Fluorescence Immunoassay System via Enzyme-Enabled in Situ

Sep 22, 2016 - The emergence of fluorescent nanomaterials with excellent performances has triggered the development of fluorescence analysis technique...
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Fluorescence Immunoassay System via Enzyme-Enabled in Situ Synthesis of Fluorescent Silicon Nanoparticles Jian Sun,† Tao Hu,†,‡ Chuanxia Chen,†,§ Dan Zhao,†,§ Fan Yang,† and Xiurong Yang*,†,‡ †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China § University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: The emergence of fluorescent nanomaterials with excellent performances has triggered the development of fluorescence analysis technique, which possesses several advantages in the research and clinical applications. However, current strategies for fluorescence immunoassay usually involve the routine fluorophore-labeled antibody and/or awkward signal generation procedure that may not be available in conventional enzyme-linked immunosorbent assay (ELISA) systems. Herein, we circumvent this problem by imparting an exquisite signal generation mechanism to commercially available alkaline phosphatase (ALP)-based ELISA platform and putting forward a conceptual fluorescent ELISA system based on an original ALP-enabled in situ synthesis of fluorescent nanomaterials. After adding target antigen, the presence of ALP labeled on antibody catalyzes the transformation of the substrate ascorbic acid 2-phosphate into ascorbic acid. Then the resultant ascorbic acid (i.e., ascorbate) interacts with amine-containing silane molecules (no fluorescence) to produce intense cyan fluorescent silicon nanoparticles. For the proof-ofconcept, alpha-fetoprotein and human immunoglobulin G are chosen as the model antigen targets, and our proposed immunoassay (designated as the nanoparticles generation-based fluorescent ELISA) enables the detection with either fluorescence spectroscopy or naked-eye readout under the ultraviolet lamp. The convincing recognition mechanism and assay performance ensure fluorescent ELISA to quantitatively evaluate the alpha-fetoprotein level in serologic test and potentially apply in the clinic diagnosis of hepatocellular carcinoma.

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immunoassays in this category refer to complicated signal generation mechanisms and procedures, such as the immunoagglutination, fluorescence anisotropy/polarization measurements, competitive immunoreaction, fluorescence labeling of antibody, and so on.12−15 Meanwhile, the generally used fluorescent materials have been already extended from simple fluorescent organic dye or conjugated polymers into the fluorescent nanomaterials, including fluorophore-doped nanoparticles,12 semiconductor quantum dots,13,16 and even nearinfrared fluorescent single-walled carbon nanotubes.17 In fact, current fluorescent nanomaterial-based immunoassays typically employ the nanomaterials as a label to substitute for the enzymatic labeling in traditional ELISA. Consequently, the enormous efforts have been focused on the development of novel enzymes or substrates, the modification or conjugation of the nanomaterials, and the optimization of the fluorescence behaviors. To the best of our knowledge, however, a

mmunoassay is a primary analytical technique, which mainly relies on the high specificity and binding affinity between the antigen and antibody.1,2 By means of the corresponding antibody, an immunoassay can be engaged in the qualitative and quantitative evaluation of a certain analyte (usually as the antigen) in biological matrixes such as urine or serum.3 Until now, the conventional enzyme-linked immunosorbent assay (ELISA) has been known as the preferred method and extensively applied in laboratory research and clinical diagnosis.4,5 The routine ELISA usually uses an enzyme, typically horseradish peroxidase (HRP) or alkaline phosphatase (ALP), to catalyze the conversion of the enzyme substrate into a colored product, during which the signal change could be monitored by absorption spectroscopy and even naked-eye readout detection.6−8 With the technical progress for fluorescent materials, several current immunoassays have referred to the fluorescent substrates or the conversion of nonfluorescent substrates to the fluorescent products.9 As a supplement or substitute of conventional colorimetric ELISA, the fluorescent one has also been widely used in several research fields.10,11 However, most © XXXX American Chemical Society

