Ratiometric SERS immunosorbent assay of allergenic proteins via

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Ratiometric SERS immunosorbent assay of allergenic proteins via covalent organic framework composite material based nanozyme tag triggered Raman signal "turn-on" and amplification Yiyun Su, Di Wu, Jian Chen, Guang Chen, Na Hu, Honglun Wang, Panxue Wang, Haoyu Han, Guoliang Li, and Yongning Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02233 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019

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Analytical Chemistry

Ratiometric SERS immunosorbent assay of allergenic proteins via covalent organic framework composite material based nanozyme tag triggered Raman signal "turn-on" and amplification Yiyun Su1a, Di Wu1d, Jian Chena, Guang Chene, Na Huc, Honglun Wangc, Panxue Wanga, Haoyu Hana, Guoliang Li ae* and Yongning Wub a.

School of Food and Biological Engineering, Shaanxi University of Science and

Technology, Xi’an 710021, China b.

NHC Key Laboratory of Food Safety Risk Assessment, China National Center for

Food Safety Risk Assessment, Beijing 100050, China c.

Key Laboratory of Tibetan Medicine Research＆Qinghai Provincial Key Laboratory

of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China d.

Yangtze Delta Region Institute of Tsinghua University, Zhejiang 314006, China

e.

Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu 273165, China

1The

authors equally contribute to this work.

*Corresponding author: E-mail: [email protected] (Guoliang Li)

1

ACS Paragon Plus Environment

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

Abstract The exploration of nanomaterials with mimic enzyme activity (named as nanozyme) has gained extensive attentions in the fields of advance analytical chemistry and material science. Herein, the gold nanoparticles-doped covalent organic frameworks (COFs) were prepared, which exhibited not only the excellent mimic nitroreductase activity but also robust stability. By replacing the traditional natural enzyme tag in enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In SERS assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both Au-S bond and electrostatic force to further produce Raman “hot spot”. Meanwhile, the Raman signal of 4-nitrothiophenol at 1573 cm-1 was weakened, and a new signal at 1591 cm-1 generated by 4-ATP was turned on, leading to the generation of ratiometric SERS signal. Based on this performance, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately, which was applied to detect β-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I1591/I1573 with a limit of detection (LOD) of 0.01 ng/mL. The linear range is 25.65-6.2×104 ng/mL, covering more than three orders of magnitude. The developed method showed many advantages such as low-cost, higher recovery and lower cross-reactivity, providing a new insight on the application of SERS technology for trace target analysis. Keywords: surface enhanced Raman scattering; nanozyme; immunosorbent assay; gold nanostars; allergenic proteins 2


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Introduction Nowadays, food allergy, which may affect about 8% of children and 5% of adults, is a growing concern all over the world.1 On January 1st, 2006, Food Allergen Labeling and Consumer Protection Act (FALCPA) took action and pointed eight most common food allergens: milk,

eggs, fish (e.g. bass, flounder and cod), crustacean shellfish (e.g. crab, lobster and shrimp),

tree nuts (e.g. almonds, pecans and walnuts), wheat, peanuts and soybeans, making it easier for consumers to identify the food allergens.2 Up to date, strict avoidance of the allergen-containing food is the only way to protect allergic individuals against the allergy reaction of food as there is no treatment for food allergies. Although the allergens information can be read from the labels of food, undeclared allergenic substances can be accidentally introduced into food by uncontrolled cross-contamination and cause accidental exposures.3 Therefore, detection of food allergies is of great importance for the allergic individuals. So far, many methods have been developed to detect food allergens: DNA detection based on polymerase chain reaction (PCR) technology,4,5 liquid chromatography tandem mass spectrometry (LC-MS/MS) detection,6,7 biosensor platforms,8,9 and immunoassay.10,11 However, methods based on instrumental analysis such as LC-MS/MS and PCR typically require well-trained professionals, high costs and complicated operations. For example, sample pre-treatment is required in LC-MS/MS analytical procedure to remove the interfering proteins, and then the enzymatic hydrolysis of the obtained protein is performed to obtain the specified peptide before further identification and detection by LC-MS/MS.12 Moreover, the feature fragments often presented at trace level and may be lost in the preprocessing operations, making analytical process more difficult. Currently, the most widely used method for detecting allergenic proteins is still enzyme-linked immunosorbent assays (ELISA).13 3



