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Metal-Polydopamine Framework: An Innovative Signal-Generation Tag for Colorimetric Immunoassay Rongrong Ren, Guoneng Cai, Zhenzhong Yu, Yongyi Zeng, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03538 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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Analytical Chemistry

Metal-Polydopamine Framework: An Innovative SignalGeneration Tag for Colorimetric Immunoassay Rongrong Ren,† Guoneng Cai,† Zhenzhong Yu,† Yongyi Zeng,*,‡ and Dianping Tang*,† †

Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350116, People's Republic of China ‡

Liver Disease Center, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, People's Republic of China *Corresponding Author: Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected]. ABSTRACT: In this work, an innovative enzyme-free colorimetric immunoassay was proposed for the sensitive detection of alpha-fetoprotein (AFP) by introducing thymolphthalein-modified metal-polydopamine framework (MPDA@TP) for the signal generation and amplification. Using zeolitic imidazolate framework (ZIF-67) as the template, the hollowstructured metal-polydopamine framework (MPDA) with high surface recovery and abundant groups was synthesized and functionalized with thymolphthalein (TP) molecules via typical π-stacking reaction. In the presence of target AFP, an MPDA@TP-linked immunosorbent assay (MLISA) was implemented on the capture antibody-modified microplate by using detection antibody-labeled MPDA-TP as the secondary antibody. Upon alkaline solution introduction, the coated hydrophobic TP on the MPDA was deprotonated into hydrophilic TP2- ion and dissolved in the solution, thereby resulting in the color change of the solution from nearly colorless to deep blue, and the increasing absorbance of the solution at 595 nm. Importantly, the MPDA@TP-based immunoassay could exhibit high sensitivity for the quantitative detection of target AFP on the basis of the absorbance within a linear range of 10 - 1000 pg mL-1 at a low detection limit of 2.3 pg mL-1. Furthermore, this system was validated preliminarily to screen human serum specimens with well-matched results for the referenced AFP ELISA kit. Taking advantages of simplicity, enzyme-free, convenience and sensitivity, MPDA@TP-linked immunosorbent assay has the potential for the application in scientific research and clinical diagnosis.

Effective but convenient techniques for the early determination of the disease-related biomarkers are highly desirable to help physicians or patients in making therapeutic decision without delay.1-5 Colorimetric immunoassay, that converts the antigen-antibody reaction into the shift in the color intensity, has been considered as a powerful tool in the point-of-care diagnosis and on-site testing thanks to its advantages, e.g., simple operation and intuitive results.6,7 To enhance the sensitivity and visibility of the colorimetric immunoassay, routine approaches are adopted by coupling enzyme labels with classical TMBH2O2 chromogenic system.8,9 Ambrosi et al. utilized gold nanoparticles as the labeling of the signal antibody for the amplification of enzyme immunoassay.10 Despite some outstanding achievements in research laboratories, the widespread adoption of colorimetric assay in practical application has been limited to some extent. Typically, enzyme immunoassays usually depend on the enzymes toward the high-efficient catalysis of the corresponding substrates in the optimum conditions (e.g., pH and temperature). Unfortunately, natural enzymes (e.g., horseradish peroxidase and alkaline phosphatase) are easily deactivated by environment interfering effects during operation process due to the inherently fragile nature, thus

increasing the cross-platform or cross-laboratory consistency.11-15 Additionally, the TMB-H2O2 chromogenic system often encounters many restrictions such as the interference of endogenous peroxide and the dependence on enzyme kinetics.16 In this regard, it takes a long-term incubation time for the generation of the colorimetric signal. Therefore, exploring an enzyme-free signalamplification colorimetric immunoassay is very advantageous for the development of effective and convenient technology in routine diagnostic practice. The tremendous advances in nano(bio)technology have provided the enormous possibility for the development of intelligent smart nanomaterials and the design of enzyme-free immunoassays to overwhelm above-mentioned bottleneck problems.17,18 Taking advantages of the highloading capacity that can encapsulate signal tags into their aqueous cavity, bio-friendly liposomes therefore have been introduced for construction of enzyme-free immunoassays.19-22 Although the introduction of liposomes into signal-amplification methods is groundbreaking, the chemical and/or physical lability of liposomes may bring about leakage issues and is detrimental to long-term preservation, which has fallen into the dilemma of the practical application.23

