Lateral Flow Immunoassay Based on Polydopamine-Coated Gold

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Lateral Flow Immunoassay Based on Polydopamine-Coated Gold Nanoparticles for the Sensitive Detection of Zearalenone in Maize Shaolan Xu, Ganggang Zhang, Bolong Fang, Qirong Xiong, Hongwei Duan, and Weihua Lai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08789 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Applied Materials & Interfaces

Lateral Flow Immunoassay Based on Polydopamine-Coated Gold Nanoparticles for the Sensitive Detection of Zearalenone in Maize Shaolan Xua, Ganggang Zhanga, Bolong Fanga, Qirong Xiongb, Hongwei Duanb,*, Weihua Laia,* a

State Key Laboratory of Food Science and Technology, Nanchang University,

Nanchang 330047, China b

School of Chemical and Biomedical Engineering, Nanyang Technological

University, Singapore 637457, Singapore

*

Corresponding authors:

a Dr. Weihua Lai Tel.: +86 13879178802 E-mail address: [email protected] b Dr. Hongwei Duan Tel.: +65 6514 1019 E-mail address: [email protected]

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ABSTRACT In this work, polydopamine-coated gold nanoparticles (Au@PDA) were synthesized by the oxidative self-polymerization of dopamine (DA) on the surface of AuNPs and were applied for the first time as the signal-amplification label in lateral flow immunoassays (LFIA) for the sensitive detection of zearalenone (ZEN) in maize. The PDA layer functioned as a linker between AuNPs and anti-ZEN monoclonal antibody (mAb) to form the probe (Au@PDA-mAb). Compared with AuNPs, Au@PDA had excellent color intensity, colloidal stability, and mAb coupling efficiency. The limit of detection (LOD) of the Au@PDA-based LFIA (Au@PDA-LFIA) was 7.4 pg/mL, which was 10-fold lower than that of traditional AuNPs-based LFIA (AuNPs-LFIA) (76.1 pg/mL). The recoveries of the Au@PDA-LFIA were 93.80%-111.82%, with coefficient of variation (CV) of 1.08%-9.04%. In addition, the reliability of the Au@PDA-LFIA was further confirmed by the high-performance liquid chromatography (HPLC) method. Overall, our study showed that PDA coating can chemically modify the surface of AuNPs through a simple method and can thus significantly improve the detection sensitivity of LFIA. KEYWORDS: lateral flow immunoassay, gold nanoparticles, polydopamine, zearalenone, sensitivity


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1. INTRODUCTION Zearalenone (ZEN), an estrogenic metabolite produced by Fusarium species, is a potential risk to human health and a widespread mycotoxin in maize, wheat, barley, and rice.1-3 Fusarium toxins are the most frequent and co-occur in contaminated food samples.4 To ensure food safety, the maximum residue limit of ZEN in maize is set at 100 μg/kg in the European Union, and 60 μg/kg in China. Various analytical methods have been developed for the sensitive detection of ZEN, including microarray assay,5 high-performance liquid chromatography (HPLC),6 ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS),7

liquid

chromatography-mass

spectrometry

(LC-MS),8

and

enzyme-linked immunosorbent assay (ELISA).9 However, these methods require skilled operators and complicated sample-preprocessing procedures, which are time-consuming and unsuitable for on-site detection. Meanwhile, many researchers have developed lateral flow immunoassays (LFIA) to detect ZEN.10-14 LFIA is widely used to detect targets in vitro diagnosis,15 food safety16 and environment monitoring17 due to its rapidity, convenience, and low cost.18-21 The signal-amplification label is a key factor that affects LFIA performance, especially sensitivity.22,

23

Gold nanoparticles (AuNPs) are the most widely used

labels for LFIA. However, the low color intensity and poor colloidal stability of traditional AuNPs result in low sensitivity of LFIA.24-26 High sensitivity is essential in on-site detection because the influences of complex sample-processing procedures and sample matrix cause the detection to be time-consuming and laborious, resulting 3 ACS Paragon Plus Environment


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in greatly decreased detection sensitivity. Therefore, a simple and sensitive method for detecting targets for LFIA is necessary to establish. Polydopamine (PDA) is an emerging biomimetic adhesive polymer and an oxidative self-polymerized form of dopamine (DA). DA is an amine synthesized by removing the carboxyl group from its precursor chemical L-DA. In recent years, PDA has attracted attention from researchers because of its high chemical reactivity, versatile adhesion capacity, and good biocompatibility.27,

