A colorimetric immunosensor based on Au@g-C3N4 doped sponge

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Biological and Medical Applications of Materials and Interfaces

A colorimetric immunosensor based on Au@g-C3N4 doped spongelike 3D network cellulose hydrogel for detecting alpha fetoprotein Fang Ma, Chun-Wang Yuan, Jian-Ni Liu, Jian-Hua Cao, and Da-Yong Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b06769 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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ACS Applied Materials & Interfaces

A colorimetric immunosensor based on Au@g-C 3 N 4 doped sponge-like 3D network cellulose hydrogel for detecting alpha fetoprotein Fang Ma†,‡,⊥, Chun-Wang Yuan§,⊥, Jian-Ni Liu†,‡, Jian-Hua Cao†, Da-Yong Wu *† Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhong-guan-cun

†

East Road, Haidian District, Beijing, 100190, P. R. China University of Chinese Academy of Sciences, Beijing, 100039, P. R. China

‡

§ Center of

Interventional Oncology and Liver Diseases, Beijing Youan Hospital, Capital Medical

University, 8 Xitoutiao, Youwai Street, Fengtai District, Beijing, 100069, P.R. China ⊥

These authors contributed equally to this work.

*

Corresponding to: [email protected]

KEYWORDS: Colorimetric Immunoassay, AFP, Au NPs, g-C 3 N 4 Nanosheets, Cellulose hydrogels

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ABSTRACT Colorimetric immunoassay is a powerful tool for detecting tumor markers, with outstanding advantages of visualization and convenience. This study designed a colorimetric immunoassay by using the antibody/antigen to control the catalytic activity to be “switched on/off”. This system, where Au NPs (18.5±3.9 nm) loaded on the gC 3 N 4 nanosheets that were fixed in a 3D porous cellulose hydrogel, was used as a binding site for antibody/antigen. After being incubated with antibody of cancer marker, the turned-off catalytic sites on Au NPs in Au@g-C 3 N 4 /MCC hydrogel would not be “turned on” until the corresponding antigen was added. The number of the recovered Au active sites was related to the amount of antigen added. The FTIR and XPS measurements did not detect the existence of Au-S bonds. Catalyzed by the “turned-on” Au NPs, 4-nitrophenol was reduced to 4-aminophenol (4-AP) accompanied by a color fading. The color and the absorption spectrum changes in the process were used as the colorimetric quantitative basis for immunoassays. The colorimetric immunoassay showed a linear relationship to the liver cancer marker (AFP) in the range of 0.1-10,000 ng/mL with the detection limit of 0.46 ng/mL. In addition, 4-nitrophenol had a significant color fading when the AFP concentration exceeded the healthy human threshold. The clinical patient’s serum test results obtained from the developed colorimetric immunosensor were consistent with that obtained from the commercial ELISA. Furthermore, the immunosensor exhibited a good selectivity, repeatability and stability, which demonstrated its potential for practical diagnostic application.

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1. INTRODUCTION The presence of some protein markers in serum can serve as an early identification signal for diseases. The early detection of cancer markers, such as carcinoembryonic antibody, alpha-fetoprotein (AFP) and prostate specific antigen has a crucial role to play in cancer diagnosis and treatment. So far, many immunoassay methods for detecting cancer marker have been reported, such as enzyme-linked immunosorbent assay,1 fluorescence

immunoassay,2-4

chemiluminescent

enzyme

immunoassay,5

electrochemical immunoassay,6-9 colorimetric immunoassay,10 phase time-resolved fluorescence immunoassay,11 and the surface enhanced Raman scattering-based sandwich-type immunoassay.12 Among many biomolecule detection methods, the colorimetric immunoassay has attracted plenty of interest because of its advantages of visualization, easy operation and fast readout. The main challenge of developing colorimetric biosensors is to convert biological signals to color changes. In typical colorimetric immunoassays, some biological enzymes such as horseradish oxidase (HRP) and alkaline phosphatase are commonly used as probes. For example, with the presence of H 2 O 2 , HRP can catalyze the reaction of 3,3,5',5'-tetramethylbenzidine to produce blue products, brown products from ophenylenediamine

and

blue-green

products

from

2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid), respectively.13-15 However, natural enzymes have limitations, including the high sensitivity to environmental changes, easy denaturation and degradation, high price, tedious steps of preparation and purification. Therefore, several kinds of nanomaterials with similar 3



functions have been gradually developed as substitutes for enzymes in the study of colorimetric immunoassay. Wang et al. 16constructed the Fe 3 O 4 @Ag-Ab 1 /CEA/ Ab 2 Ag 3 PO 4 /Ag "sandwich" system accomplishing the efficient colorimetric immunoassay to detect CEA antigen by applying the Ag+ on the detector antibody to oxidize TMB into blue products. Among numerous nanomaterials, gold nanoparticles (Au NPs), which can accelerate color change reactions to give out visual signals (such as the reduction of 4-nitrophenol), are widely used in colorimetric immunoassay due to their good biocompatibility and catalytic properties.17-18 However, with high surface energy, Au NPs tend to agglomerate, change the shape, destroy the surface state, resulting in a significant decrease in its catalytic activity.19 The common solution is to immobilize Au NPs on a suitable carrier, such as polymers, metal oxides (TiO 2 , MgO, Al 2 O 3 ),20-23 metal organic framework materials24 and nanosheets with topological structure. Using dopamine-loaded Au NPs (PDA-Au) as catalytic label, Zhao et al.25 applied Fe 3 O 4 nanoparticles as immunosensor probe. With the color change from yellow 4nitrophenol to colorless 4-AP reduced by this “sandwich” system, CA125 was successfully detected. The results showed that the PDA-Au was twice as active as Au NPs. Xiao et al.26 immobilized Au NPs on Bi 2 Se 3 nanosheets and used it to catalyze the reduction of 4-nitrophenol. The activity parameter K of the model reaction reached 386.67 s-1g-1, while it was 0.35 s-1 g-1 when Au NPs were used in a control test. In addition, some carbon materials such as graphene and graphite-like carbon nitride (gC 3 N 4 ) have been used as Au NPs carrier as well.27-28 g-C 3 N 4 shows advantages, like chemical stability, strong thermal stability, nontoxicity and so on when it is used as the 4