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Scheme 1. Schematic representation of MPDA@TP-linked immunsorbent assay (MLISA) for alpha-fetoprotein (AFP) on anti-AFP capture antibody (CAb)-modified microplate by using anti-AFP detection antibody (DAb)-labeled MPDA@TP with a sandwich-type immunoreaction mode: (A) preparation procedure of MPDA@TP-DAb and (B) the deprotonation of hydrophobic TP into hydrophilic TP2- ion under alkaline condition for the signal generation (MPDA: metal-polydopamine framework; TP: thymolphthalein; ZIF-67: zeolitic imidazolate framework; Ag: antigen). Metal-organic frameworks (MOFs; a class of materials with perpetual porosity) can form three-dimensional (3D) coordination networks by modular self-assembly of nodes (metal ions or clusters) and organic bridging ligands.24 The captivating structural topology and functional adjustability can provide unique merits for synthesis of various functional materials by using MOFs as both the template and precursor.25,26 Moreover, its derivatives, especially MOFs-derived porous or hollow nanostructures, have been widely applied in gas adsorption, energy storage and conversion, biosensing, catalysis, and drug delivery.25-28 In contrast, polydopamine (PDA), prepared from the self-oxidation of the catecholamine neurotransmitter dopamine, displays remarkable mussel-inspired materialindependent adhesion characteristics and bioconjugation properties, thereby holding considerable application promise in the materials sciences, biosensors, and nanomedicine.29 By benefiting from rich functional groups (e.g., amine, catechol, and imine), PDA allows the facile graft of functional molecules on the different materials, controlling the surface properties and conferring the new functionalities to materials. In addition, aromatic ring-rich PDA has been well exploited as chemical drugs and dyes carrier by π-stacking and/or hydrophobic-hydrophilic interactivity.30-32 In contrast, metal–polydopamine (MPDA) frameworks with hollow metal-organic materials can be synthetized by using MOFs as templates, involving the oxidation-induced in-situ polymerization of dopamine into PDA under alkaline conditions and competition between organic ligands and dopamine toward metal ions, as well as synchronous release of organic ligands.33,34 MPDA frameworks are novel hybrid materials that combine the advantages of PDA and MOFs, perfectly. MPDAs inherit some original structural characteristics of parent MOFs, such as rich 3D morphology, high specific surface

area, which greatly promote the loading capacity of the materials. Furthermore, the inside/outside surface of hollow nanostructures polymerizes a plentiful of functional groups-rich PDA with surface functionalization availability for loading or modification with the biomolecules. Ravikumar’s group applied MPDA as strong fluorescence quencher to electively identify Hg2+ and Ag+ ions.35 To the best of our knowledge, there is no report focusing on MPAD utilizing its rich graphite-like π-electron backbones for development of enzyme-free colorimetric immunoassays until now. Using a prototypical MOF (zeolitic imidazolate framework: ZIF-67) as the template, hollow MPDA frameworks with rich π-electrons are synthesized herein. Owing to the rich graphite-like π-electron, MPDA can be modified with abundant hydrophobic allochroic dye by π-stacking reaction (e.g., thymolphthalein: TP; phenolphthalein: PP; methyl red: MR), a new signal-amplified MPDA@TPlinked immunosorbent assay (termed as MLISA) is constructed. The MLISA-based colorimetric immunoassay synchronizes pH induced-thymolphthalein chromogenic system with hollow MPDA-based amplification strategy, which results in an enzyme-free breakthrough to the inherent limitation of traditional ELISA immunoassay (Scheme 1). As a proof-of concept, alpha-fetoprotein (AFP) is chosen as a model target. The assay is performed in anti-AFP-coated microplate with a sandwich-type immunoreaction model by using the TP–loaded MPDA (MPDA@TP) labeled anti-AFP detection antibody (DAb) as the signal-generation tag (MPDA@TP-DAb). The detection mostly involves the formation of a sandwiched immunocomplex and pH-induced visual color rendering process. Upon addition of target AFP, the MPDA@TPDAbs with specific antigen-antibody