Received: July 25, 2016 Accepted: September 13, 2016

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DOI: 10.1021/acs.analchem.6b02847 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry straightforward fluorescence immunoassay protocol directly taking advantage of the classical ELISA platform and commercially available enzyme conjugates (HRP or ALP) is still in its infancy.15,18 Moreover, there is even no report that a fluorescent ELISA system allows accurate measurement and naked-eye readout via ALP (or HRP)-enabled synthesis of fluorescent nanomaterials. As a promising kind of quantum dots, fluorescent silicon nanoparticles (Si NPs) are of emerging interest recently based on the superior optical properties and biocompatibility.19−21 The Si NPs prepared by the previously reported methods, however, hardly possess outstanding fluorescence property and aqueous dispersibility in the same case. This problem has already been circumvented by a recent pioneering study. By using amine-containing silane molecules, typically (3-aminopropyl) trimethoxysilane (APTMS), as the silicon source and trisodium citrate as the reductant, He et al. has developed an aqueous synthetic method to prepare fluorescent Si NPs under microwave irradiation (160 °C).22,23 Subsequently, a more convenient and gentle synthetic protocol for Si NPs can be carried out at ambient temperature and pressure, just using sodium ascorbate instead of trisodium citrate.24 Being a common reductant, ascorbic acid (ascorbate) has participated in several analytical systems during which it acts as either a target analyte or signal transmission medium.25,26 Therefore, the involvement of such vigorous reagent and the accessible reaction conditions enable that the synthetic process of Si NPs possesses an enormous potential in the development of a novel fluorescent assay. ALP is responsible for catalyzing the dephosphorylation of copious amounts of phosphate esters, and the ALP assay itself plays an important role in the diagnosis of many diseases.27,28 By means of its broad substrate specificity and high catalytic activity, a large number of routine ALP-based ELISA systems have been accessed in the laboratory research and clinical diagnosis.3,29 Recent advances provide several nanomaterialrelated colorimetric or plasmonic ELISAs based on the commercially available ALP-antibody conjugates and a specific substrate, ascorbic acid 2-phosphate (AA2P).26,30,31 In this regard, ALP triggers the removal of the phosphate group from AA2P, and the AA2P is transformed into the ascorbic acid. As a typical reductant, the ascorbic acid (ascorbate) can easily influence the morphology of the noble metal nanostructures and corresponding color of the solution through the redox reaction. However, it is relatively scarce for paying attention to the AA2P or ascorbate-triggered fluorescence intensity and morphologic changes of the fluorescent nanomaterials,15 especially from none to appearance of fluorescent nanoparticles. Inspired by the aforementioned AA2P-based colorimetric ELISAs and ascorbate-triggered synthesis of Si NPs, we present a novel conceptual fluorescent ELISA system based on ALPenabled in situ synthesis of fluorescent Si NPs herein. The developed assay has an unambiguous sensing mechanism and been first applied in the ALP activity sensing and inhibitor screening. In the subsequent proof of concept of fluorescent ELISA, alpha-fetoprotein (AFP) and human immunoglobulin G (IgG) are chosen as the model antigen targets.11 In the case of adding increased levels of antigen into the ELISA system, there should be more primary antibody and more ALPsecondary antibody conjugates binding with the antigen target proportionately. More significantly, the level of ALP activity (i.e., the amount of ALP-secondary antibody conjugates) is

directly related to the generated ascorbic acid (or ascorbate), likewise is indirectly related to the fluorescence intensity of the resultant solution (i.e., the production of Si NPs). Through monitoring the fluorescence of the resultant Si NPs solution, therefore, we have developed a synthetic process-related fluorescent ELISA for the first time, as far as we know. Meanwhile, the fluorescent change of the solutions also allows a straightforward and naked-eye readout under the ultraviolet lamp. In addition, AFP is the best-studied serologic marker for hepatocellular carcinoma (HCC) diagnosis and prognosis.32,33 The convincing assay performance ensures that our developed fluorescent ELISA can quantitatively analyze target AFP in real human serum samples in a fluorescence turn-on way. The serologic test results of normal adults and patients with HCC, being correlated well with those from the pNPP-based standard ELISA method, clearly indicate that this nanoparticles generation-based fluorescent ELISA enables a fluorescence diagnosis of HCC.