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a carrier of catalyst.29 More importantly, g-C 3 N 4 is a polymer semiconductor that can function as a photocatalyst under natural light conditions. Based on this feature, g-C 3 N 4 nanosheets have been reported to load Au NPs to achieve synergistic catalysis.30-31 While the Au NPs/g-C 3 N 4 hybrid suspended in the solution system is hard to separate and reuse. Besides, the scattering effect of the suspension will affect the sensitivity of UV-vis spectrometer when the concentration of substrates is measured. 25 In order to make the catalyst probe separable, several approaches have been achieved, for example, magnetic Fe 3 O 4 nanoparticles are used to form composite catalyst with Au NPs.32 Chang et al.33 reported a novel recyclable gold nanocatalyst for the reduction of 4-nitrophenol, where the Au3+ ions were reduced by the chitosan-coated Fe 3 O 4 . The results showed that the hybrid catalyst was well separated by using a magnet. In addition to magnetic materials, 3D bulk materials (hydrogels and aerogels) have been developed recently. Hydrogel has a polymeric cross-linked 3D network structure which has the capacity to hold water within its porous structure.34 It has been widely used in absorbents, biomedical devices, cosmetics, catalysts and other fields.35 In this study, a 3D porous cellulose hydrogel-immobilized Au@g-C 3 N 4 system was designed and prepared to detect the cancer marker. The AuCl 4 - added to the cellulose was reduced in situ to Au NPs and partially loaded on the surface of g-C 3 N 4 nanosheets. Based on the good catalytic activity and biocompatibility of Au NPs in the hydrogel, a colorimetric immunoassay with “switching” performance was designed. The excessive amount of alpha-fetoprotein (AFP) in adult blood indicated the presence of certain cancers, especially in the liver, stomach, pancreas, ovaries or testes. This 5



immunoassay provided an easy separation for detector probe from the detection mixture, avoiding the interference to the detection signal. The developed colorimetric immunoassay based on Au@g-C 3 N 4 /MCC hydrogel had prospects to be applied in the detection of AFP in clinical serum.

2. EXPERMENTAL SECTION 2.1 Materials and Reagents. Microcrystalline cellulose (MCC) was supplied by Sigma-Aldrich. Sodium hydroxide and urea were purchased from Beijing Chemical Works. Ethanol and Chloroauric acid (HAuCl 4 .4H 2 O) were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (China). 4-Nitrophenol was purchased from Beijing InnoChem Science & Technology Co., Ltd. (China). Sodium borohydride (NaBH 4 ) was purchased from Tianjin Jinke Fine Chemical Research Institute (Tianjin, China). Melamine was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Alpha-fetoprotein antibody (Anti-AFP) was purchased from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing, China). Alpha-fetoprotein antigen (AFP) was purchased from National Institute for Food and Drug Control (Beijing, China). Bovine serum albumin (BSA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS; 0.1 M, pH 7.2–7.4) and PBST (contain Tween-20, 0.1 M, pH 7.2–7.4) were purchased from Solarbio (Beijing, China), diluting to 0.01M with ultrapure water before using. Clinical serum samples were from Beijing Youan Hospital. Commercial enzyme-linked immunosorbent assay (ELISA) kit was purchased from Autobio Diagnostics Co., Ltd. (China). Ultrapure water obtained from a Millipore water purification system (≥18 6


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MΩ, Milli-Q, Millipore) was used in all assays. All other reagents were of analytical grade and used as received.

2.2 Preparation of the g-C 3 N 4 nanosheets The g-C 3 N 4 nanosheets were prepared according to the previously reported literature.28 10 g of melamine was added into a crucible with a lid, and the crucible was subsequently placed in a tube furnace, which was heated up to 500℃ (4 ℃/min,) and maintained for 3 h under air condition. A pale yellow solid, bulk g-C 3 N 4 was obtained. A further ultrasonic treatment in water exfoliated the product into g-C 3 N 4 nanosheets. Typically, 100 mg of bulk g-C 3 N 4 was dispersed in 200 mL of ultrapure water and treated by ultrasonic for 5 h to make the bulk g-C 3 N 4 exfoliated. The obtained suspension was centrifuged at 5,000 rmp for 10 min to remove the unexfoliated bulk gC 3 N 4 as the precipitate. The supernatant was further centrifuged at 13,000 rmp to get the g-C 3 N 4 nanosheets on the bottom. The product was collected and freeze-dried for subsequent experiments.

2.3 Preparation of the MCC-Au and Au@g-C 3 N 4 /MCC Preparation of the Microcrystalline Cellulose (MCC) solution: In this procedure, a typical method was used to dissolve microcrystalline cellulose in a NaOH/urea solution.36 7 g of NaOH and 12 g of urea were dissolved in 76 g of ultrapure water. Then 5 g of cellulose was added in, obtaining a cellulose dispersion of 5 wt%. The dispersion was frozen at -20℃ for 8 h and dissolved at room temperature with a quick 7



stirring. After defoaming by applying a centrifugation treatment at 2,000 rmp, the obtained solution was saved at 4℃ for later use. The rapid dissolution of cellulose in NaOH/ urea aqueous solution is due to the formation of urea-NaOH-cellulose complex formed by self-assembly of small solvent molecules (NaOH hydrate, urea hydrate, water) and cellulose macromolecules through hydrogen bonds induced by low temperature, which is in a relatively stable state at low temperature, resulting in rapid dissolution of cellulose at low temperature.37 Preparation of MCC-Au hydrogel and the optimization of preparation conditions: 1 g of the cellulose solution was mixed with a various amount of chloroauric acid solution (1 w/v % HAuCl 4 ) listed in Table 1 to investigate the best ratio of the two materials. After being stirred on a vortex mixer for 1 min, the mixed solution was added into a 24-well plate at 0.5 g per well. The plate was placed in liquid nitrogen for freezing for 3 min, in order to avoid the damage of morphology when adding ethanol. Subsequently, the frozen hybrid solutions were coagulated by ethanol (1 mL at each well) to get the regenerated cellulose hydrogels for 5 h at -20℃. Such a low temperature was to reduce the reduction activity of cellulose on Au ions. While residual ethanol was removed, several pieces of hydrogel were added into 100℃ ultrapure water (50 mL) and boiled for 15 min. During the heating process, Au ions could be reduced in situ by a plenty of active hydroxyl groups of the cellulose to produce Au NPs. NaOH and urea which are present in the cellulose must be washed out by using ultrapure water until the hydrogel was turned to neutral. The obtained MCCAu hydrogel (12 mm in diameter and 3 mm in height) was kept in water at 4℃. 8


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Table 1. The preparation of MCC-Au hydrogels. Samples

MCC Solution（g）

1% HAuCl 4 Solution（μL）

MCC

1

0

MCC-Au 20

1

20

MCC-Au 40

1

40

MCC-Au 60

1

60

MCC-Au 80

1

80

MCC-Au 100

1

100

MCC-Au 120

1

120

MCC means microcrystalline cellulose hydrogel. Au means the Au NPs. The numbers (20, 40, 60, 80, 100, 120) stand for the volume of added 1% HAuCl 4 (20 μL, 40 μL, 60 μL, 80 μL, 100 μL and 120 μL) in 1 g of MCC solution, respectively.