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Figure 1. (A) TEM image and photograph (right inset) of ZIF-67; (B) HAADF-STEM image and elemental mapping for Co, N, and O of an individual ZIF-67; (C) TEM image and photograph (right inset) of MPDA; (D) HAADF-STEM image and elemental mapping for Co, N, and O of an individual MPDA; (E) XRD patterns of ZIF-67 and MPDA; (F) XPS spectra of ZIF-67 and MPDA; and (G) FT-IR spectra of TP (blue), MPDA (green) and MPDA@TP (orange). recognition are introduced in the well. Numerous hydrophobic TP molecules absorbed on the inner/outer surface of the MPDA shell is transformed into hydrophilic TP2ions (TP2-) in the presence of alkaline solution, and released into the solution, thereby causing the change of solution from nearly colorless to deep blue. The study aims to emphasize that the proposed MLISA platform is simple to operate but sensitive to detection and can be used as an alternative in ultrasensitive colorimetric immunoassays. EXPERIMENTAL SECTION Synthesis of Zeolitic Imidazolate Framework (ZIF67). ZIF-67 nanocubes were synthesized on the basis of previous work with minor modification.36 Initially, 2methylimidazole (3.632 g) was dispersed into 56-mL aqueous solution under vigorous stirring. Thereafter, cetrimonium bromide (CTAB) aqueous solution (8.0 mL, 0.1%, w/v) containing 232-mg Co(NO3)2·6H2O was quickly added into the above mixture, and reacted for 20 min at room temperature under the same conditions. Finally, the resulting purple ZIF-67 nanostructures were collected by filtration and washed three times with ethanol, and dried at 60 °C for further use. Preparation of Hollow Metal-polydopamine Framework (MPDA). Hollow metal-polydopamine framework was synthesized by using the above-prepared ZIF-67 as the template similar to the literature.37 Prior to synthesis, the dried ZIF-67 (20 mg) was dispersed in an ethanol-water solution (35 mL, 3 : 4, v/v) under sonication. Following that, dopamine hydrochloride (8.0 mg) was injected into the mixture under violent stirring. Subsequently, 25ml of Tris-HCl buffer solution (10 mM, pH

8.5) was thrown in the mixture and reacted for 24 h in dark with gentle stirring. Finally, the resulting product (denoted as “MPDA”) was collected by centrifugation (10,000g, 10 min), and washing with ultrapure water and ethanol alternately. Preparation of Thymolphthalein-Modified MPDA Framework (MPDA@TP). Thymolphthaleinfunctionalized MPDA framework was prepared by a simple mixture reaction process. Initially, the above-collected MPDA was dispersed into 1.0-mL ethanol solution containing thymolphthalein (TP, 15 mM). Then, the resulting mixture was gently shaken on a shaker for 120 min at room temperature. During this process, TP molecules were physically adsorbed on the MPDA through πstacking reaction. Following that, excess TP molecules were removed by centrifugation (10,000g, 10 min) and washing with ultrapure water. Finally, the as-prepared MPDA@TP was used for the labeling of monoclonal mouse anti-human AFP detection antibody (denoted as MPDA@TP-DAb), and colorimetric measurement for target AFP on antibody-coated microplate (Please see the detailed process in the Supporting Information). RESULTS AND DISCUSSION Synthesis and Characterization of MPDA@TP. As described above, the as-synthesized MPDA@TP was used as the signal-generation tag for the signal amplification of the colorimetric immunoassay. ZIF-67 was first utilized as the template for the preparation of MPDA. To verify the successful synthesis of ZIF-67 with good morphology, we used transmission electron microscopy (TEM) to characterize the nanostructures. As seen from Figure 1A, the