For traditional ELISA, detecting antibodies were typically labeled with native enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), which showed many advantages such as high efficiency and commercial availability. However, high production cost, short shelf life, sensitivity to environmental change and decrease of activity during conjugation reactions of natural enzymes create an increasing demand for cheap and more stable enzymes.14 Recently, the rapid development of nanoscience has brought many opportunities.15,16 Some nanomaterials showed fascinating mimic enzyme activity, which were named as nanozymes. Now, nanozyme is also a very important, interesting and exciting branch of biomimetic chemistry. Compared with native enzymes, nanozymes are low-cost, better in stability and easy to prepare.17 Covalent organic frameworks (COFs) as a new star of crystalline porous material were constructed by organic building blocks via covalent bonds, which have attracted immense attentions in adsorbing gases,18,19 separating,20,21 and drug delivery.22,23 Moreover, COF shows great potential applications in catalysis. Recently, COF-supported ultrafine Pd nanoparticles (PdNPs) and Au nanoparticles (AuNPs) were proved to possess the superior catalytic activity in nitrophenol reduction and Suzuki−Miyaura coupling reaction.24-26 The high catalytic activity of metal NP@COF composite materials may be attributed to large specific surface area and adsorptivity of COF, which increase the concentration of substrate around the NP@COF. In addition, the introduction of non-aggregating catalytically active Au nanoparticles not only greatly improves catalytic performance of the composite, but also provides easy binding site for detecting antibody in ELISA. Surface enhanced Raman scattering (SERS) based sensing as an ultrasensitive sensing strategy has attracted wide attentions, which has been rapidly developed for biosensing. SERS technique 4


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shows many advantages including convenience, stability, little damage to biological samples and high sensitivity.27 In recent years, SERS has been widely used in bioimaging,28 and detecting disease biomarkers,29-31 toxins,32,33 bacteria,34,35 viruses,36,37 and cells.38 Typically, in SERS based immunoassay, antibody-functionalized SERS tag was employed to directly reflect analytic signal, which showed satisfactory sensitivity.37,39,40 However, in these sensing methods, the lack of secondary amplification of the analyte signal by enzyme catalytic would limit the ultra-trace detection to a certain degree. Moreover, background interference of antibodies may bring out various characteristic peaks in the final Raman spectrum, resulting in complex and indistinguishable signals, which are also disadvantageous in performing trace analysis. Therefore, in SERS-based immunoassay, it is critical to reduce the background interference and purify the signal of the Raman reporter. Meanwhile, the secondary amplification of detecting signal will be a great progress for achieving ultrasensitive detection. Herein, we proposed a novel ratiometric SERS immunosorbent assay for highly sensitive detection of allergenic proteins via the covalent organic frameworks (COF) composite material based nanozyme tag triggered Raman signal "turn-on" and amplification. Firstly, the nanozyme (AuNPs

doped

COFs)

were

prepared

using

1,3,5-tris(4-aminophenyl)benzene

and

2,5-dimethoxyterephaldehyde as COFs ligands, which not only exhibited the excellent mimic nitroreductase activity but also robust stability. Secondly, by replacing the traditional natural enzyme tag, we employed the proposed nanozyme to label the detecting antibody. According to the mimic nitroreductase activity property, 4-nitrothiophenol (4-NTP) was delicately introduced as the nanozyme substrate, which can be quickly reduced to 4-aminothiophenol (4-ATP) in the presence of NaBH4. In SERS assay, gold nanostars have exhibited excellent enhancement factors 5