Preparation of the Au@g-C 3 N 4 /MCC hydrogel: 2 mg of g-C 3 N 4 nanosheets were added to 4 mL of a HAuCl 4 solution (1 w/v %) and treated with ultrasonic for 30 min. 400 μL of the obtained g-C 3 N 4 /HAuCl 4 mixture was added into 5 g of MCC solution. A short stirring was needed to form a uniform mixture of HAuCl 4 , g-C 3 N 4 and MCC. -

The stirring should not be longer than 1 minute, otherwise AuCl 4 ions would be reduced by cellulose during this step. The following steps were the same as the preparation of MCC-Au hydrogel mentioned above. The formed Au@g-C 3 N 4 /MCC hydrogel (12 mm in diameter and 3 mm in height) was kept in water at 4℃.

2.4 Photocatalytic performance tests of the MCC-Au and Au@g-C 3 N 4 /MCC The photocatalytic performance of the samples was measured by employing the reaction of reducing 4-nitrophenol to 4-aminophenol (4-AP). The yellow 4-nitrophenol 9



would gradually turn into colorless 4-AP under natural light in the presence of reducing agent NaBH 4 and a catalyst. Typically, 80 mL of water, 10 mL of 1.6 mmol/L of 4nitrophenol and 10 mL of 0.42 mol/L NaBH 4 solution were sequentially added into a 200 mL beaker to obtain a 4-nitrophenol/NaBH 4 mixture solution. 10 mL of 4nitrophenol/NaBH 4 solution was added into a 10 mL of glass bottle. As soon as a piece of MCC-Au or Au@g-C 3 N 4 /MCC hydrogel was added into the glass bottle, timer started. The UV-Vis spectrum measurement indicated that the absorption peak of 4nitrophenol decreased gradually at 400 nm, while the absorption peak of product 4-AP increased at 300 nm. At certain intervals, 1 mL of the reaction mixture was taken out, diluted with 2 mL of water and measured by UV-Vis spectrometer (Cary 5000, Varian).

2.5 Immobilization of anti-AFP on the Au@g-C 3 N 4 /MCC The filter paper was used to remove residual water on the surface of the Au@gC 3 N 4 /MCC which was taken out from cold water. One piece of Au@g-C 3 N 4 /MCC hydrogel was put into a 10 mL centrifuge tube, and mixed with 500 μL of anti-AFP solution (10 μg/mL). The mixture was incubated together for 1 h at 37℃ with gently shaking to get the anti-AFP-loaded Au@g-C 3 N 4 /MCC hydrogel (anti-AFP/Au@gC 3 N 4 /MCC). The PBST solution was used to wash the anti-AFP treated hydrogel three times, removing the unattached or weakly attached antibodies. And the filter paper was used to remove excess water. Subsequently, 500 μL of bovine serum albumin (BSA, 1.0 w/v %, 0.01 M PBS) was employed to block the nonspecific sites of Au NPs at 37℃ for 0.5 h. The excess BSA could be removed by washing the resultant hydrogel for three 10


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times. Finally, the blocked anti-AFP/Au@g-C 3 N 4 /MCC hydrogel was obtained and saved at 4℃ for later use.

2.6 AFP detection with the colorimetric immunoassay One piece of anti-AFP/Au@g-C 3 N 4 /MCC was incubated with 400 μL of different concentrations (0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1,000 ng/mL, 10,000 ng/mL) of AFP for 1 h at 37℃ with gently shaking. AFP bound specifically to anti-AFP to form a conjugate that detached from the surface of Au, followed by three washing steps with PBST. Residual water was removed by filter paper and then the hydrogel was put into 10 mL 4-nitrophenol/NaBH 4 hybrid solution for catalytic reaction at room temperature. The changes of the absorption peak of 4-nitrophenol at 400 nm was recorded. The preparation of hybrid solution and the catalytic detection process were same as before described in §2.4.

2.7 Application test with the samples from clinical patients To further validate the performance of the anti-AFP/Au@g-C 3 N 4 /MCC in real patient serum testing, we collected serum from three patients. Before the colorimetric immunoassay test, a commercial AFP ELISA kit was used for quantitative determination as the control group and the process is presented in the Supporting Information. The developed colorimetric immunoassay was carried out as follows. 50 μL of each serum sample was mixed with 450 μL PBS solution to get the serum dilution. The blocked anti-AFP/Au@g-C 3 N 4 /MCC was incubated with 400 μL above serum 11



diluent for 1 h at 37℃ with gentle shaking. After washing three times with PBST solution to remove the anti-AFP/AFP conjugates and removing residual water by using filter paper, the hydrogel was then transferred to a 4-nitrophenol/NaBH 4 solution. A UV-Vis absorption spectrometer was used to detect changes in absorption peak at 400 nm. The preparation of hybrid solution and the catalytic detection process were same as before described in §2.4.

3. RESULTS AND DISCUSSION 3.1 Characterization of g-C 3 N 4 As shown in Fig. 1a, g-C 3 N 4 exhibited its typical sheet-like morphology after ultrasonic treatment. The energy-dispersive X-ray images (Fig. 1a, inset) were confirmed that carbon and nitrogen were uniformly distributed over the nanosheets. Fig. 1b shows the X-ray diffraction (XRD) pattern of the bulk g-C 3 N 4 . There were two characteristic diffraction peaks at 13.8° and 27.2°. The peak of 27.2° is associated with the (002) plane causing by the interplanar stacking of aromatic systems with an interplanar distance of 0.324 nm, while the peak of 13.8°is indexed to (100) plane arising from in-planar ordering of tri-s-triazine units with distance of 0.675 nm.38-39 The AFM image (Fig. S1a) has proved that the prepared g-C 3 N 4 was sheets in nanoscale and well separated. The randomly measured 3 nanosheets gave a consistent thickness 3.86 ± 0.18 nm (Fig. S1b).

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Figure 1. (a) The TEM image of the ultrathin g-C 3 N 4 nanosheets and the corresponding elemental mapping, (b) the XRD patterns of bulk g-C 3 N 4 .