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EXPERIMENTAL SECTION Chemicals and Materials. Sodium L-ascorbate, (3aminopropyl)trimethoxysilane, L-ascorbic acid 2-phosphate trisodium salt, sodium orthovanadate, magnesium chloride, hydroxylammonium chloride, hydrazine monohydrate, and sodium hydroxide were purchased from Aladdin Industrial Corporation (Shanghai, China). Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa, acetylcholinesterase, bovine serum albumin, casein, choline oxidase, glucose oxidase, lysozyme, human serum albumin, human immunoglobulin G (IgG), peroxidase from horseradish, trypsin, tetrakis(hydroxymethyl)phosphonium chloride, sodium borohydride, sodium citrate tribasic dihydrate, diethanolamine, and 4nitrophenyl phosphate disodium salt hexahydrate (pNPP) were purchased from Sigma-Aldrich (St. Louis, MO). Alphafetoprotein (AFP) was bought from ProSpec (Ness Ziona, Israel). Rabbit anti-AFP was purchased from Proteintech (Wuhan, China). Rabbit anti-IgG was purchased from Santa Cruz (CA). ALP-conjugated secondary antibody was purchased from Abcam (Cambridge, MA). Both the wash buffer and antibody diluent buffer for ELISA were purchased from Boster (Wuhan, China). All reagents were analytical grade and used as received without any further purification. Ultrapure water from a Millipore system was used in all aqueous solution. Apparatus and Characterization. Fluorescence excitation and emission spectra of all samples were recorded on a Hitachi F-4600 spectrofluorometer (Tokyo, Japan). TEM and HRTEM measurements were taken using a FEI Tecnai G2 F20 S-TWIN (OR). Absorption spectra were obtained with a CARY 500 UV−Vis-NIR Varian spectrophotometer (CA). FT-IR spectra were obtained on a Bruker Optics VERTEX 70 spectrometer (Ettlingen, Germany) in the transmission mode. Synthesis and Purification of Fluorescent Si NPs. The silicon nanoparticles were prepared by a modified protocol described previously.24 In brief, APTMS (1.6 mL) and a solution of sodium ascorbate (2.0 mL, 100 mM) were successively added into water (6.4 mL). Then the mixture was left to stand for 30 min at room temperature without stirring, during which the colorless solution gradually turned pale yellow. The as-prepared transparent solution was purified with a 3000 Da cutoff dialysis bag against ultrapure water for more than 24 h, and then the precipitation emerged in the dialysis procedure was removed by using a membrane filter (pore size, 0.45 μm). B

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Figure 1. (A) Schematic representation of the preparation of fluorescent Si NPs from APTMS and sodium ascorbate. (B) Absorption (black), fluorescence excitation (red), and emission (blue) spectra of the Si NPs in aqueous solution. Inset shows the corresponding photographs under ambient (left) and 365 nm light (right), respectively. (C) Typical TEM image of the as-prepared Si NPs. Insets show the HRTEM image and size distribution histogram.

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Sensing ALP Activity. A fluorescent alkaline phosphatase activity assay was performed using the following procedures. Volumes of 200 μL of diethanolamine buffer (DEA, 2.0 M, pH 9.8), 100 μL of AA2P (100 mM), 100 μL of MgCl2 (5 mM), and 240 μL of water were injected into a 1.5 mL microcentrifugal tube. The 200 μL of freshly prepared ALP aqueous solutions with different activities ranging from 0 to 750 mU/mL were then added into the mixtures, respectively. Following incubation at 37 °C for 60 min, these ALP-treated AA2P solutions were mixed with APTMS (160 μL), and then the above mixture solutions were left to stand at room temperature for 20 min before the fluorescence spectra measurements. The selectivity of this sensing system for ALP activity was assessed by using other control proteins/enzymes instead of ALP. Fluorescence Immunoassay Strategy for the Model Protein. Prior to the fluorescence immunoassay, the 96-well microplates were modified and functionalized as follows: first, 100 μL of AFP standards with various concentrations were added into wells and incubated at 4 °C overnight. (Note: After each following step until the enzyme reaction, the wells were rinsed with the wash buffer three times.) Second, the wells were blocked with 5% BSA at 37 °C for 1 h. Third, 100 μL of diluted rabbit anti-AFP (1:500) in antibody diluent buffer was added. After incubation for 30 min, we added 100 μL of goat antirabbit secondary antibody labeled with ALP (1:500), and the microplates were incubated at 37 °C for 30 min. Subsequently, 50 μL of DEA (2.0 M, pH 9.8), 25 μL of AA2P (100 mM), 25 μL of MgCl2 (5 mM), and 110 μL of water were added into the wells for an enzyme reaction at 37 °C for 1 h. And then 40 μL of APTMS was mixed with the above solution in the wells and reacted at room temperature for 20 min before the fluorescence spectra measurements. The detailed procedures for sensing another model protein, human IgG, were based on that for the AFP by just changing the addition of AFP standards and rabbit anti-AFP to IgG standards and rabbit anti-IgG, respectively. Fluorescence Immunoassay for AFP in Real Human Serum Sample. The developed fluorescence immunoassay system was applied to detect the level of AFP in the clinical blood samples (containing three human normal serum samples and five human hepatocellular carcinoma serum samples), which were kindly supplied by The Second Hospital of Jilin University. The detailed procedures of the fluorescence immunoassay referred to that for the model protein by just adding the human serum instead of the AFP standard solution.