with ultra-sensitivity that even can realize the single molecule detection, which were employed as SERS unit. The produced 4-ATP was capable of acting as a powerful bridge to connect gold nanostars by both Au-S bond and electrostatic force to further produce Raman “hot spot”. Meanwhile, the Raman signal of 4-NTP at 1573 cm-1 was weakened, and a new signal at 1591 cm-1 generated from 4-ATP was turned on. Finally, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately. With the aid of the nanozyme mediated ratiometric signal amplification output and the further nanozymatic products triggered Raman “hot spot”, the new sensing strategy enabled the anti-interference, improved accuracy, high sensitivity and selectivity for target analysis. The proposed strategy was further employed to detect β-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I1591/I1573, which yielded an ultra-low limit of detection (LOD) of 0.01 ng/mL with a wide linear range of 25.65-6.2×104 ng/mL. The developed method showed many advantages such as low-cost, excellent applicability, higher recovery and lower cross-reactivity, showing great prospect for immunosorbent assay of other target proteins. It is also expected the new sensing strategy would open a new avenue for taking control of nanozyme and SERS for trace target analysis. EXPERIMENTAL SECTION Chemicals and Materials. β-LG, rabbit anti-β-LG polyclonal antibody (pAb), mouse anti-β-LG monoclonal antibody (mAb), α-casein (α-CN), β-casein (β-CN), κ-casein (κ-CN), egg protein, peanut protein, soybean protein and wheat protein were purchased from yuanye Bio-Technology Co. Ltd. (Shanghai, China). Chloroauric acid (HAuCl4·4H2O), sodium borohydride (NaBH4), 4-NTP, 4-ATP, silver nitrate (AgNO3), ascorbic acid (AA) and bovine serum albumin (BSA) were obtained from 6


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Sigma-Aldrich. Milk, yoghurt, cookie, candy and infant formulas were purchased from local supermarkets. The 96-well polystyrene plates were purchased from Jincanhua (Shenzhen, China). All other reagents were of analytical grade and used without further purification. All solutions were prepared with deionized water (DI). Characterization and Measurements. Transmission electron microscope (TEM) images were obtained with FEI Tecnai G20 (FEI Company, USA). Energy Dispersive Spectrometer (EDS) and Energy Dispersive X-Ray Spectroscopy (EDX) mapping were performed with X-Max (Oxford，UK). The X-ray diffraction (XRD) curve was recorded using a Mini Flex 600 diffractometer instrument (Rigaku, Tokyo, Japan). FTIR spectra were recorded on a Vector-22 Fourier-transform spectrophotometer (Bruker, Germany). Brunauer-Emmett-Teller (BET) specific surface area was performed at 77 K under N2 sorption on a TriStar II Plus 2.02 physical equipment (Micromeritics, USA). Ultraviolet (UV) measurements were performed using a UV-visible spectrophotometer (Thermo evolution 201, USA). SERS measurements were performed on a portable Raman spectrometer (BWS465-785, B&W Tek. Inc., USA). A 785nm laser line was used for all the measurements. Synthesis of AuNPs Doped COF Nanozyme. The COF was synthesized according to a previously reported method with minor modification.41 In brief, 0.030 mmoL 1,3,5-tris(4-aminophenyl)benzene and 0.045 mmoL 2,5-dimethoxyterephaldehyde were added to 4.5 mL 1,4-dioxane-butanol-methanol (v/v/v, 4:4:1) cosolvent system. Then, the mixture was sonicated for 20 min to get a homogenous solution. After 0.050 mL of 12 moL/L aqueous acetic acid was added dropwise, the mixture was left undisturbed at room temperature for 2 h. Subsequently, 0.45 mL of 12 moL/L aqueous acetic acid was added 7



and the solution was heated at 70 °C for 24 h. Finally, the product was cooled to room temperature and the resultant COF was washed 3 times with tetrahydrofuran and acetone before dried in vacuum for 24 h. The AuNPs doped COF nanozyme was synthesized according to a previously reported method with some modifications.42 Typically, COF (0.060 g) was suspended in 30 mL methanol and treated with hydrochloric acid to adjust the pH to about 4. Then，320 μL 1% HAuCl4·4H2O was added dropwise into the above mixture under vigorous stirring, and the mixture was continuously stirred for 5 h at 0 °C. Afterwards, 2 mL freshly prepared NaBH4 methanol solution (0.20 mol/L) was added dropwise at the same synthesis condition for another 3 h. The obtained precipitate was centrifuged and washed with methanol. Finally, the yellow powder was dried in a vacuum oven at 70 °C for 24 h. Preparation of Antibody-Modified AuNPs Doped COF Nanozyme. 1 mg of AuNPs doped COF nanozyme was dispersed in 5 mL of phosphate buffer saline (PBS 10 mmoL/L, pH 7.4) and sonicated for 10 min to get a homogenous solution. Then, 200 μL of pAb solution (10 mg/mL) was added under slightly stirring for 2 h. The unconjugated antibody was removed by centrifugation. Finally, the nanozyme tag was re-dispersed in 5 mL of PBS solution (10 mmoL/L, pH 7.4, containing 1% BSA), and diluted 200 times for further use in NELISA. Synthesis of Gold Nanostars. Gold nanostars were synthesized according to a previously reported method with some modifications.43 Briefly, 5 mL of 1% citrate solution was added to 30 mL of boiling 1 mmoL/L HAuCl4 solution under vigorous stirring for 15 min, which was then cooled at room temperature 8