3.2 Promoting effect of g-C 3 N 4 on the formation of porous hydrogel Fig. 2 is the SEM images of MCC, MCC-Au, Au@g-C 3 N 4 /MCC hydrogels. The SEM images revealed that the surface of the MCC hydrogel was relatively dense with a few observable pores (Fig.2a). A large number of small pores (260 ± 80 nm) appeared in the hydrogel where the HAuCl 4 was in situ reduced to Au nanoparticles (Fig. 2b). More significant changes appeared when the g-C 3 N 4 was introduced into the Au/MCC mixture. The obtained Au@g-C 3 N 4 /MCC hydrogel showed a so called loofah spongelike 3D network structure (Fig. 2c). The holes of Au@g-C 3 N 4 /MCC were curved with an average pore diameter of 1.29 ± 0.57 μm. Among many factors affecting the formation of a loofah sponge-like structure, which is extremely advantageous for a heterogeneous catalytic system, the boiling process is the most important one. Such a structure cannot be obtained without a boiling treatment (Fig. S2).

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Figure 2. The SEM images of (a) MCC hydrogel, (b) MCC-Au 80 hydrogel and (c) Au@g-C 3 N 4 /MCC hydrogel.

3.3 In situ reduction of Au NPs in the cellulose Usually, Au NPs act as active centers of catalysis reactions, and their quantity, size and size distribution are important control factors. The shape, size and particle size distribution of Au NPs depend on the amount of chloroauric acid added to a certain amount of cellulose. The size of prepared Au NPs under different added volumes of HAuCl 4 had been studied. When the volumes of HAuCl 4 were 20 μL，40 μL，60 μL， 80 μL，100 μL and120 μL , the corresponding diameters of Au NPs were 5.9±1.9 nm， 8.8±1.8 nm，11.2±1.2 nm，37.8±20.0 nm，39.2±5.3 nm and 42.0±20.8 nm, respectively (Fig. S3a). Obviously, as the volume of HAuCl 4 added increased, the particle size and size distribution of Au NPs increased. The size of Au NPs in the MCCAu 20 , MCC-Au 40 and MCC-Au 60 hydrogels were similar, while the diameter and distribution increased dramatically in MCC-Au 80 . The TEM image of Au NPs in MCCAu 80 hydrogel (Fig. S3b) shows that the lager Au NPs (blue region) had a diameter of several tens of nanometers, while the smaller Au NPs (orange region) had a size below 10 nm, which could be attributed to that in a system in which the cellulose concentration and the number of reducing hydroxyl groups were determined. When HAuCl 4 was 14


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added in a small amount, HAuCl 4 was rapidly reduced to small Au particles; when HAuCl 4 was continuously added, since the concentration of the reducing group was lowered, HAuCl 4 was easily deposited on the existing Au particles to form larger particles. In this study, the introduction of g-C 3 N 4 also had a remarkable effect on the formation of Au nanoparticles. The Au NPs loaded on the g-C 3 N 4 nanosheets in Au@gC 3 N 4 /MCC hydrogel (Fig. S3c) were evenly distributed on the g-C 3 N 4 nanosheets, and its size was relatively uniform (18.5±3.9 nm, the last data column in the histogram Fig. S3a). The results showed that the addition of g-C 3 N 4 could effectively reduce the size of Au NPs and the size distribution was decreased as well. The uncondensed –NH 2 and -NH- groups on g-C 3 N 4 can act as good ligands to coordinate with metal ions and thus efficiently stabilize metal NPs. The strong coordination of the surface functional groups to the metal ions finally results in the formation of uniform NPs with small size.40 In addition, a part of the Au NPs were directly loaded in the MCC (Fig. S3d) and the diameter was 22.9±5.0 nm, which was slightly larger than the Au NPs loaded on the nanosheets.

TEM investigation provided the information of surface loading of Au NPs on gC 3 N 4 nanosheets and the elemental composition of Au@g-C 3 N 4 /MCC. Fig. 3a shows the Au NPs uniformly distributed over the surface of g-C 3 N 4 nanosheets. Fig. 3b reveals an enlarged TEM image of a single Au nanoparticle, which exhibits a spherical shape. The lattice fringe of the obtained Au NPs was 0.204 nm, corresponding to (200) plane of face-centered cubic (fcc) Au NPs (Fig. 3b, inset). It is difficult to distinguish 15



the difference between MCC and g-C 3 N 4 nanosheets in TEM image. The obtained hybrid nanosheets were further characterized by HAADF-STEM element mapping. Fig. 3c-f indicates the uniform distribution of C (red) and O (yellow) over the whole nanosheet, and decoration of Au NPs (purple) of the surface of nanosheet. Besides, the N (green) from g-C 3 N 4 was distributed on the whole nanosheets as well, revealing that the hybrid nanosheets was consist with g-C 3 N 4 nanosheets, MCC and Au NPs.

Figure 3. (a) The TEM image of Au@g-C 3 N 4 /MCC hydrogel, (b) high-magnification TEM image and corresponding HRTEM image (inset) of one single Au NP, and (c-f) the corresponding elemental mapping of Au@g-C 3 N 4 /MCC hydrogel.

XRD was applied to characterize the crystal form of MCC cellulose in the dissolution and regeneration process, and confirmed the existence of Au NPs. In Fig. 4a, the diffraction peaks at 14.5°, 16.3° and 22.4° represents the (110), (110) and (200) planes of cellulose I that are the characteristic peaks of the MCC raw material. The 16


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XRD pattern of the freeze-dried cellulose hydrogel (MCC hydrogel) showed diffraction peaks at 12.2°, 20.1° and 21.7°, corresponding to (110) , (110) and (200) planes of cellulose II.41 After the deposition of Au NPs in MCC hydrogel, new peaks at 38.2° and 44.4° were observed, which were indexed to the (111) and (200) crystal face of Au NPs. The characteristic peak of Au NPs still existed in the Au@g-C 3 N 4 /MCC hydrogel, but no obvious diffraction peaks of g-C 3 N 4 appeared, indicating that the amount of g-C 3 N 4 was very low. In order to prevent the Au ions from being reduced at room temperature (Fig. S4), it needed to be maintained at -20℃ in the regeneration process of MCC. Lin et al.36 demonstrated that a small part of Au ions diffused into ethanol and water during the regeneration of cellulose into a hydrogel and reduction process. ICP (Table S1) measurements detected the amount of Au overflowing from the hydrogel. By calculating the difference between the added total amount and the overflow amount, we found that about 90% of added gold retained in each hydrogel.