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produced ZIF-67 showed good uniformity with a size of

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about

Figure 2. (A) UV-Vis absorption spectra of (a) MPDA, (b) MPDA@TP, (c) MPDA-OH- and (d) MPDA@TP-OH- (insets: the corresponding photographs); (B) the standard curve between the TP concentration and the absorbance in alkaline solution; and (C) the absorbance of MPDA@TP toward different-concentration TP in alkaline solution. 200 nm. Meanwhile, the relatively smooth and regular surface was observed from the high-angle annular darkfield scanning transmission electron microscopy (HAADF-STEM) image of one single nanoparticle (Figure 1B, white). The EDX elemental mapping images (Figure 1B) of the typical products were recorded that the even distribution of Co (yellow), N (green) and O (blue) elements throughout the whole of the nanocube. After reaction with a certain proportion of dopamine, the color of the suspension would generally change from purple (Figure 1A, top right inset) to black (Figure 1C, top right inset). The hollow-structured MPDA were obtained in the process of in-situ transformation. The TEM image (Figure 1C) of the obtained products revealed that the hollowstructured particles maintained the cubic shape and size of the ZIF-67 templates and the cubic cavity inside the product could be observed from the comparison of the shell and the inner void of nanoparticle, and the average thickness of the shell was about 21 nm. HAADF-STEM image (Figure 1D) of an individual MPDA further observed its rough and porous feature. The EDX element mapping image of a single MPDA (Figure 2D) revealed the variation in the distribution of Co, N, and O elements to confirmed the MPDA hollow structures of the final products. Compared to ZIF-67, the amount of these elements in the central region were significantly reduced, but homogeneously distributed throughout the shell. This formation mechanism of the hollow-structured products was attributed to the hydrolysis of ZIF-67 in water and the competitive reaction between 2-methylimidazole and PDA toward Co2+.38 The characteristic absorption of zif-67 in the FT-IR spectrum (Figure S1) between 500 and 1500 cm-1 disappeared, accompanied by the occurrence of the PDA characteristic bands, further confirming the formation mechanism. In addition, the characteristic peaks belonging to the ZIF-67 disappeared in the X-ray powder diffraction (XRD) patterns of MPDA (Figure 1E), demonstrating that the ZIF-67 was completely transformed into armorphous polymer MPDA. To confirm the formation of MPDA, the X-ray photoelectron spectroscopy (XPS) spectra of Co 2p were recorded (Figure 1F). The peaks were fitted into Co 2p1/2 at 796 eV and Co 2p3/2 peak at 780 eV for both samples. The two satellite peaks of the oxidation state of Co2+ in ZIF-67 located at around 786 eV and 803