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and stored at 4 °C for storage. In order to synthesize gold nanostars, 100 µL of the above citrate-stabilized seed solution was added to 10 mL of 0.25 mmoL/L HAuCl4 solution (with 10 µL of 1 moL/L HCl) in a 20 mL glass vial at room temperature under moderate stirring (700 rpm). 100 µL of AgNO3 (2 mmoL/L) and 50 µL of AA (100 mmoL/L) were quickly added. The solution was stirred for 30 s as the color rapidly turned from light red to blue or greenish-black. Finally, the gold nanostars were centrifuged at 10000 × g for 15 min to halt the nucleation. Process of the SERS-NELISA. The 96-well microliter plates were coated with mAb (3.0 μg/mL in 50 mmoL/L pH 9.6 carbonate buffer solution) and incubated overnight at 4 °C, and then washed three times with 10 mmoL/L, pH 7.4 PBS containing 0.1% Tween-20 (PBST). Next, 100 μL of 1 % BSA in PBS was added and incubated at 37 °C for 1 h to block unbound sites. After being washed three times with PBST, 100 μL of β-LG or food samples was added to each well, incubated at 37 °C for 1 h and washed again. PBST containing 1% BSA was used as control. The diluted nanozyme tag was added and incubated at 37 °C for 2 h. After further washing, 100 μL of 4-NTP solution was added to each well, and set at 40 °C in the dark for 15 min. Finally, 20 μL 3 mol/L hydrochloric acid was used to stop the reaction. For further SERS analysis, 10 μL of the reacted solution from the 96-well plate was mixed with 100 μL of gold nanostars solution. Gently stirred for 5 min and 10 μL of the mixed solution was dropped on the silicon wafer for SERS measurement with 10 mW of laser power and 20 s of acquisition time. Analysis of Food Samples. The sample pretreatment was carried out according to a reported study with minor modification.44 The liquid sample was fortified with β-LG standard solution at a concentration of 9



1 or 5 μg/mL, and then was agitated for 1 h at 4 °C. The mixture was centrifuged at 6000 ×g for 15 min, and the supernatant was collected for further usage. Cookie, candy and infant formula samples were crushed into powder before use. 1 g of the powder was fortified with β-LG at a concentration of 1 or 5 μg/g and then dissolved in 20 mL 20 mM Tris-HCl (pH 8.0, containing 2% Tween-20). The mixture was agitated overnight at 4 °C and then centrifuged at 6000 ×g for 15 min. The aqueous layer was obtained for further usage. RESULTS AND DISCUSSIONS Material Characterization. The morphology of AuNPs doped COF nanozyme was characterized by TEM. As shown in Figure 1A, COF was nearly spherical with an average diameter of approximately 250 nm. It can be clearly noticed that AuNPs were well dispersed on COF due to the coordination of the unsaturated amino groups of COF (Figure 1C and D). Figure 1E showed results of XRD. And the results indicated that additional peaks at 38.21◦, 44.41◦ and 64.61◦ were in good accordance with the reflections from (111), (200) and (220) planes of Au.42 In addition, by comparing the results before and after the introduction of AuNPs, we can find that the original structure of the COF was not destroyed by dope of AuNPs (Figure 1A and C). To further understand the performance of the nanozyme, N2 adsorption-desorption experiment was performed and results were shown in Figure 1F. The BET surface area of COF and AuNPs doped COF is 1060.2 and 901.5 m² g-1, respectively. The pore volume of COF (0.774 cm³ g-1) was decreased to 0.662 cm³ g-1 after the dope of AuNPs. The decreased surface area and pore volume of AuNPs doped COF demonstrated the highly dispersed AuNPs on the surface of COF. The presence of Au element on the nanozyme is further demonstrated by EDX imaging and EDS (Figure S1). To verify the 10