Figure 4. (a) XRD pattern of MCC raw material (purple line), MCC hydrogel (green line), MCC-Au 80 hydrogel (red line), and Au@g-C 3 N 4 /MCC hydrogel (brown line)， (b) UV-vis absorption spectra of MCC raw material (black line), MCC hydrogel (red line), g-C 3 N 4 (blue line), MCC-Au 80 hydrogel (purple line), and Au@g-C 3 N 4 /MCC hydrogel (green line). 17



The absorption spectra of five samples (Fig. 4b) indicated that the absorption of the MCC raw material (MCC) and MCC hydrogel were very weak in visible light range (380-700 nm), while an obvious absorption edge of g-C 3 N 4 appeared at about 460 nm, which was related to the band gap of 2.7 eV implying its visible-light-induced photocatalytic activity.42-44 Due to the introduction of Au nanoparticles, new peaks at 544 nm and 543 nm in MCC-Au and Au@g-C 3 N 4 /MCC hydrogel appeared for MCCAu hydrogel sample and Au@g-C 3 N 4 /MCC hydrogel sample, which could be attributed to the localized SPR effect of Au nanoparticles, showing an efficient plasmon resonance in the visible region. Compared with MCC hydrogel and g-C 3 N 4 as the catalyst alone, the absorption within visible light range was significantly extended for the MCC-Au and Au@g-C 3 N 4 /MCC hydrogels. Relative to MCC-Au 80 hydrogel, the intensity of the maximum absorption peak of Au@g-C 3 N 4 /MCC was significantly enhanced, and the position was blue-shifted. In the MCC-Au 80 and Au@g-C 3 N 4 /MCC systems, although the amount of the initial raw material HAuCl 4 was the same, the presence of g-C 3 N 4 nanosheets caused the Au NPs in the Au@g-C 3 N 4 /MCC system smaller in particle size and relatively larger in number. The wider light absorption range and increased intensity of absorption allowed the Au@g-C 3 N 4 /MCC hydrogel photocatalytic system to accelerate the generation of more electron-hole pairs, thereby significantly increasing the catalytic efficiency.

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Figure 5. (a) The full XPS spectrum of the Au@g-C 3 N 4 /MCC hydrogel, highresolution XPS of (b) Au 4f peaks in MCC-Au 80 and Au@g-C 3 N 4 /MCC hydrogel, respectively, (c) N 1s peaks in Au@g-C 3 N 4 /MCC hydrogel.

With the help of X-ray photoelectron spectroscopy (XPS) test, we determined the elemental composition and its valence of MCC-Au 80 and Au@g-C 3 N 4 /MCC. Full-scan XPS spectrum of Au@g-C 3 N 4 /MCC (Fig. 5a) suggested that hybrid hydrogel was composed of C (MCC and g-C 3 N 4 ), N (g-C 3 N 4 ), O (MCC and the surrounding environment) and Au element. Fig. 5b presents the high-resolution XPS spectrum of Au 4f doublet (4f 5/2 and 4f 7/2 ) for the Au NPs in the MCC-Au 80 and Au@g-C 3 N 4 /MCC hydrogel, respectively. In the MCC-Au 80 hydrogel, the Au 4f 5/2 and 4f 7/2 peaks appeared at 87.7 eV and 84.0 eV, 31 which are characteristic peaks for metallic Au0. It indicates that the Au NPs were reduced, consistent with XRD spectra and TEM images. For Au@g-C 3 N 4 /MCC hydrogel, the Au 4f 5/2 and 4f 7/2 peaks appeared at 87.5 eV and 83.8 eV. The negative shift (-0.2 eV) obtained in the above two samples came from the electron transfer from the g-C 3 N 4 nanosheets to Au NPs. The g-C 3 N 4 is an N-type semiconductor, its work function (4.65 eV) is lower than that of Au (5.31-5.47 eV). Therefore, the electrons generated on g-C 3 N 4 nanosheets can be transported to Au nanoparticles in the surface of Au-loaded g-C 3 N 4 .28 Fig. 5c shows XPS spectrum of N 19



1s. The peak at 398.5 eV is attributed to C-N=C coordination, which originates from the sp2-bonded N in triazine rings. The other two peaks at higher binding energies (around 399.9 eV and 402.1 eV) were assigned to N-(C) 3 and C-N-H species, respectively. All of those peaks are the typical peaks of g-C 3 N 4 nanosheets.39

3.4 Catalytic efficiency of the MCC-Au hydrogel The various volumes of HAuCl 4 (1 w/v %, 0, 20 μL, 40 μL, 60 μL, 80 μL, 100 μL, 120 μL) were added to 1 g cellulose solution to study the catalytic performance. After taking the natural logarithm of the absorbance (ln(Abs)) of 4-nitrophenol at different reaction times, we found that ln(Abs) was linear with the reaction time (Fig. 6a). Apparently, the MCC hydrogel had no catalytic activity for the model reaction. As the amount of Au added was increasing, the catalytic effect of the MCC-Au samples gradually increased to the maximum value and then began to decline. Fig. 6b shows the UV-vis absorption spectra of 4-nitrophenol reduced by NaBH 4 in the presence of Au@gC 3 N 4 /MCC under natural light condition at different reaction times. The absorption of 4-nitrophenol at 400 nm gradually decreased, while a new absorption peak of product 4-AP at 300 nm gradually increased. Simultaneously, the color of solution gradually changed from bright yellow to colorless (Fig. 6b, inset). Fig. 6c shows the rate constant k (min-1) value of each hydrogel samples, where the k is the slope of the fitting linear. The k values of MCC, MCC-Au 20 , MCC-Au 40 , MCC-Au 60 , MCC-Au 80 , MCC-Au 100 and MCC-Au 120 were 0.22 ×10-3 min-1，1.77 ×10-3 min-1，3.26 ×10-3 min-1，3.38 ×103

min-1，4.86 ×10-3 min-1，3.26 ×10-3 min-1 and 2.33 ×10-3 min-1, respectively. When 20


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the amount of HAuCl 4 added was increased up to 80 μL, the catalytic activity of the Au/MCC reached a maximum. In general, the catalytic activity of Au NPs is positively correlated with the number of Au particles and negatively dependent on the particle size. The final catalytic effect depends on the balance between the above two factors. Although the diameter of Au NPs in MCC-Au 20 , MCC-Au 40 , MCC-Au 60 was small, their gold particle density did not provide enough catalytic sites. While the amount of HAuCl 4 was excessive in the MCC-Au 100 and MCC-Au 120 to obtain the larger size of Au NPs，resulting in a decrease in catalytic efficiency.45

Figure 6. (a) The plot of natural logarithmic value of 4-nitrophenol absorption (ln (Abs)) at 400 nm over time catalyzed by MCC, MCC-Au 20 , MCC-Au 40 , MCC-Au 60 , MCCAu 80 , MCC-Au 100 , and MCC-Au 120 hydrogel, (b) the UV-vis absorption spectra of the reduction of 4-nitrophenol by NaBH 4 in the presence of Au@g-C 3 N 4 /MCC hydrogel, (c) the calculated rate constant (k) of MCC, MCC-g-C 3 N 4 , MCC-Au 20 , MCC-Au 40 , MCC-Au 60 , MCC-Au 80 , MCC-Au 100 , MCC-Au 120 and Au@g-C 3 N 4 /MCC hydrogels, respectively.

