of the fluorescent ELISA, we further employed this assay in determining AFP in real clinical samples. The AFP levels in six different human serum samples were measured by using our ALP-enabled ELISA sensing system and commercialized pNPP-based standard ELISA kit (Table S4). Considering that the AFP concentrations in human serums of patients are outside of the linear range of the proposed protocol, the clinical samples were diluted 20 times in order to reduce AFP to a suitable concentration. It was noted that AFP concentration ranges in these clinical samples measured here coincided well with those obtained by other developed AFP systems.13,18-23 In addition, the data analyzed by our proposed system coincided well with that by using commercial pNPPbased standard ELISA kit. It is unambiguously demonstrated that our proposed assay manifested high AFP assessing capability in real samples, and thus possessed great prospect in clinical diagnosis of disease biomarkers with excellent performance. CONCLUSION

In summary, we have developed a straightforward and extendable (or even universal) strategy to detect ALP activity, ALP inhibitor, and immunoassay of AFP with simple signalgeneration process and extraordinary analytical performance. By coupling with the ALP-triggered hydrolysis of AA2P into AA and the in-situ formation of the fluorescent quinoxaline derivative originated from the reaction between OPD and AA, a well-defined label-free fluorescent assay for ALP activity has been rationally developed. ALP has been employed as an enzyme-labelled tracer to construct a fluorescent ELISA for the sensitive quantitation of AFP. Benefiting from the excellent sensing performance for ALP with LOD of 0.06 mU/L, the fluorescent immunoassay achieved a detection limit of as low as 0.21 ng/mL of AFP. Besides, our proposed assay could be successfully utilized to assess the AFP activity in serums, showing feasible potential in constructing extremely sensitive and specific clinical assays for cancer biomarkers detection. More importantly, the proposed assay is convenient, flexible, versatile and possesses unequivocal sensing mechanism and simple signal-generation procedures. We believe that this work could pave a new pathway to generate more versatile ELISA platforms of other cancer biomarkers in a similar fashion with the aid of their corresponding antibodies or antigens.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed fluorescence emission and absorption spectra of the sensing system, the fluorescence spectra and intensities of OPD in addition of varied concentrations of sodium ascorbate, fluorescence responses of OPD and OPD+AA


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Analytical Chemistry during the continuous six days, optimization of pH values, temperature, the concentrations of AA2P and MgCl2, enzymolysis time and fluorogenic reaction time for ALP detection, the interfere study of different concentrations of UC and DA, the influence of antibody or antibody-ALP conjugates on the fluorescence of the as-resulted fluorophore, comparison of performance of different ALP and AFP sensors, analytical results for ALP and AFP in the human serum samples. (PDF).

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

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Education Key Projects of Henan Provincial Department (19A150002), the Scientific Research Foundation of Luoyang Institute of Science and Technology (2017BZ16), the National Natural Science Foundation of China (Nos. 21605139, 21435005), and the Youth Innovation Promotion Association, CAS (No. 2018258).

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Fluorescence Immunoassay Based on the Alkaline Phosphatase

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