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successful modification of the pAb on nanozyme, the AuNPs doped COF before and after modification with pAb were characterized by FTIR spectrometry (Figure S2). The characteristic peak at 1650 cm-1 (the C=O stretching vibration) were observed at pure pAb, which was corresponded to the amide group in antibody.45 For AuNPs doped COF, a characteristic peak of C=N was appeared at 1614 cm-1. When pAb was modified onto AuNPs doped COF, the absorption bands for amide groups at 1650 cm-1 and C=N at 1614 cm-1 were simultaneously observed, indicating the successful modification of antibody on the surface of nanozyme. TEM images of the prepared Au nanostars were shown in Figure 2A and it can be seen that as-prepared AuNPs were well dispersed and possessed sharply-pointed tips. After introduce of 4-ATP, plasmonic Au nanostars were obviously aggregated (Figure 2C). Mechanism of Ratiometric SERS Immunosorbent Assay. As shown in Scheme 1, AuNPs doped COF nanozyme was employed as the signal amplification tag to catalyze the reduction of 4-NTP by NaBH4. The reaction of 4-NTP to 4-ATP has been widely used as a model reaction to evaluate the activity of nanomaterial in the field of material chemistry.46 To investigate nitroreductase activity of the AuNPs doped COF nanozyme, apparent steady-state kinetic parameters for 4-NTP reduction were determined by varying the concentration of 4-NTP. The data were fitted to the Michaelis−Menten kinetic model by a nonlinear least-squares fitting to obtain the Michaelis−Menten constant (Km) and maximum velocity (Vmax). Figure 3 showed a typical Michaelis-Mentenlike curve and a Lineweaver−Burk double-reciprocal plot (1/Vmax [Vo] vs 1/substrate concentration [S]). The kinetic parameters of AuNPs doped COF nanozyme were calculated from the Lineweaver−Burk double-reciprocal plot. We also systematically investigated the Km and Vmax of COF itself. The Vmax of COF and AuNPs 11



doped COF is 1.77 × 10-5 and 2.81 × 10-5 M min-1, respectively. Moreover, Km indicates the affinity of the enzyme for the substrate, and a lower Km value represents a stronger affinity between enzymes and substrates. The Km for AuNPs doped COF nanozyme was 0.0927 mM which was lower than that of COF (0.71 mM). The value of Km and Vmax indicated the AuNPs doped COF nanozyme showed higher catalytic activity. The excellent mimic enzyme property may be attributed to the following reasons. Firstly, it can be seen from Figure 1 that Au nanoparticles less than 10 nm in diameter, which has been proven to possess highly catalytic ability.47 The uniform pore structure and large specific surface area of COF are beneficial to the perfect distribution of AuNPs on the COF surface, ensuring well disperse of catalytic active site. Secondly, AuNPs doped COF nanozyme is a composite material, which combined the advantages of COF and AuNPs. Thus, the synergistic catalytic effect plays a critical role for ensuring the ultra-high activity in catalyzing 4-NTP reductive reaction. Thirdly, COF materials as a new type of crystalline porous material not only have catalytic activity but also have superior adsorption ability to the substrate 4-NTP, greatly facilitating high concentration of substrate around the nanozyme. Meanwhile, the multi-layered network structure can provide efficient access to the catalytic sites and fast mass-transport of the substrates.48 The powerful catalytic property of nanozyme can ensure the effective signal amplification for trace analytes in the further immunosorbent assay. Meanwhile, AuNPs on the nanozyme facilitate the antibody modification by the Au-S bonding. When the newly produced 4-ATP solution is mixed with the Au nanostars, one end of the 4-ATP is to connect Au nanostar via Au-S bond, and the amino group at the other end can attract the other Au nanostar by electrostatic adsorption and forms a sandwich structure, resulting in “hot 12