3.5 Improvement of catalytic performance of g-C 3 N 4 nanosheets for Au/MCC system Under the condition of fixed MCC/g-C 3 N 4 ratio (1g: 40 μg), we evaluated the catalytic 21



performances of the Au NPs/g-C 3 N 4 /MCC when different amounts of HAuCl 4 solution were employed (supporting information, Fig. S5). The results showed that 80 μL was the best value. For the same model reaction, the k value of Au@g-C 3 N 4 /MCC was calculated 39.54×10-3 min-1. It was remarkably increased up to 8.1 times compared with that obtained from MCC-Au 80 hydrogel (k=4.86×10-3 min-1). Meanwhile, a control test (MCC-g-C 3 N 4 hydrogel) presented a k value 0.75×10-3 min-1, demonstrating that g-C 3 N 4 alone had no obvious catalytic effect. The addition of g-C 3 N 4 increased the catalytic activity of Au NPs in MCC significantly. The reason can be summarized as the following three aspects. Firstly, the porous structure facilitates the substrate accessing to the active sites inside the Au@gC 3 N 4 /MCC. Secondly, the Au NPs supported on the g-C 3 N 4 nanosheets had a small particle size and were uniformly dispersed without aggregation. Last but not the least, the electron transfer from g-C 3 N 4 to Au NPs accelerated the production of superoxide ion radicals (𝑂𝑂2∙− ) (Fig. S6a) and further promoted the reduction of 4-nitrophenol to 4AP.46

3.6 Mechanism of colorimetric assay Normally, the methods of immunoassay have been developed on the basis of the “sandwich structure”.1, 47-48 Generally, when the Au NPs are used as a carrier to bind the protein, the thiol group (-SH) bond of protein is cleaved to form the Au-S bond.49 In order to investigate the mechanism of this colorimetric assay, spectroscopic methods such as FTIR and XPS spectroscopy were used to detect the interaction between the 22


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Au@g-C 3 N 4 /MCC hydrogel and antibody. The position of amide I (1600-1700 cm-1) and amide II (1500-1600 cm-1) of the FTIR bands provide detailed information on the secondary structure of the polypeptide chain and can serve as an indicator of conformational changes in the protein.50 The signals of amide I and II bands of the antiAFP/Au@g-C 3 N 4（1539 cm-1 and 1633 cm-1）and anti-AFP/Au@g-C 3 N 4 /MCC（1543 cm-1）were similar to those of anti-AFP (1539 cm-1and 1635 cm-1), indicating that the anti-AFP was successfully attached to Au@g-C 3 N 4 and Au@g-C 3 N 4 /MCC (Fig. S7). The anti-AFP/Au@g-C 3 N 4 /MCC shows the –SH stretching vibrations at 2550-2600 cm-1 (Fig. 7a, blue line). Disappearance of this peak in spectrum of anti-AFP/Au@gC 3 N 4 suggested the bonding of Au-S (Fig. 7a, purple line).51 To further demonstrate the presence of Au-S bond, the oxidation state and binding properties of Au NPs labeled to anti-AFP were determined by XPS. Fig. 7b shows an obvious new peak appeared at 85.2 eV in the spectrum of anti-AFP/Au@g-C 3 N 4 , which is assigned to Au (I) attributed to Au-S bond, while this peak didn’t appear when the anti-AFP/Au@g-C 3 N 4 /MCC was measured. The XPS results were in accordance with the FTIR measurement. The above results illustrated that the MCC hindered the Au-S bond between Au NPs and anti-AFP, and the anti-AFP was attached to Au NPs surface by non-covalent bond. This judgment is in agreement with the proposed mechanism by Xiao et al in 2017.26

23



Figure 7. (a) The FTIR spectra of anti-AFP/Au@g-C 3 N 4 and anti-AFP/Au@gC 3 N 4 /MCC, (b) the XPS spectra of Au 4f in anti-AFP/Au@g-C 3 N 4 and anti-AFP/Au@ g-C 3 N 4 /MCC.

The suggested detection mechanism is demonstrated in Scheme 1. Au@g-C 3 N 4 /MCC is able to catalyze the conversion of 4-nitrophenol to 4-AP, with the substrate’s color gradually fading from yellow into colorless. When the Au@g-C 3 N 4 /MCC hydrogel was incubated with anti-AFP, the catalytic sites of the Au NPs surface were covered by anti-AFP，causing the catalytic performance to be “ switched off ”. Since AFP and antiAFP were specific antigen-antibody pairs, the addition of AFP could specifically bind to anti-AFP absorbed on Au NPs surface. When the anti-AFP/AFP complex was formed, anti-AFP was supposed to undergo conformational change, resulting in a weak affinity between the complex and Au@g-C 3 N 4 /MCC. Finally, the anti-AFP/AFP complex dissociated from the surface of Au NPs in the Au@g-C 3 N 4 /MCC, whose catalytic site could be “switched on” and the catalytic performance was recovered again. Theoretically, when the amount of anti-AFP immobilized on the Au@g-C 3 N 4 /MCC surface was sufficient and quantitative, the recovered catalytic sites of Au NPs surface increased and the color change of the catalytic substrate was more obvious at the same 24


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time with the increasing of AFP concentration. The qualitative and the quantitative detection of AFP can be achieved by using the developed theory.

Scheme 1. Schematic illustration of the colorimetric immunoassay for the detection of alpha-fetoprotein antigen (AFP) based on the catalytic performance of Au@gC 3 N 4 /MCC: (a) the mechanism of the electron transfer between g-C 3 N 4 and Au NPs, (b) the color of 4-nitrophenol was fading almost completely at the 40th minute catalyzed by Au@g-C 3 N 4 /MCC, (c) the incubate procession of antibody/antigen with Au@g-C 3 N 4 /MCC, (d) the color of 4-nitrophenol was fading partly at the 40th minute catalyzed by incubated Au@g-C 3 N 4 /MCC, (e) the color and structure of 4-nitrophenol (yellow) and 4-AP (colorless).