28

Taking advantage of

similar catecholamine groups, PDA can easily adhere onto the NP surface via electrostatic interaction or covalent linkage.29 A notably higher level of UV-vis absorption was observed after coating the surface of NPs (such as silica and magnetic NPs) with the PDA layer compared with pure NPs.30, 31 Therefore, we also applied this feature of PDA on the AuNPs to enhance the color intensity of the NPs, so as to facilitate test strip detection. When the PDA is used for enzyme immobilization, the biocompatibility between the PDA layer and enzyme was excellent and enzyme activity was only slightly affected.32,

33

These excellent physicochemical properties

are the reason for the popularity of PDA-coated nanoparticles in the field of biomedical therapeutics.34, 35 In this study, PDA-coated AuNPs (Au@PDA) were synthesized by the oxidative self-polymerization of DA on the surface of AuNPs and first proposed as a signal-amplification label for the LFIA detection of ZEN in maize. After PDA layer coated AuNPs with an appropriate thickness, Au@PDA became more stably dispersed in the solution and did not easily aggregate. In addition, Au@PDA also had 4 ACS Paragon Plus Environment

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a stronger color intensity than AuNPs. These advantages of Au@PDA render them suitable for application in LFIA to improve sensitivity, indicating the potential of Au@PDA-based LFIA (Au@PDA-LFIA) for the simple and sensitive detection of other mycotoxins in food safety monitoring.

2. MATERIALS AND METHODS 2.1. Materials and equipment DA

hydrochloride

(DA·HCl),

catechol

(CC),

4-ethylcatechol

(ECC),

3,4-dimethoxyphenethylamine (DMPEA) and glycine (Gly) were purchased from Aladdin Co, Ltd. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Beijing Biotech Co, Ltd. (Beijing, China). Chloroauric acid (HAuCl4), trisodium citrate, casein, γ-polyglutamic acid (γ-PGA), gelatin, ZEN, fumonisin B1 (FB1), aflatoxin B1 (AFB1), ochratoxin A (OTA), patulin (PAT), citrinin (CIT), and deoxynivalenol (DON) were purchased from Sigma-Aldrich Chemical Co, Ltd. (St. Louis, MO, USA). The sample pad, conjugate pad, nitrocellulose (NC) membrane, and absorbent pad were supplied by Millipore (Bedford, MA, USA). ZEN-BSA, anti-ZEN-monoclonal antibody (mAb), and goat-anti-mouse antibody were obtained from Zodolabs Bioengineering Co, Ltd. (Nanchang, Jiangxi, China). Phosphate buffer saline (PBS; 0.01 M, pH 7.4), borate buffer (0.2 M, pH 8.0), potassium carbonate (K2CO3) and the other reagents were analytical grade and purchased from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). ZEN-free maize, as validated by HPLC, was purchased from Rain-bow Supermarket (Nanchang, Jiangxi, China). 5 ACS Paragon Plus Environment


Twenty real maize samples were collected from the local market. Transmission electron microscopy (TEM) analyses were conducted on a JEM-2100 electron microscope operated at 100 kV (Tokyo, Japan). An XYZ-3050 platform was supplied by BioDot (Irvine, CA,USA). The zeta potential and hydrodynamic diameter of AuNPs and Au@PDA were measured by dynamic light scattering (DelsaMax PRO, Beckman Coulter). The immunochromatographic test strip (ITS) reader was obtained from Fenghang Science Instrument Co, Ltd. (Zhejiang, China). 2.2. Preparation of AuNPs and Au@PDA A total of 1 mL of 1% HAuCl4 solution (w/w) was mixed with 99 mL of ultrapure water, and the resulting solution was heated to boiling. After adding 1.45 mL of 1% trisodium citrate solution (w/v), the solution was continued to be heated. After 20 min, the color of the mixture turned red, indicating the formation of AuNPs. The prepared AuNPs were cooled to room temperature and stored at 4 °C for further use. Au@PDA was synthesized according to a previous report36 with minor modifications. Briefly, the pH of the prepared AuNPs (1 mL) was adjusted to 7, 8, 9, and 10, and then different volumes (0, 5, 10, 15, 20, and 25 μL) of 3% H2O2 were added to the solution. After vigorous stirring for 5 min, 10 mg/mL (5, 10, and 15 μL) DA·HCl solution was added to the mixture, which was then blended and reacted overnight. After centrifugation at 8,000 r/min for 30 min, and removing the supernatant, the prepared Au@PDA was washed with ultrapure water twice and 6 ACS Paragon Plus Environment