eV disappeared in the spectrum of MPDA, which matched well with the XPS of MPDA33 and testified the existence of MPDA. The porous structure of MPDA was determined by nitrogen adsorption–desorption isotherms of MPDA (Figure S2), and a typical type IV isotherm with a significant hysteresis loop was recorded, simultaneously, the related pore size distribution of MPDA was observed (Figure S2, inset), confirming the porous structure of hollow PDA shells.39 The MPDA had a Brunauer-EmmettTeller (BET) surface area of 532 m2 g-1. These results indicate that the successful synthesis of the porous hollowstructured MPDA with high specific surface area, which provides a necessary platform for the material modification. Finally, the prepared MPDA absorbed massive TP molecules via π-stacking interactions to form the signal material of MPDA@TP. The FT-IR spectra were carried out to confirm the composition of MPDA@TP. (Figure 1G). Pure TP exhibited five characteristic peaks at 3374, 29622868, 1736, and 1110 cm-1 corresponding to the stretching vibration of O-H group, stretching in methyl and methenyl of C-H group, stretching of esoteric carbonyl group, and stretching of esoteric C-O group, respectively(curve blue).40 In contrast, the FTIR spectrum of MPDA had three peaks at 3200-3500 cm-1 and 1585 cm-1 assigned to the stretching vibrations of O-H/N-H and ring vibration of indole, respectively (curve green).41 Significantly, these characteristic peaks for TP and MPDA simultaneously appeared in MPDA@TP (curve orange). Favorably, the above results proved that MPDA@TP were successfully synthesized by our design. Feasibility Verification of MPDA@TP-Based MLISA Method. In this study, the generation and amplification of signal mainly derived from alkaline-induced the intelligent release of TP molecules from the surface of signal tags, accompanying the change in the color. To realize our design, an important precondition was whether the alkaline solution could cause desorption of TP and the color change in the solution. To verify this point, the changes in the solution before and after the reaction with alkaline solution were investigated. As seen from the insets in Figure 2A, the as-synthesized MPDA (photograph 'a') and MPDA@TP (photograph 'b') exhibited the light brown under natural light. Moreover, no characteristic absorbance peaks were observed at the MPDA (curve 'a')


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Analytical Chemistry and MPDA@TP (curve 'b') alone. Favorably, a strong characteristic peak at 595 nm appeared when the asprepared MPDA@TP reacted with the alkaline solution (curve 'b'). Furthermore, the solution after reaction became a deep blue (photograph 'b'). Logically, one question arises as to whether the change in the color and absorbance got from the MPDA framework. As control test, the newly synthesized MPDA directly reacted with the alkaline solution. Clearly, UV-vis absorption spectra and the photograph were almost the same as that of MPDA alone (curve 'c' vs. curve 'a'; photograph 'c' vs. photograph 'a'). These results confirmed that (i) TP molecules adhered on the surface of MPDA could be released by alkaline solution for the generation of the visual color; and (ii) TP molecules was successfully immobilized onto the surface of MPDA by π-π stacking interaction. The phenomenon could be explained as follows: the deprotonation of the carboxyl and hydroxyl groups of TP molecule by OHresulted in the conversion of TP in neutral hydrophobic state to a hydrophilic state of TP2- (see Figure S3 in supporting information). Since the interaction between TP2ion and the surface of MPDA was relatively weak, TP2- ion was easily separated from the nanomaterial, thus resulting in the formation of blue solution.

colorless, whereas it is blue above. The change in the color is ascribed to the transition from hydrophobic TP to hydrophilic TP2- ion. In this regard, we investigated the effect of pH of alkaline solution on the sensitivity of MPDA@TP-based MLISA by using 1.0 ng mL-1 AFP as an example. As displayed in Figure 3A, the absorbance intensities did not change nearly from pH 9.0, then increased rapidly with the increasing pH value of alkaline solution at the range of pH 9.0 – 13.0, and then reached a plateau after pH 13. The maximum absorbance could be obtained at ≥pH 13. The reason was attributed to the fact that the amount of TP molecules heavily depended on pH value of alkaline solution for the color development and the absorbance change.42 A high-concentration thymolphthalein molecule usually requires a higher pH solution. Moreover, TP molecules were conjugated onto the nanomaterials through π-stacking reaction, which was different from thymolphthalein solution alone. At ≥pH 13, the coated TP molecules on the MPDA framework could be completely dissolved to the solution. However, too high pH solution might affect the safety of experimental operation. Considering this issue, pH 13 of alkaline solution was selected for deprotonation of thymolphthalein in this work.