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spot” and the enhanced Raman intensity (Figure 2C). To investigate the enhancement effect of the “hot spot”, Raman intensity at 1080 cm-1 was selected, which is a mixture peak of νC-S and νC-C vibrations presented in both 4-ATP and 4-NTP.49 As shown in Figure S3, the Raman intensity of 4-ATP at 1080 cm-1 was much stronger than 4-NTP, indicating the significant enhancement effect of the “hot spot”. In this work, accompany with the nanozyme participated catalytic reaction, 4-NTP was continuously transformed into 4-ATP. Meanwhile, the Raman signal of 4-NTP at 1573 cm-1 (4-NTP C=C phenyl ring stretching modes) was weakened, and a new signal at 1591 cm-1 (the characteristic band of phenyl ring modes of 4-ATP) was generated and gradually strengthened, leading to the turn on of the ratiometric signal of I1591/I1573. Based on the above design, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately. In presence of the target antigen, the target signal can be finally transformed into the ratiometric signal of I1591/I1573, realizing high sensitive, anti-interference, and accurate SERS analysis. Optimization of Detection Conditions. To obtain the maximum catalytic efficiency of AuNPs doped COF nanozyme tag, parameters including incubating temperature and incubating time were optimized by response surface methodology (RSM) via three-level, two variable BBD method. Compared with single factor experiment, RSM can assess the interactive influence between the tested factors, which could provide more suitable reaction conditions for the nanozyme tag. Due to the maximum UV absorption of 4-NTP at 400 nm, the catalytic efficiency of the nanozyme tag was monitored by UV absorption spectrum. The decrease of absorbance value in 400 nm ΔA was selected as the response variable. The predicted second-order polynomial model was as follows: 13



Y = 13.16 + 0.03A +0.05B − 2.51×10-5AB − 4.11×10-3A2 − 1.6×10-3B2 where, Y is the decrease of absorbance value; A and B are the values of incubating temperature (A) and incubating time (B), respectively. Three-dimensional response surface curves were presented in Figure 4a. The analysis of variance (ANOVA) showed the fitness was significant at the level of p < 0.0001. Also, the optimal conditions were given by RSM as follows: incubating time of 16 min and temperature of 40 °C. The suitability of the proposed optimal conditions was verified by 6 experiments, and the average ΔA max was 0.4416, indicating the feasibility of the response model to reflect the expected optimization. Figure 4b shows the conversion rate under different concentrations of 4-NTP. And the conversion of 4-NTP was expressed by the ratio of the final absorbance and the initiate absorbance. The concentration rate of 4-NTP at 0.1 mmoL/L showed a better conversion rate. Lower or higher concentration would affect the detection range. Thus, 0.1 mmoL/L 4-NTP was chosen as the most suitable substrate concentration in the following experiment. Moreover, since different pH may affect the electrostatic adsorption between 4-ATP and Au nanostars, we explored the effect of pH on Raman intensity. As shown in Figure S4 and S5, the Raman intensity gradually enhanced with the increase of pH from 3-5, and reached a maximum value at pH 5. The further increase of pH will lead to the decrease of Raman intensity. Thus, pH 5 was selected for the gold nanostar solution. Analytical Performance of Ratiometric SERS Immunosorbent Assay To evaluate the performance of ratiometric SERS immunosorbent assay, β-LG was chosen as the target analyte. We optimized the concentration of nanozyme tag and Au nanostars, respectively. Results indicated the best nanozyme tag concentration was obtained by diluting 200 times of the stock solution (Figure S6). And the direct usage of undiluted Au nanostar solution 14


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yielded the best analytical performance (Figure S7). As shown in Figure 5a, with an increase in the β-LG concentration from 1×10-3 ng/mL to 1×105 ng/mL, an increasing SERS intensity at 1591 cm-1 was observed, which is the characteristic band of phenyl ring modes of 4-ATP.50 At the same time, SERS intensity of 4-NTP C=C phenyl ring stretching modes at 1573 cm-1 decreased. By monitoring the value of I1591/I1573, the calibration curve of the method is obtained at a range of 4.16×10-2-3.85×106 ng/mL (Figure 5b). And a wide linear dynamic detection range was obtained from 25.65 to 6.2×104 ng/mL with R2 = 0.9936. The LOD was as low as 0.01 ng/mL. Table 1 compared the linear dynamic detection range and LOD of the proposed method with several newly reported methods for β-LG detection. It can be seen from Table 1 that the LOD and linear dynamic range of the proposed sensing strategy were more superior, showing great prospect for β-LG detection. Method Selectivity, Stability and Recovery. The specificity of the method was evaluated by testing the cross-reactivity with different allergenic proteins at the concentrations of 1, 5 and 10 μg/mL. As shown in Figure 6a, the BSA, egg proteins, peanut proteins, soybean proteins and wheat proteins showed no cross-reactivity with β-LG. However, α-CN, β-CN and κ-CN had cross-reactivity with β-LG, which might be due to the residue of β-LG in these caseins. The stability of the proposed nanozyme tag was also evaluated, and 5 μg/mL of β-LG was used as antigen. As shown in Figure 6b, after 35 days stored at 4 °C in the refrigerator, the value of I1591/I1573 still reached 75% of the initial value. And the average value of I1591/I1573 is 2.07, which remained 90% of the initial value. All the results indicated the robust stability of the proposed nanozyme tag. Recovery tests were performed by spiking 1 and 5 µg/mL of β-LG in milk and yoghurt, and spiking 1 and 5 µg/g in cookie, candy, PHF and EHF. Recovery was calculated by using the following formula: R =（C2－C1）/ C3× 100% R is recovery; C1 is the concentration of sample; C2 is the concentration of the spiked sample; 15