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resuspended in borate buffer (0.2 M, pH 8.0). 2.3. Preparation of Au@PDA-mAb The pH of the Au@PDA solution was adjusted with 0.2 M K2CO3, and then 100 μL of mAb was added dropwise to 1 mL of Au@PDA solution. After gentle shaking at room temperature for 1 h, the mixture was blocked with 100 μL of five capping agent (1% casein, 10% Gly, 10% γ-PGA, 10% gelatin, and 10% BSA) for 30 min. After centrifugation at 8,000 r/min for 30 min, Au@PDA-mAb was resuspended in 100 μL of PBS for further use. 2.4. Evaluation of the mAb coupling efficiency of Au@PDA-10 and AuNPs The mAb coupling efficiency of Au@PDA-10 and AuNPs was evaluated. Firstly, the standard curve of OD450 versus mAb concentrations was established. Afterwards, 100 μL of different concentrations of mAb was added to 1 mL of Au@PDA and AuNPs solution at the final concentrations of 1, 5, 10, 15, and 20 μg/mL. After centrifugation, the supernatant was collected and then diluted by 100 times to detect the mAb coupling efficiency. The coupling efficiency = [(Total mAb) − (Supernatant mAb)]/(Total mAb) × 100%. 2.5. Preparation of LFIA The sample pad was pretreated with 0.01 M PBS containing 1.0% BSA (w/v), 0.5% Tween-20 (v/v), and 0.05% NaN3 (w/v) and then dried at 60 °C for 2 h. The test line (T line) was sprayed with ZEN-BSA, while the control line (C line) was sprayed with 0.5 mg/mL goat-anti-mouse antibody. The sample pad, conjugate pad, NC membrane,

and

absorbent

pad

were

sequentially


assembled

as

the


immunochromatographic test strip (Scheme 1B a). 2.6. Optimization of key parameters To optimize the pH of conjugation, the pH of the Au@PDA solution was adjusted to 5, 6, 7, 8, and 9 with 0.2 M K2CO3. To optimize the mAb concentration, 100 μL of mAb was added dropwise to 1 mL of Au@PDA solution at final concentrations of 1.5, 2.0, 2.5, 3.0, and 3.5 μg/mL. To optimize the ZEN-BSA concentration, the T line was sprayed with ZEN-BSA at concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 mg/mL. To optimize the volume of Au@PDA-mAb, Au@PDA-mAb with volumes of 1.5, 2.0, 2.5, 3.0, and 3.5 μL was added to an microplate well for optimization. In all of the above optimization experiments, PBS spiked with ZEN at 0 ng/mL and 1 ng/mL was used to optimize the key parameters. All experiments were performed in triplicate. 2.7. Immunological kinetics analysis Briefly, 0, 0.1, and 1 ng/mL ZEN in PBS were used to evaluate the immunological kinetics analysis. The signal intensity of the T line was measured every minute for 40 min with the ITS reader. 2.8. Establishment of standard curve A total of 100 μL of ZEN solution diluted with PBS was added to Au@PDA-mAb, and then the resulting solution was incubated for 5 min. Subsequently, 100 μL of solution was added to the sample pad of the test strip. Then, the signal intensity of the T line was read with the ITS reader. Under optimal conditions, the standard curve was established by plotting the signal intensities of the 8 ACS Paragon Plus Environment