RESULTS AND DISCUSSION Synthesis and Characterization of Si NPs. As presented in Figure 1A, incorporating APTMS as the silicon source and sodium ascorbate as the reductant, fluorescent Si NPs can be facilely prepared based on the previous report with several modifications.24 After mixing APTMS and sodium ascorbate in aqueous solution without stirring, the nearly colorless mixture has gradually turned pale yellow and generated a spectacularly intense cyan emission under ultraviolet light within minutes (inset of Figure 1B). The typical absorption spectrum of the aspurified Si NPs in aqueous solution shows that there is an obvious absorption band around 350−400 nm and the absorbance gradually increases with a decrease in the wavelength. Likewise in Figure 1B, the fluorescence spectra of the Si NPs exhibit maximum emission wavelength around 515 nm and excitation wavelength around 400 nm, which correlates well with the aforementioned absorption spectrum. The relative quantum yield of the as-purified fluorescent Si NPs in aqueous solution was measured to be approximately 19.0% with quinine sulfate as the reference, using a modified method described previously for similar Si NPs.35 Figure 1C presents a representative transmission electron microscopy (TEM) micrograph of the well monodisperse Si NPs with a high-resolution image of an individual particle displayed in the inset. The lattice planes in the high-resolution TEM (HRTEM) image are separated by ∼0.31 nm, corresponding to the (111) lattice spacing of the cubic crystalline silicon.23,34 The size distribution shows that the mean diameter of spherical particles is 2.38 nm with a standard deviation of 0.34 nm, as estimated from TEM image analysis of 100 individual particles. In addition, the Fourier transform infrared (FT-IR) spectrum of the freeze-dried Si NPs is shown in Figure S1. It exhibits the characteristic peaks of Si−O−Si and Si−O−C stretching vibration (at 1000−1200 cm−1), further confirming the presence of silicon atoms in the as-prepared sample.23,35 At the same time, it is illustrated that the Si NPs possess several other functional groups such as the N−H (bending vibration around 1600 cm−1, stretching vibration at 3300 cm−1), C−H (stretching vibration between 2850 and 2950 cm−1), and O−H (stretching vibrations at 3400 cm−1) on the surface. Assessment of Sensing Strategy for ALP Activity Based on Si NPs. Under the similar experimental conditions, no fluorescent product can be achieved if we use other common reductants such as NaBH4, THPC, N2H4·H2O, NH2OH, or trisodium citrate instead of sodium ascorbate to react with APTMS (Figure 2A). These results illustrate that C