C3 is the spiked β-LG standard concentration. As shown in Table 2, the average recoveries of β-LG from spiked milk, yoghurt, cookie, candy, PHF, and EHF were 101.49, 98.98, 99.92, 99.37, 98.89 and 98.81%, respectively. And the relative standard deviation was in the range of 0.20−6.35%. These results confirmed that the developed sensing method is capable of detecting trace β-LG in real samples with high recoveries. Application to Food Samples Screening To further validate the performance of the proposed approach, different samples were submitted for β-LG analysis. Table 2 shows the β-LG contents in milk, yogurt, cookie and candy were 4.26 μg/mL, 2.75 μg/mL, 3.42 μg/g and 0 μg/g, respectively. We also measured allergenic residues in partial hydrolyzed infant formula (PHF) and extensively hydrolyzed infant formula (EHF) samples. The protein hydrolyzed infant formulas are classified into PHF and EHF according to the degree of proteolysis. PHF is generally recommended for primary intervention of infants with high risk of milk protein allergy. EHF is deeply hydrolyzed protein formulations which produces a mixture of free amino acids, dipeptides, tripeptides and short peptides. Thus, EHF is suitable for infants who are allergic to milk proteins and have severe enteritis. However, according to relevant literature reports, there may be a small amount of residual β-LG in EHF, which may cause allergic reactions.51 As shown in Table S1, β-LG was not detected in EHF, but was found in PHF (54.23 and 98.16 μg/g). Although PHF reduces the risk of allergies in infants, the residues still possess a certain threat to the health of the baby. In general, the above results indicated the proposed method showed excellent applicability for β-LG residue detection in food samples. CONCLUSION In summary, a novel ratiometric SERS immunosorbent assay for highly sensitive detection of allergenic proteins has been successfully established based on AuNPs doped COF nanozyme, which is conceptually different from the previously reported SERS immunosorbent assays. Benefiting from the nanozyme mediated ratiometric Raman signal amplification output and the further nanozymatic products triggered Raman “hot spot”, the new sensing strategy showed many 16


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remarkable advantages including low-cost, anti-interference, improved accuracy, high sensitivity and selectivity. The present study provides a potential platform for allergenic protein sensing in food safety screening. The designed ratiometric SERS immunosorbent assay also showed great promising in various applications such as on-site food safety screening, environmental monitoring, and so on. Moreover, we also expect the powerful and versatile AuNPs doped COF nanozyme would be employed for development of many other sensing approaches.

Supporting Information Supporting Information Available: Table S1 β-LG contents in hydrolyzed infant formulas; Figure S1, EDX mapping of AuNPs doped COF and EDS results of COF and AuNPs doped COF; Figure S2, FTIR spectrum of AuNPs doped COF (blue), pAb-modified AuNPs doped COF (red) and pAb (black); Figure S3, Raman spectra of 4-ATP mixed with Au nanostar (red), 4-NTP mixed with Au nanostar (black) and Au nanostar (blue); Figure S4, Raman spectra of 4-ATP mixed with Au nanostar at different pH; Figure S5, Raman intensity at 1080 cm-1 of 4-ATP mixed with Au nanostar at different pH; Figure S6, Optimization of nanozyme tag concentration; Figure S7, Optimization of Au nanostars concentration. This material is available free of charge via the Internet at http://pubs.acs.org

Acknowledgement This work was supported by the National R&D Key Programme of China (No. 2017YFE0110800), the Natural Science Foundation of Shandong Province (ZR2017JL012), the National Natural Science Foundation of China (21677085 and 31801454), the Science and Technology Nova Plan of Shaanxi Province (2019KJXX-010), the Project funded by China Postdoctoral Science Foundation (2018M640574), and the Youth Innovation Team of Shaanxi

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Universities (Food Quality and Safety).