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T line against the logarithm of a series of ZEN concentrations (0, 0.01, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng /mL). All experiments were performed in triplicate. The limit of detection (LOD) is defined as the ZEN concentration that causes a 10% (IC10) reduction in the signal intensity of the T line compared with that of the PBS.37 2.9. Specificity experiment The specificity of LFIA was evaluated by testing FB1, AFB1, OTA, PAT, CIT, and DON at a concentration of 10 ng/mL, whereas the concentration of ZEN was 1 ng/mL. Each experiment was performed in triplicate. 2.10. Recovery experiment Maize samples were pretreated according to the method used in a previous study.38 Dry grated samples (1.0 g) were spiked with ZEN at 0.05, 0.1, 0.5, and 1 ng/mL and extracted with 3.0 mL of 60% methanol in water (v/v). The mixture was then placed on a vortex mixer and vibrated for 5 min. After centrifugation at 8,000 r/min for 10 min, the supernatant solution was stored at 4 °C and further diluted five-fold with PBS for LFIA analysis. 2.11. Detection of real maize samples To evaluate the reliability of Au@PDA-LFIA, 20 real maize samples were detected by Au@PDA-LFIA and HPLC. Sample extraction, purification, and HPLC determination were performed in accordance with the Chinese national standard GB 5009.209-2016 with slight modifications. The grated sample (40.0 g) was extracted with 100.0 mL of acetonitrile/water (90:10, v/v) by vigorous stirring for 10 min. Then, the mixture was filtered with filter paper. Afterwards, 9 ACS Paragon Plus Environment


10.0 mL of filtrate was diluted five-fold with 40.0 mL of deionized water, and then filtered with glass fiber filter paper. Chromatographic separation was conducted with a Kinetex C18, Phenomenex column (250 mm × 4.6 mm, 2.6 µm) by using acetonitrile/methanol/water (46:46:8, v/v/v) as the mobile-phase solvent. The flow rate of the mobile phase was 0.6 mL/min, and the injection volume was 10 μL. The separation column was maintained at room temperature and the wavelength of excitation and emission was set at 275 nm and 440 nm, respectively. 2.12. Evaluation of the developed labeling system under conventional LFIA condition The experiments were conducted to compare two LFIA mode—the developed labeling system in this work (the probe was added in the microplate wells) and the conventional LFIA condition (the probe was sprayed on the conjugate pad). Under conventional LFIA condition, the probe (Au@PDA-mAb, 8 μL/cm) was sprayed on the conjugate pad and dried at 37 ° C in vacuum drying oven for 2 h. Then, the conjugate pad was assembled into lateral flow test strips for ZEN detection. Four concentrations of ZEN (0, 0.1, 1, and 5 ng/mL) were selected to compare the two LFIA mode.

3. RESULTS AND DISCUSSION 3.1. Immunoassay principle Scheme 1 showed the schematic illustration of ZEN detection by 10 ACS Paragon Plus Environment

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Au@PDA-LFIA . Scheme 1A showed the process of preparing Au@PDA-mAb. PDA coating served as a linker of mAb and the surface of AuNPs to form the probe (Au@PDA-mAb). With the PDA coating, the Au@PDA was more stable and did not easily aggregate. The work of Lin et al.30 and Liu et al.31 proved that the color brightness of Fe3O4@PDA and SiO2@PDA was stronger than that of Fe3O4 and SiO2, respectively. We speculate that the color brightness of Au@PDA may also be stronger than that of AuNPs. Considering these advantages, Au@PDA-LFIA was constructed by indirect competitive immunoassay. Scheme 1B showed the principle of ZEN detection by LFIA. In the presence of high-concentration ZEN (as shown in Scheme 1B b), Au@PDA-mAb-ZEN was formed and a purple color did not appear on the T line but appeared on the C line . In the absence of ZEN (as shown in Scheme 1B c), a purple T line was formed for the probe bound to ZEN-BSA. The color intensity was recorded with the ITS reader. A higher concentration of ZEN resulted in a weaker color intensity of the T line.



Scheme

1.

Principle

of

Au@PDA-LFIA.

(A)

Preparation

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procedure

of

Au@PDA-mAb. (B) Schematic of the proposed Au@PDA-LFIA for ZEN detection: (a) structure of immunochromatographic test strip, (b) detection mode in the presence of ZEN, and (c) detection mode in the absence of ZEN.