To investigate the stability of the coated TP molecules on the MPDA framework, the different components of the as-prepared MPDA@TP was monitored by using UVvis absorption spectroscopy before and after centrifugation. As seen from Figure S4 in the Supporting Information, almost no absorbance was achieved at the obtained supernatant after centrifugation of MPDA@TP suspension. In contrast, the collected precipitates exhibited a strong absorbance intensity in comparison with original uncentrifuged MPDA@TP suspension. The results suggested that TP molecules could firmly attach onto the MPDA framework by π-π stacking interaction. In addition, we also studied the loaded capacity of MPDA toward TP molecules on the basis of the obtained MPDA by using 20-mg ZIF-67 as an example. The judgment was carried out by assaying the absorbance after MPDA@TP reacted with alkaline solution (pH 13). As shown in Figure 2B, the absorbance intensities increased with the increasing TP molecules, and a good linear relationship was found between the increasing absorbance at 595 nm and the concentration of TP in the range of 1.0 to 50 μM. The detection limit was as low as 0.4 μM. The results clearly demonstrated the amount of TP loaded on the MPDA could be quantified. Following that, we also investigated the absorbed amount of TP on the MPDA (100 μg used in this case). As seen from Figure 2C, the absorbance reached the maximum absorbance value when TP concentration was up to 1.5 mM. The maximum loading capacity of MPDA toward TP was estimated to be 412.52 mg g-1 with the assistance of the above-mentioned standard curve.

Except for pH of alkaline solution, the analytical performance of MPDA@TP-based MLISA also relies on the incubation time of alkaline solution with MPDA@TP. A short-incubation time is unfavorable for the deprotonation of TP molecules, thereby resulting in a low sensitivity. Moreover, it is not conducive for the color development. Figure 3B gives the dependence of absorbance intensity on the different incubation times toward this system. With the incubation time aged, the absorbance initially increased, and then tended to level off after 120 s. To ensure the adequate progression of the deprotonation reaction, we chose 180 s for alkaline solution-triggered color development.

Optimization of Analytical Conditions. As described above, the color change and the absorbance increase originated from the deprotonation of thymolphthalein under the alkaline condition. Typically, the transition of thymolphthalein is around pH 9.3 – 10.5. Below this pH, it is

Figure 3. Effects of (A) pH value of alkaline solution and (B) incubation time for alkaline solution-triggered color development on the absorbance of MLISA-based colorimetric as-1 say by using 1.0 ng mL AFP as an example.

Dose Responses of MPDA@TP-Linked Immunoassay toward Target AFP. Under the optimal conditions, we applied MPDA@TP-linked immunosorbent assay (MLISA) for qualitative or quantitative determination of target AFP on the basis of the detection process, described in Scheme 1B. In this system, the detection antibody (DAb) was covalently conjugated onto the MPDA@TP by typical idiopathetic Schiff base and/or Michael addition reaction between the abundant quinone


5


groups of polydopamine polymerized on the MPDA shell and the amine groups on antibodies.43-45 AFP standards with various levels were determined in a high-binding microplate (Figure 4A) and quantified by using a Tecan Infinite 200 PRO (TECAN, Switzerland). The gradually deepened blue solution in the wells stemmed from the products of TP deprotonation process induced by alkaline solution.46 Thanks to the high loading ability of MPDA for TP molecules, the absorbance at 595 nm increased with the increment of target AFP concentration (Figure 4B). To quantify the AFP concentration in the sample, a sigmoidal ‘S’ relationship between the absorbance at 595 nm and AFP level is plotted (Figure 4C, plots ‘a’). A good linear relationship between absorbance (a.u.) and the decimal logarithm of AFP concentration (pg mL-1) was acquired within the dynamic range 0f 10 – 1000 pg mL-1 (Figure 4C, inset). The regression equation can be fitted as y = 0.4537 × logC[AFP] – 0.4163 (pg mL-1; r = 0.9993; n = 7). The limit of detection (LOD) was calculated to be 2.3 pg mL-1 at a signal-to-noise ratio of 3SB (where SB is the standard deviations from the blank sample, n = 13). To further elucidate the advantage of MPDA@TP-based MLISA with high sensitivity, traditional immunoassay by using horseradish peroxidase (HRP)-labeled anti-AFP detection antibody was also used for the colorimetric detection of target AFP relative to 3,3',5,5'tetramethylbenzidine (TMB)-H2O2 system with a sandwich-type immunoassay format (i.e., HRP-based ELISA). As shown Figure S5 in the Supporting Information, the linear range and LOD of using HRP-based ELISA were 0.2 – 20 ng mL-1 and 52 pg mL-1 AFP, respectively. Obviously, the LOD of our strategy was ~20-fold higher than that of conventional HRP-based ELISA. Such a high sensitivity can be utilized to determinate low-abundance biomarkers.