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and ascorbic acid as a transducer component of the Si NPsbased fluorescence signal. Prior to the ALP activity sensing, we have evaluated the effect of sodium ascorbate concentration on the synthesis of fluorescent Si NPs at a constant APTMS concentration. The results in Figure 2B and Figure S2 show that the absorbance and fluorescence of resultant Si NPs solution gradually enhance with the increasing concentration of sodium ascorbate, and the concentration-dependent fluorescence intensity provides a possibility for the subsequent quantitative ALP activity assay. As well-known, diethanolamine buffer (DEA, pH 9.8) is usually used as the buffer in the enzymatic assay of ALP.31 Fortunately, such strongly alkaline solution and DEA molecule do not influence the generation of fluorescent Si NPs from APTEM and sodium ascorbate (Figure 2C). Subsequently, several control experiments have been undertaken to further evaluate our proposal. As indicated from Figure 2D, there are similar experimental results in the absence and presence of the target ALP alone, substrate AA2P alone, and Mg2+ ions alone (typical activater for enzyme activity of phosphatase36). Therefore, the fluorescent bioassay for ALP activity based on ascorbate (i.e., ascorbic acid, the hydrolysate of AA2P)-triggered synthesis of Si NPs could be developed and utilized in the standard DEA buffer containing Mg2+ ions (Figure 3A). Selective and Sensitive Fluorescence Turn-On Assay for ALP Activity. The assay of ALP activity has been carried out by using the AA2P−APTMS sensing system in 400 mM DEA buffer at pH 9.8 containing 500 μM Mg2+ ions. Under these conditions, the reaction time of both ALP-induced AA2P hydrolysis and ascorbate-controlled generation of fluorescent Si NPs should be investigated and optimized in detail. Comprehensively considering the resultant fluorescence intensity of the analytical solution in Figure S3 and the bioassay efficiency, we choose 60 min as the optimum enzymatic reaction time and 20 min for the subsequent in situ synthesis reaction. The fluorescence of the AA2P−APTMS sensing system in the presence of other nonspecific proteins/ enzymes such as acetylcholinesterase (AChE), bovine serum

Figure 2. (A) Fluorescence emission intensities at 515 nm and photographs under 365 nm light (inset) of the APTMS mixed (1) without reductant, with (2) NaBH4, (3) THPC, (4) N2H4·H2O, (5) NH2OH, (6) trisodium citrate, and (7) sodium ascorbate, respectively. (B) Fluorescence intensities of APTMS toward sodium ascorbate with different concentrations. (C) Fluorescence emission spectra and photographs (inset) of APTMS (1), APTMS + DEA (2), APTMS + sodium ascorbate (3), and APTMS + sodium ascorbate + DEA (4). (D) Fluorescence emission spectra and photographs (inset) of the APTMS mixed without (1−4) and with (5−8) sodium ascorbate in the presence of no additional agent (1, 5), Mg2+ (2, 6), ALP (3, 7) and AA2P (4, 8), respectively, in DEA buffer (pH 9.8).

sodium ascorbate plays a pivotal role in this convenient Si NPs synthesis. In fact, ascorbic acid or ascorbate, being an essential nutrient, has been not only involved in several biological and physiological processes but also used as the target analyte or signal transmission medium in a range of analytical system of biologically relevant molecules.25,26 Inspired by the fact that the ALP is capable of catalyzing the hydrolysis of AA2P into ascorbic acid, we have designed and developed a fluorescent sensing assay for ALP activity by using AA2P as the substrate

Figure 3. (A) Schematic representation of the fluorescent biosensor based on ALP-enabled synthesis of Si NPs. (B) Fluorescence responses of the sensing system against the control enzymes/proteins (10 μg/mL) in absence and presence of ALP (50 mU/mL). The fluorescence emission spectra (C), fluorescence intensities at 515 nm (D), and the corresponding photographs under 365 nm light (E) of the sensing system toward ALP standards with various activities. Inset in part D presents the linear response of the sensing system to ALP activity. D