3.2. Synthesis of Au@PDA The effects of pH and volume of 3% H2O2 on Au@PDA formation were discussed. The pH is a key factor affecting the self-polymerization of DA on the surface of AuNPs. As shown in Figure S1, the synthesized Au@PDA had the highest absorbance and the maximum red-shift at pH 7 compared with uncoated AuNPs. After adjusting the pH of the AuNP solution to 6, the AuNPs were immediately aggregated when DA·HCl was added, resulting in failure of formation of the PDA layer. Accordingly, pH 7 was the optimal pH for the synthesis of Au@PDA. Sodium 12 ACS Paragon Plus Environment

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citrate on the surface of AuNPs has reducing ability and thus inhibits the oxidative self-polymerization of DA, therefore, a certain amount of oxidant needs to be added to oxidize sodium citrate. Au@PDA had the highest absorbance after adding 5 μL of 3% H2O2 to AuNPs solution (Figure S2), therefore, 5 μL was selected as the optimal volume of 3% H2O2. To further illustrate the advantages of PDA coating compared with other PDA derivative molecules, several structural analogues of DA (CC, ECC, and DMPEA, slightly different from those of DA in functional groups) were selected for comparison. The absorbance of pure citrate-capped AuNPs was lower than that of AuNPs+DA·HCl but higher than that of AuNPs+CC, AuNPs+ECC, AuNPs+DMPEA (Figure S3). These results indicated that the color intensity of DA · HCl was the strongest than that of three other DA derivative molecules coated AuNPs. Therefore, AuNPs+DA·HCl (Au@PDA) were selected for further experiment. 3.3. Characterization and evaluation of AuNPs, Au@PDA and Au@PDA-mAb TEM images in Figure 1 exhibited the morphology and size of AuNPs and Au@PDA, respectively. The synthesized AuNPs had good dispersity and uniformity with an average size of 21.5 nm (Figure 1A). Figures 1B, 1C, and 1D showed that the thicknesses of the PDA layer of Au@PDA-5, Au@PDA-10, and Au@PDA-15 was 2.5, 5.5, and 13.6 nm when 5, 10 and 15 μL of DA·HCl (10 mg/mL) was added, respectively. With the addition of DA·HCl monomer, the deposition of PDA occurred, and the PDA layer became thicker, resulting in larger changes in the size of the NP. The thickness of the PDA layer on the surface of AuNPs can be controlled by adjusting the volume of DA·HCl. With increased DA·HCl volume, the PDA layer 13 ACS Paragon Plus Environment


thickened.

Figure 1. TEM images of AuNPs and Au@PDA. (A) AuNPs, AuNPs with the addition of (B) 5 μL of DA·HCl (Au@PDA-5), (C) 10 μL of DA·HCl (Au@PDA-10), and (D) 15 μL of DA·HCl (Au@PDA-15). All scale bars at high magnification are 10 nm. As shown in the zeta potential analysis (Figure 2A), a lower zeta potential was observed for Au@PDA-5 (-29.17 mV) and Au@PDA-10 (-34.73 mV) than for AuNPs (-24.73 mV) due to the abundance of hydroxyl groups on the PDA layer. With an excess amount of DA added to citrate-capped AuNPs, the aggregation of NPs was emerged, resulting in a decrease in NP number. Therefore, the zeta-potential increased in the Au@PDA-15 (-18.00 mV) case. These results indicated that the negatively charged Au@PDA-10 was the most stable. An appropriate thickness of the PDA layer can enhance the dispersity of NPs, whereas an excessively thick PDA layer can cause NP aggregation, which is not conducive to the subsequent mAb-coupling process. Given the colloidal stability of Au@PDA, we further investigated their optical 14 ACS Paragon Plus Environment

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properties. In the UV-vis absorption spectrum (Figure 2B), the maximum absorption peaks of AuNPs, Au@PDA-5, Au@PDA-10, and Au@PDA-15 were 524, 534, 540, and 631 nm, respectively. The color of Au@PDA solution is the composite color of two primary colors (red from citrate-capped AuNPs, and black from PDA NPs self-polymerized by DA), which is deeper than that of pure citrate-capped AuNPs, resulting in changes of the maximum absorption wavelength of NP. These results indicate that the PDA coating on the surface of the AuNPs led to an obvious red-shift from 524 nm to 534, 540, and 631 nm because of the deposition of the PDA coating on the surface of AuNPs. Moreover, with an increased volume of DA·HCl from 5 μL to 10 μL, the maximum absorbance of Au@PDA increased from 1.126 to 1.411. With a further increase in the volume of DA·HCl to 15 μL, the maximum absorbance of Au@PDA decreased to 1.181. An appropriate thickness of the PDA layer on the surface of AuNPs can enhance the absorbance of Au@PDA, while an excessively thick PDA layer can cause the aggregation of Au@PDA, leading to a wider absorption peak and lower absorbance. Thus, the color brightness of Au@PDA-10 was the strongest among the three samples. The color brightness of the labels is essential in LFIA because it determines the signal intensity and detection sensitivity. First, εAu@PDA-10/ εAuNPs was calculated to be 1.68 through the Beer–Lambert law (ε = A/bc,39 where A is the absorbance, ε is the molar extinction coefficient, b is the optical path length through the sample, and c is the particle concentration). Then, the grayscale intensities of Au@PDA-10 and AuNPs on the NC membrane were compared. The grayscale intensity of Au@PDA-10 (615.0) was 1.87-fold stronger 15 ACS Paragon Plus Environment