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125, 10 ng mL CA 19-9, and 10 ng mL HIgG, respectively; and (E) comparison of the results for human serum specimens obtained between MLISA method and the commercial ELISA kit.

Reproducibility, Selectivity and Specificity. The precision and reproducibility of MPDA@TP-based MLISA were monitored toward AFP standards with replicates of the assay by using MPDA@TP-DAb from a single batch and different synthetic batches, respectively. As seen from plots 'b' in Figure 4C, the coefficient of variations (CVs, n = 3) over the whole concentration range was 3.2-8.6% by using the MPDA@TP-DAb from a single batch. Also, the synthesis-to-synthesis reproducibility of TP@MPDA-DAb was monitored by using the nanostructures from different synthetic batches for the fabrication of the immunosensing platform. The CVs were 11.5%, 8.7%, 10.2%, 11.2%, 9.6%, 8.8% and 9.8% for 10, 20, 50, 100, 200, 500 and 1000 pg mL-1 AFP, respectively. These results revealed that TP@MPDA-based MLISA had a good reproducibility. Next, we also investigated the specificity of MPDA@TPbased MLISA toward target AFP and other biomarkers (e.g., prostate-specific antigen, PSA; carcinoembryonic antigen, CEA; cancer antigen 125, CA 125; cancer antigen 19-9, CA 19-9; human immunoglobulin G, HIgG). As shown in Figure 4D, all the absorbance intensities of our systems were close to zero toward the nontarget analytes including PSA, CEA, CA 125, CA 19-9 and HIgG at a concentration of 10 ng mL-1. In contrast, a low-concentration target AFP (1.0 ng mL-1) could cause the high absorbance. More importantly, the absorbance intensities of this system containing target AFP and nontarget did not significantly increase relative to AFP alone. Hence, the MPDA@TP-based MLISA exhibited high specificity and selectivity toward target AFP.

Table 1. Evaluation of MLISA-Based Method Accuracy for Human Serum Specimens Using Human AFP ELISA Kit as the Reference detection method (concentration: mean ± SD, pg mL-1)a sample no.

MLISA assay

ELISA kitb

texp

1

8763.21 ± 132.12

8523.12 ± 342.45

1.13

2

12371.43 ± 243.40

12543.76 ± 312.31

0.75

3

2314.56 ± 89.11

2378.81 ± 67.19

1.01

4

926.53 ± 56.48

858.52 ± 48.03

1.59

5

743.65 ± 40.70

762.75 ± 46.05

0.54

6

545.93 ± 19.92

503.85 ± 27.63

2.14

a

Figure 4. (A) Representative photographs taken from the MLISA-based visual assay for AFP standards with different concentrations; (B) absorbance intensity and (C) the calibration plots (a) and the imprecision profile (b) of MLISA method toward different-concentration AFP standards (inset: linear curve); (D) the selectivity of MLISA method against 1.0 -1 -1 -1 -1 ng mL AFP, 10 ng mL CEA, 10 ng mL PSA, 10 ng mL CA

Each sample was determined in triplicate, and the highconcentration AFP samples were calculated according to the dilution ratio when the amount of AFP exceeded the linear range of MPDA@TP-based MLISA. b The linear range of commercial human AFP ELISA kit is 0.31 - 20 ng mL-1.