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The nanoparticles generation-based ALP activity assay can be further extended in assessment of the enzyme inhibitor efficiency in vitro by using a common inhibitor (sodium orthovanadate, Na3VO4) as a model.37,38 The control experiments have ascertained that the addition of Na3VO4 alone does not significantly affect both the enzymatic reaction and the subsequent synthesis reaction (data not shown). The procedures of the ALP inhibition assay are based on that of ALP activity sensing assay by just changing 200 μL of ALP solutions into 100 μL of 300 mU/mL ALP activities incubated with 100 μL of various concentrations Na3VO4. As depicted in Figure S4, with the Na3VO4 concentrations increasing from 0 to 40 mM, the ALP activity has been efficaciously inhibited and the fluorescence of the sensing system exhibits a gradual decrease. A typical sigmoidal profile has been observed from the plots of fluorescence intensities versus the logarithm of the Na3VO4 concentration (Figure 4B). Under current assay conditions, the IC50 value (the inhibitor concentrations where ALP activity is inhibited by 50%) is calculated to be approximate 201 μM, which is of the same order of magnitude as those previously reported by other ALP activity assays.37,39 Fluorescent ELISA Strategy via ALP-Enabled Synthesis of Si NPs. Inspired by the successful and extensive application of ALP in ELISA, the highly selective and sensitive ALP activity sensing system has a great potential to be extended into the ALP-labeled immunoassay for target antigen. Figure 5A schematically illustrates the recognition principle of the proposed fluorescent ELISA, which has been used to detect a model antigen protein (AFP). The adopted antigen, primary antibody, and secondary antibody correspond to AFP, rabbit anti-AFP, and goat antirabbit IgG labeled with ALP, respectively. Meanwhile, we have used AA2P as the substrate and ALP as the readout enzyme. The control experiments clearly illustrate that the presence of our used antibody alone and antibody−ALP conjugates alone do not affect the generation and fluorescence of Si NPs (Figure S5). Therefore, a Si NPs-related fluorescent ELISA can be established and developed. The procedures of the proposed fluorescent ELISA are similar to that of conventional ones. Initially, the tested sample solution containing AFP is added into the well of microplate, where it is given time to adhere to the well through nonspecific physical adsorption. Subsequently, on the basis of the specific antigen−antibody recognition, the rabbit anti-AFP antibody can bind specifically to the AFP adsorbed on the microplates and the goat antirabbit IgG antibody labeled with ALP binds specifically to the primary antibody as well. After immobilization of the antigen and antibodies on the 96-well microplates, AA2P has been introduced into the wells. Through the hydrolysis of AA2P by the labeled ALP, the hydrolysate ascorbic acid (i.e., ascorbate) acts as the reductant to in situ generate fluorescent Si NPs in the presence of APTMS, thus emerging an antigen-dependent change in the fluorescence of the resultant solution. With the AFP concentrations increasing from 0 to 150 ng/mL, the fluorescence emission of the reaction solution exhibited a gradual enhancement (Figure 5B). The fluorescence intensities at 515 nm plotted against AFP concentration are shown in Figure 5C, presenting a quasilinear relationship over the range of 1−60 ng/mL, and a plateau at higher activity levels (typically >70 ng/mL). The fitted linear equation is described as I = 0.719 + 0.138CAFP (ng/mL), R2 = 0.979. Our proposed fluorescent ELISA strategy is quite sensitive, where 1 ng/mL AFP can be easily detected by using

albumin (BSA), choline oxidase (COD), glucose oxidase (GOx), peroxidase from horseradish (HRP), lysozyme, and trypsin were explored to investigate the specificity of the ALP assay. The fluorescent results in Figure 3B have unambiguously elucidated that none of these control proteins/enzymes induces convincing fluorescence response in the Si NPs generationbased sensing system and interferes in the recognition response of ALP. We have further evaluated the sensing performance for fluorescence turn-on sensing of ALP and the fluorescence spectra of the obtained Si NPs after introducing ALP standards with various activities from 0 to 150 mU/mL are shown in Figure 3C. With the increase of the added ALP activity, the fluorescence emission intensity at 515 nm of the resultant solution gradually increased (Figure 3D). In this regard, there is a quasi-linear relationship between fluorescence intensities and ALP activities over the range from 0.2 to 30 mU/mL, where the fitted linear equation can be described as I = 0.844 + 0.532CALP (mU/mL), R2 = 0.986. It is noteworthy that 0.2 mU/mL of ALP activities can be easily identified by the fluorescence spectrometer. Besides, the fluorescent color change of the solutions induced by 1 mU/mL of ALP activities can be found out with naked-eye readout under the ultraviolet lamp (Figure 3E). Furthermore, our developed Si NPs generation-based fluorescent sensing system has been compared with the pNPP-based standard colorimetric assay for testing ALP activity in the diluted fetal bovine serum (2%). As shown in Figure 4A, the experimental results in the diluted biological samples obtained from two types of assays are similar, which have clearly demonstrated that our proposed fluorescence assay owns great potential to monitor the level of ALP activity in real biological samples.