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Table 1 Comparison of the reported analytical methods for β-LG determination. Analytical methods

Linear range (ng/mL) 2.8-100 5-4×103 125-4×103 1.225×103-1.61×106 31.25-8×103 20-500 480-3.125×104 1.11-1.111×103 31.25-64 ×103 0.49-1.6 × 104 25.65-6.2 ×104

Electrochemical magnetoimmunosensing Surface plasmon resonance Fluorescence sandwich ELISA Sandwich ELISA Sandwich ELISA Microcantilever resonator arrays LC-MS LC-MS AuNPs probe-based ELISA PtNPs probe-based ELISA SERS-ELISA

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LOD (ng/mL) 0.8 5.54 0.49 33.95 1.96 80 200 1.11 0.49 0.12 0.01

Reference 52 53 54 55 56 57 58 59 44 60 This study


Table 2 The analytical results of β-LG in food samples. Sample

Spiked concentration Detected concentration Recovery RSD (µg/mL or µg/g)a (µg/mL or µg/g)a (%) (%) Milk 0 4.26±0.11 2.58 1 5.25±0.09 99.81 1.71 5 9.41±0.21 103.16 2.23 Yoghurt 0 2.75±0.03 1.09 1 3.73±0.03 98.74 0.80 5 7.81±0.17 101.22 2.18 Cookie 0 3.42±0.03 0.88 1 4.41±0.28 99.72 6.35 5 8.42±0.34 100.11 4.04 Candy 0 ND 1 0.99±0.01 99.03 1.01 5 4.98±0.01 99.70 0.20 PHFb 0 12.50±0.23 1.84 1 13.50±0.50 100.13 3.70 5 17.38±0.36 97.64 2.07 EHFc 0 ND 1 0.98±0.02 98.22 2.04 5 4.99±0.02 99.79 0.40 a The “µg/mL” was the unit for milk and yoghurt, and “µg/g” was the unit for cookie, candy, PHF and EHF. b Partial hydrolyzed infant formula (PHF). c Extensively hydrolyzed infant formula (EHF). ND: Not detected

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Scheme 1 Schematic illustration of the AuNPs doped COF nanozyme-based SERS immunosorbent assay.

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Figure 1. TEM images of (A,B) COF and (C,D) AuNPs doped COF nanozyme. (E) XRD image and (F) N2 adsorption-desorption curves of (a) COF and (b) AuNPs doped COF nanozyme.

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Figure 2. TEM images of (A) as prepared Au nanostars, (B) single Au nanostar and (C) the aggregation of plasmonic Au nanostars induced by 4-ATP.

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Figure 3. Steady-state kinetic analyses using the Michaelis−Menten model (main panel) and Lineweaver−Burk model (inset panel) for (A) COF and (B) AuNPs doped COF nanozyme respectively.

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Figure 4. (A) Optimization of AuNPs doped COF nanozyme tag catalytic conditions through response surface methodology. (B) The conversion of 4-NTP with different concentration.

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Figure 5. (A) SERS spectra of 4-NTP at different concentrations of β-LG from 1×10-3 ng/mL to 1×105 ng/mL (a-i) and (B) calibration curve (main panel) and linear dynamic range (inset panel) of the method.

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Figure 6. (A) Cross-reactivity of the developed SERS-ELISA with α-CN, β-CN, κ-CN, BSA, egg proteins, peanut proteins, soybean proteins and wheat proteins. (B) Storage stability of AuNPs doped COF nanozyme tag stored at 4 °C.

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Schematic illustration of the AuNPs doped COF nanozyme-based SERS immunosorbent assay. 298x166mm (96 x 96 DPI)


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Ratiometric SERS immunosorbent assay of allergenic proteins via

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