than that of AuNPs (328.6) at the same concentration in LFIA (Figure 2C). These results indicated that the optical performance of Au@PDA-10 was better than that of AuNPs. The hydrodynamic diameter of Au@PDA clearly increased after the PDA coating on the surface of AuNPs (Figure S4). The hydrodynamic diameters of AuNPs, Au@PDA-5, Au@PDA-10, and Au@PDA-15 were 27.2, 37.1, 47.4, and 446.7 nm, respectively. The value of the particle distribution index (PDI) is a crucial index for measuring the dispersion performance of NP. The PDI of AuNPs, Au@PDA-5, Au@PDA-10, and Au@PDA-15 was 0.073, 0.065, 0.057, and 0.438, respectively. The results indicated that Au@PDA-5 and Au@PDA-10 were not aggregated with a reasonable size, while Au@PDA-15 was seriously aggregated because its hydrodynamic diameter and PDI was larger than that of AuNPs. The results can also be verified by zeta potential and UV-vis spectra. Due to the serious aggregation of Au@PDA-15, the number of NPs decreased. Therefore, its absorbance decreased while its zeta potential increased. Consequently, Au@PDA-10 was selected as the label of LFIA and compared with AuNPs for colloidal stability evaluation. The optical density (OD) of Au@PDA-10 and AuNPs was compared to evaluate the stability (OD(Au@PDA-10) is the absorbance of Au@PDA-10 at 540 nm, and OD(AuNPs) is the absorbance of AuNPs at 524 nm). OD(Au@PDA-10) barely changed within pH 6-14, whereas OD(AuNPs) obviously decreased at pH 10-14 (Figure 2E). This result proved that Au@PDA-10 had stronger tolerance to pH than AuNPs. Many LFIA are performed in buffers to make the environment suitable for the 16 ACS Paragon Plus Environment

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biological ingredients. However, common buffers contain a certain amount of salts, which causes the salt ion concentration increase in the solutions. With the increase of the salt ion concentration, the instability and aggregation of AuNPs emerged. Therefore, before the NPs were used to the LFIA, the stability of both the Au@PDA and citrate-capped AuNPs was estimated by adding different volume of 10% NaCl. Next, 10% NaCl (0-100 μL) and Au@PDA-10 or AuNPs (900 μL) were mixed with water (final volume: 1 mL) and then OD(Au@PDA-10) or OD(AuNPs) was obtained. As shown in Figure 2F, OD(Au@PDA-10) exerted only a slight reduction when 100 μL of 10% NaCl was added. However, the AuNPs turned purple and OD(AuNPs) obviously decreased after adding 10 μL of 10% NaCl (Figure 2D d), confirming that Au@PDA-10 resisted the high salt buffer environment. These results demonstrated that Au@PDA-10 was undisturbed by salt ion concentration during the LFIA tests, whereas the conventional citrated-capped AuNPs were suffered from aggregation while the sample was detected by the LFIA analysis.



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Figure 2. Characterization and evaluation of AuNPs and Au@PDA. (A) Zeta potential of (1) AuNPs, (2) Au@PDA-5, (3) Au@PDA-10, and (4) Au@PDA-15. (B) UV-vis

absorption

spectrum

of

AuNPs,

Au@PDA-5,

Au@PDA-10,

and

Au@PDA-15 (Inset: 1, 2, 3, and 4 represent AuNPs, Au@PDA-5, Au@PDA-10, and Au@PDA-15). (C) Comparison of grayscale intensity of Au@PDA-10 and AuNPs at the same concentration on the NC membrane. (D) Images of Au@PDA-10 and AuNPs at different pH values and volumes of 10% NaCl (μL): tolerance to pH of Au@PDA-10 (a) and AuNPs (b); and tolerance to NaCl of Au@PDA-10 (c) and AuNPs (d). Absorbance of Au@PDA-10 and AuNPs at different pH values (E) and 18 ACS Paragon Plus Environment

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volumes of 10% NaCl (F).