Figure 4. (A) Comparison of the assay results toward ALP in 2% fetal bovine serum by using the proposed fluorescent assay and the standard pNPP-based colorimetric method, respectively. (B) Kinetic plots of the fluorescence intensity of sensing system in the presence of 30 mU/ mL ALP against the logarithm of the Na3VO4 concentration. E

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Figure 5. (A) Schematic representation of the fluorescent ELISA strategy via ALP-enabled in situ synthesis of Si NPs. The fluorescence emission spectra (B), fluorescence intensities at 515 nm (C), and corresponding photographs under 365 nm light (D) of the ELISA toward AFP standards with various concentrations. (E) Fluorescence responses of the developed ELISA against AFP or other control enzymes/proteins (100 ng/mL).

the fluorescence spectroscopy. Particularly noteworthy is that the fluorescent color of the solutions in the wells gradually transfers from almost colorless to bright cyan with increasing AFP concentrations from 0 to 150 ng/mL. As depicted in Figure 5D, the fluorescent color change of the solutions allows a straightforward and naked-eye readout sensing of AFP with the detection limit lower than 5 ng/mL in virtue of the handheld ultraviolet-lamp. It is generally known that ELISA is based on a particular antibody with specificity for the target antigen (AFP in here). Therefore, it is almost certain that our immunoassay system has outstanding capability of the selective recognition and other nonspecific proteins, such as human IgG, HSA, BSA, casein, and lysozyme, have induced negligible influence on the resultant fluorescence (Figure 5E). To further evaluate the universal applicability of our proposed fluorescent ELISA, we have utilized this strategy to detect another common model antigen protein, human IgG, just by choosing IgG standards and rabbit anti-IgG instead of AFP standards and rabbit anti-AFP. The fluorescence results in Figure S6, accompanied by that in Figure 5, unambiguously illustrate that our Si NPs generation-based fluorescent ELISA system not only can sensitively detect human IgG like AFP but also has the potentiality in being extended to fluorescently determine several other target analytes. Determination of AFP in Real Human Serum Sample. It is a well-known fact that our model protein, AFP, is an important and acknowledged serologic marker used for hepatocellular carcinoma (HCC) surveillance.11,32,40 The serum AFP level for normal adults is generally below 20 ng/ mL, even lower than 5 ng/mL in several previous reports.32,41 However, abnormally elevated AFP is usually observed in the serum of patients with HCC (typically >400 ng/mL). The feasibility of our developed system for potential clinical application has been assessed by using real human serum samples from three normal adults and five patients with HCC. Using our fluorescent ELISA, the AFP concentrations in eight serum samples were detected and calculated. As shown in Table

S1, the results are consistent with the results from the clinical diagnosis and pNPP-based standard ELISA method, suggesting the great potential of our Si NPs generation-based fluorescent ELISA as an alternative to the conventional ELISA to be applied in the immunoassay.

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CONCLUSION Inspired by the ALP-triggered hydrolysis of the substrate ascorbic acid 2-phosphate as well as the product ascorbatecontrolled generation of Si NPs with intense cyan fluorescence, we have first developed a selective and sensitive fluorescence turn-on assay for the ALP activity sensing and inhibitor screening. On the basis of the conventional ELISA platform and commercially available antibody−ALP conjugates, a rational conceptual fluorescent ELISA system has been presented for the first time, employing the ALP-enabled in situ synthesis of Si NPs. Choosing AFP as the model antigen target, our proposed fluorescent ELISA can effortlessly evaluate 1 ng/mL of analytes by using fluorescence spectroscopy and 5 ng/mL with a nakedeye readout under an ultraviolet lamp. The efficacious, quantitative, and sensitive detection of AFP could be useful for the fluorescence diagnosis of HCC by serologic test. We envision that the methodology of Si NPs-based fluorescent ELISA could become a versatile tool for clinical medicine examination and disease diagnosis by using a fluorescence spectrometer and naked-eye readout in the near future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02847. FT-IR spectrum of the as-prepared Si NPs, the fluorescence and absorption spectra of the APTMS in addition of sodium ascorbate, the fluorescence intensity of the sensing system as a function of the enzymolysis F

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time for ALP and AA2P and the reaction time for the mixture and APTMS, fluorescence spectra of the sensing system in the presence of Na 3VO4-treated ALP, fluorescence spectra and intensities of the ELISA system toward human IgG standards, and analytical results for 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.

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ACKNOWLEDGMENTS We thank the financial supports by the National Natural Science Foundation of China (Grant No. 21435005) and the Development Project of Science and Technology of Jilin Province (Grant No. 20150520011JH)

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DOI: 10.1021/acs.analchem.6b02847 Anal. Chem. XXXX, XXX, XXX−XXX