3.7 Immobilization of anti-AFP on the Au@g-C 3 N 4 /MCC hydrogel The Au NPs in Au@g-C 3 N 4 /MCC lose high catalytic activity and become "inactive" after being incubated with antibody. Fig. 8 shows the logarithmic value of absorption 25



of 4-nitrophenol (400 nm) over time catalyzed by different anti-AFP-incubated Au@gC 3 N 4 /MCC (anti-AFP-Au@g-C 3 N 4 /MCC), indicating that the catalytic activity of Au@g-C 3 N 4 /MCC gradually decreased with the increase of anti-AFP concentration. The k value of 10 μg/mL (9.0×10-3 min-1) was quite close to that of 100 ug/mL (8.5×103

min-1), so we chose a concentration of 10 μg/mL as the incubation concentration in

the subsequent experiments in order to improve its economy. The catalytic activity of the obtained anti-AFP-Au@g-C 3 N 4 /MCC was reduced by 77.2%. The non-specific sites of anti-AFP-Au@g-C 3 N 4 /MCC were further blocked with 1% BSA, and the catalytic efficiency was reduced by 88.8%. The catalytic sites of catalyst were almost “turned off” with the “inactive” catalytic performance.

Figure 8. Time-dependent absorbance changes at 400 nm of 4-nitrophenol reaction solution catalyzed by Au@g-C 3 N 4 /MCC hydrogel with different concentration of antiAFP.

3.8 Analytical performance and selectivity of the colorimetric immunoassay 26


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When the AFP antigen was added, it captured anti-AFP from the surface of Au NPs to form the anti-AFP/AFP conjugates, releasing the active sites of Au surface, thereby the catalytic activity of Au@g-C 3 N 4 /MCC was “turned on” again. Fig. 9a shows the relationship between the catalytic rate k value of recovered Au@g-C 3 N 4 /MCC and the concentration of AFP. The k values gradually increased with the increasing AFP concentration, which was constant with the above theory. The anti-AFP/AFP conjugates detached from the anti-AFP-Au@g-C 3 N 4 /MCC surface with the increase of AFP concentration, resulting in that the more catalytic active sites are “uncovered” with a higher k value appearing. The immunoassay exhibited a good linear correlation between k and the logarithm values of AFP concentration range from 0.1 ng/mL to 10,000 ng/mL. The linear equation was y=4.813x+10.320 (x=lg[c AFP (ng/mL)], R2=0.995) with a detection limit of 0.46 ng/mL (LOD=3σ blank /S, where S is the slope of the line and σ is the standard deviation of the blank sample, n=3). When the reaction proceeded to the 40th minute, the color of 4-nitrophenol changed from yellow to colorless gradient with the increase of AFP concentration (Fig. 9b). Significantly, when the AFP concentration exceeded 10 ng/mL, the color of 4-nitrophenol had a visually distinguishable change. Since the threshold values in normal human serum is 10 ng/mL for AFP, the sensitivity

of the developed colorimetric immunoassay can be appropriate for clinical diagnosis.16

Compared with the results of other reported methods in the determination of AFP (Table S2), the method we present here is one of the most excellent immunosensors for AFP.

27



Figure 9. Detection of AFP using the proposed colorimetric immunosensor: (a) The calibration curve of the reaction constant k and (b) photographs in the presence of different concentration of AFP antigen, (c) the selectivity of the immunoassay.

Since the signal factors are diverse in serum for clinical application, a good specificity for target analysis is necessary for a new detection sensing system. To inspect the selectivity, the responses of other disruptors, such as prostatic specific antigen (PSA, 10 ng/mL), luteinizing hormone (LH, 112 mIU/mL), immunoglobulin G (IgG, 10 ng/ mL) were detected by the developed colorimetric immunoassay under the same conditions. The comparison results were given in Fig. 9c. When the target AFP was added, the k value reached 14.1 min-1, which was significantly restored compared to other disruptors. The k values of PSA, LH, and IgG were not obviously different from the blank sample. The existence of interfering proteins had no effect on the detection of the target AFP protein, indicating that the selectivity of the immunoassay is quite acceptable.

28


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3.9 Clinical diagnosis, repeatability and stability of the colorimetric immunoassay To investigate the possibility of the colorimetric immunoassay in clinical applications, this biosensor was used to detect the actual serum samples from three patients, and the results were compared with commercial enzyme-linked immunosorbent assay. As illustrated in Table 2, the results obtained from the two methods were in consistence with each other. The relative error range was from -4.3% to 2.8%, indicating that the developed immunoassay was reliable.

Table 2. Comparison of the assay results by using the colorimetric immunoassay and the referenced ELISA method in real clinic samples. ELISA method

Colorimetric immunoassay

Samples

(c/ng mL-1)

(c/ng mL-1)

Relative error (%)

1

60.11

57.51

-4.3

2

1290.53

1326.77

2.8

3

2807.74

2758.51

-1.8

The reproducibility of the assay was determined by measuring the AFP-contained sample (10 ng/mL) for three times and comparing the results. The relative standard deviation was 6.0% based on three replicates, indicating a good accuracy and repeatability of the immunoassay. In addition, after storing at 4℃ in a PBS solution (0.01M, pH 7.4) for a week, the hydrogel had maintained its catalytic activity (37.35× 10-3 min-1)，less than 10% compared with the fresh Au@g-C 3 N 4 /MCC (39.54×10-3 min-1).

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4. CONCLUSIONS A highly efficient, selective hybrid hydrogel (Au@g-C 3 N 4 /MCC) for the detection of AFP was successfully prepared. Applying the model reaction (from yellow 4nitrophenol to colorless 4-AP) catalyzed by Au@g-C 3 N 4 /MCC, the quantitative detection of AFP in the range of 0.1-10,000 ng/mL was achieved on the basis of the linear relation between the k values and the AFP concentration, y=4.813x+10.320 (R2=0.995, LOD=0.46 ng/mL). When the concentration of AFP exceeded the cutoff value (10 ng/mL), an apparent color fading of reaction substrate 4-nitrophenol occurred. It was a visible basis for the diagnosis of liver cancer without a further instrument measurement. The Au NPs were reduced in situ by hydroxyl groups on the MCC without another reducing agent. The g-C 3 N 4 nanosheets efficiently reduced the diameter and the distribution of Au NPs, and made the Au NPs uniformly dispersed. The investigation results suggested that the anti-AFP was attached to Au@gC 3 N 4 /MCC by non-covalent force. Remarkably, the catalytic efficiency of Au@gC 3 N 4 /MCC was 8.1 times higher compared with MCC-Au system. Considering various advantages, this colorimetric immunoassay is promising for the target cancer markers’ detection.