Screening of Human Serum Specimens. By using the developed MPDA@TP-based detection system, 6 human serum specimens, provided by our First Affiliated Hospi-


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Analytical Chemistry tal of Fujian Medical University of China on the basis of the local ethical committee, were detected with the same assay mode, respectively. To evaluate the accuracy of the newly developed MLISA method, the collected data were compared with those obtained by using commercial human AFP ELISA kit as the reference. Table 1 shows the analyzed results for these serum samples by these two methods. As indicated in Table 1, all the experimental texp values were less than the critical value of tcrit, (tcrit[0.05,2] = 4.30) with an t-test statistical analysis. However, the low-concentration serum samples were difficultly determined by using commercial human AFP ELISA kit due to the limitation of the linear range (0.31 – 20 ng mL-1). To further embody the advantage of our system toward the low-abundance AFP, the recovery experiments were carried out by spiking AFP standards into blank human serum samples at five concentrations including 20, 50, 100, 200 and 300 pg mL-1. The final results obtained by MPDA@TP-based MLISA were 22.1, 48.7, 94.6, 234.9 and 329.2 pg mL-1 toward the above-mentioned five samples, respectively. The recovery was 94.6 – 117.45%. Moreover, the slope of the regression equation on the basis of the average values between two methods for the real serum specimens (y = 1.007x – 35.431; where x and y stand for the results obtained from the colorimetric immunoassay and commercial AFP ELISA kit, respectively) was close to idea '1' (Figure 4E). Therefore, n0 significant differences could be observed at the 0.05 significance level between the two methods for the monitoring of human serum samples, suggesting that our strategy could be considered as an optional scheme for quantitative determination of target AFP in the complex samples.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.0000. Material and reagent, bioconjugation of MPDA@TP with DAb antibody (MPDA@TP-DAb), colorimetric immunoassay for target AFP, FTIR spectra (Figure S1), N2 adsorption-desorption isotherm (Figure S2), photographs of TP 2and TP (Figure S3), absorbance intensities of the obtained supernatant and precipitate after centrifugation of MPDA@TP suspension (Figure S4), and calibration plots of HRP-based ELISA method (Figure S5) (PDF)

AUTHOR INFORMATION Corresponding Author * Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mails: [email protected] (Y. Zeng) & [email protected] (D. Tang). (iD) ORCID Dianping Tang: 0000-0002-0134-3983

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge the financial supports from the National Natural Science Foundation of China (21675029, 21874022, 21475025), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R11) REFERENCES (1)

CONCLUSIONS In summary, this contribution introduces an enzyme-free MPDA@TP-linked immunosorbent assay (MLISA) protocol for the sensitive detection of cancer biomarker, AFP, by using π-electron-rich hollow-structured MPDA for the signal amplification with a sandwich-type immunoreaction mode. Compared with conventional colorimetric immunoassays, this system can be highlighted as follows: (i) the visual color is readily produced in the process of introducing alkaline solution to induce TP dissolution, thereby decreasing the steps without the need of multiple reactions for the signal generation; (ii) the signal amplification is carried out by using MPDA with high specific surface area and rich functional groups as carriers to load numerous hydrophobic TP molecules, thus avoiding the participation of natural enzymes; and (iii) MPDA frameworks with hollow nanostructures are for the first time utilized as the carriers of color-changing agents to develop high-efficient colorimetric immunoassay. Moreover, the MLISA-based colorimetric strategy for the detection of cancer biomarker synchronizes the pH inducedthymolphthalein chromogenic system with MPDA-based amplification, hence making the detection process relatively simple, rapid, and economic. Importantly, the conception of enzyme-free signal-amplification strategy based on MLISA may open up a new way to develop more sensitive and convenient diagnostics.

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