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ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publication website at DOI:***. Methods and Characterization. The AFM image of g-C 3 N 4 nanosheets and the corresponding height image of three randomly tested nanosheets (Figure S1); The SEM image of Au NPs/g-C 3 N 4 /MCC prepared under the condition of no contact with boiling water (Figure S2); (a) The diameter of Au NPs in MCC-Au and Au@gC 3 N 4 /MCC hydrogels, the TEM images of Au NPs in (b) MCC-Au 80 hydrogel, (c) Au@g-C 3 N 4 /MCC hydrogel, and (d) the TEM image of Au NPs dispersed in the cellulose of Au@g-C 3 N 4 /MCC hydrogel (Figure S3); Photographs of colored MCCAu hydrogels before heating at room temperature for 30 min (Figure S4); The calculated rate constant (k) with the different amounts of 1% (w/v) HAuCl 4 solution (Figure S5); The EPR spectra of 𝑂𝑂2∙− and hole of Au@g-C 3 N 4 /MCC and the relevant g factor under visible light irradiation (Figure S6); The FTIR spectra of the anti-AFP, MCC, g-C 3 N 4 , anti-AFP/Au@g-C 3 N 4 and anti-AFP/Au@g-C 3 N 4 /MCC (Figure S7). Loading amount of Au atoms in MCC hydrogels (Table S1); Information of the developed colorimetric method and some reported immunoassay methods for AFP detection (Table S2).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Fang Ma and Chun-Wang Yuan contributed equally to this article. Notes The authors declare no competing financial interest.

31



ACKNOWLEDGMENTS This study was supported by the following grants: National Natural Science Foundation of China (No.51733009)；Capital Health Research and Development of Special Fund (No.2018-2-2182); Young Talent Incubation Project of Beijing Youan Hospital (No.YNKT20160032); Beijing Municipal Science & Technology Commission (No.Z181100001718070) and Beijing Higher Education Interdisciplinary Training Plan for High Level Talents (2017).

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TOC

39



TOC


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Figure 1. (a) The TEM image of the ultrathing g-C3N4 nanosheets and the corresponding elemental mapping, (b) the XRD patterns of bulk g-C3N4.



Figure 2. The SEM images of (a) MCC hydrogel, (b) MCC-Au80 hydrogel and (c) Au@g-C3N4/MCC hydrogel.


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Figure 3. (a) The TEM image of Au@g-C3N4/MCC hydrogel, (b) high-magnification TEM image and corresponding HRTEM image (inset) of one single Au NP, and (c-f) the corresponding elemental mapping of Au@g-C3N4/MCC hydrogel.



Figure 4. (a) XRD pattern of MCC raw material (purple line), MCC hydrogel (green line), MCC-Au80 hydrogel (red line), and Au@g-C3N4/MCC hydrogel (brown line)， (b) UV-vis absorption spectra of MCC raw material (black line), MCC hydrogel (red line), g-C3N4(blue line), MCC-Au80 hydrogel (purple line), and Au@g-C3N4/MCC hydrogel (green line).


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Figure 5. (a) The full XPS spectrum of the Au@g-C3N4/MCC hydrogel, highresolution XPS of (b) Au 4f peaks in MCC-Au80 and Au@g-C3N4/MCC hydrogel, respectively, (c) N 1s peaks in Au@g-C3N4/MCC hydrogel.



Figure 6. (a) The plot of natural logarithmic value of 4-nitrophenol absorption (ln (Abs)) at 400 nm over time catalyzed by MCC, MCC-Au20, MCC-Au40, MCC-Au60, MCCAu80, MCC-Au100, and MCC-Au120 hydrogel, (b) the UV-vis absorption spectra of the reduction of 4-nitrophenol by NaBH4 in the presence of Au@g-C3N4/MCC hydrogel, (c) the calculated rate constant (k) of MCC, MCC-g-C3N4, MCC-Au20, MCC-Au40, MCC-Au60, MCC-Au80, MCC-Au100, MCC-Au120 and Au@g-C3N4/MCC hydrogel, respectively.


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Figure 7. (a) The FTIR spectra of anti-AFP/Au@ g-C3N4 and anti-AFP/Au@gC3N4/MCC, (b) the XPS spectra of Au 4f in anti-AFP/Au@g-C3N4 and anti-AFP/Au@ g-C3N4/MCC.



Scheme 1. Schematic illustration of the colorimetric immunoassay for the detection of alpha-fetoprotein antigen (AFP) based on the catalytic performance of Au@gC3N4/MCC: (a) the mechanism of the electron transfer between g-C3N4 and Au NPs, (b) the color of 4-nitrophenol was fading almost completely at the 40th minute catalyzed by Au@g-C3N4/MCC, (c) the incubate procession of antibody/antigen with Au@g-C3N4/MCC, (d) the color of 4-nitrophenol was fading partly at the 40th minute catalyzed by incubated Au@g-C3N4/MCC, (e) the color and structure of 4-nitrophenol (yellow) and 4-AP (colorless).


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Figure 8. Time-dependent absorbance changes at 400 nm of 4-nitrophenol reaction solution catalyzed by Au@g-C3N4/MCC hydrogel with different concentration of antiAFP.



Figure 9. Detection of AFP using the proposed colorimetric immunosensor: (a) The calibration curve of the reaction constant k and (b) photographs in the presence of different concentration of AFP antigen, (c) the selectivity of the immunoassay.


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Table 1. The preparation of MCC-Au hydrogels. Samples

MCC Solution（g）

1% HAuCl4 Solution（μL）

MCC

1

0

MCC-Au20

1

20

MCC-Au40

1

40

MCC-Au60

1

60

MCC-Au80

1

80

MCC-Au100

1

100

MCC-Au120

1

120

MCC means microcrystalline cellulose hydrogel. Au means the Au NPs. The numbers (20, 40, 60, 80, 100, 120) stand for the volume of added 1% HAuCl4 (20 μL, 40 μL, 60 μL, 80 μL, 100 μL and 120 μL) in 1 g of MCC solution, respectively.



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Table 2. Comparison of the assay results by using the colorimetric immunoassay and the referenced ELISA method in real clinic samples. ELISA method

Colorimetric immunoassay

Samples

(c/ng mL-1)

(c/ng mL-1)

Relative error (%)

1

60.11

57.51

-4.3

2

1290.53

1326.77

2.8

3

2807.74

2758.51

-1.8


A colorimetric immunosensor based on Au@g-C3N4 doped sponge

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