As revealed by the UV-vis absorption spectrum, coupling mAb on the surface of Au@PDA-10 led to an obvious red-shift of the maximum absorption peak from 541 to 556 nm (Figure S5), indicating that the probe (Au@PDA-10-mAb) was successfully prepared. The mAb coupling efficiency of Au@PDA-10 and AuNPs was evaluated (Figure 3). Firstly, the standard curve of OD450 versus mAb concentrations was established (Figure 3A). As shown in Figure 3B, Au@PDA-10 exhibited the highest coupling efficiency (93%) at mAb concentration of 10 μg/mL, while the highest coupling efficiency of AuNPs was 81% at mAb concentration of 5 μg/mL. It indicated that Au@PDA-10 could enhance the coupling efficiency of mAb compared with uncoated AuNPs. The result may be caused by the following reason: the quinone groups in PDA coating would actively take part in nucleophilic addition reaction with –SH and –NH2 of amino acid residues of protein that lead to mAb being immobilized on Au@PDA40.



Figure 3. Evaluation of mAb coupling efficiency of Au@PDA-10 and AuNPs. (A) The standard curve of OD450 versus mAb concentration (0.005, 0.01, 0.05, 0.1, 0.5, 1, and 2.5 μg/mL) by ELISA. (B) The mAb coupling efficiency of Au@PDA-10 and AuNPs under different concentrations of mAb (1, 5, 10, 15, and 20 μg/mL). 3.4. Optimization of key parameters in Au@PDA-LFIA To quantitatively detect ZEN, ∆T was used to analyze the results for the optimization of key parameters (∆T = T0 − T1, where T0 is the intensity of T line of the negative samples, and T1 is the intensity of T line of samples spiked with ZEN at 1 ng/mL). A higher ∆T meant a higher competitive inhibition ratio, thereby resulting in higher sensitivity for LFIA detection. Therefore, ∆T was used as the standard for selecting the optimal parameters.

Figure 4. Optimization of key parameters. Intensity of the T line at different (A) pH values, (B) mAb concentrations, (C) ZEN-BSA concentrations, and (D) volumes of Au@PDA-10-mAb. 20 ACS Paragon Plus Environment

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3.4.1. Optimization of pH of conjugation Figure 4A showed the effect of pH on T0 and T1. With increasing pH from 5 to 7, T0 gradually increased from 168.5 to 888.5, T1 gradually increased from 46.0 to 210.7, and ∆T gradually increased from 122.5 to 677.8. With further increases in pH to 9, T0 decreased to 131.2, T1 decreased to 41.0, and ∆T gradually decreased to 90.2. To obtain a higher ∆T, pH 7 was selected as the optimal pH of conjugation. 3.4.2. Optimization of mAb concentration Figure 4B showed the effect of mAb concentration on T0 and T1. With increased mAb concentration from 1.5 μg/mL to 2.5 μg/mL, T0 gradually increased from 626.9 to 897.7, T1 gradually increased from 144.5 to 231.0, and ∆T gradually increased from 482.4 to 666.7. With further increasing mAb concentration to 3.5 μg/mL, T0 slowly increased to 971.6, T1 increased to 378.7, whereas ∆T slowly decreased to 592.9. For the achievement of a higher ∆T, 2.5 μg/mL was regarded as the optimal mAb concentration. 3.4.3. Optimization of ZEN-BSA concentration Figure 4C showed the influence of ZEN-BSA concentration on T0 and T1. With increased ZEN-BSA concentrations from 1.0 mg/mL to 2.5 mg/mL, T0 increased from 567.3 to 887.5, T1 increased from 162.5 to 220.1, and ∆T gradually increased from 404.8 to 667.4. With further increased ZEN-BSA concentration to 3.0 mg/mL, T0 slowly increased to 912.7, T1 increased to 290.6, whereas ∆T slowly decreased to 622.1. To obtain a higher ∆T, 2.5 mg/mL was set as the optimal concentration of ZEN-BSA. 21 ACS Paragon Plus Environment


3.4.4 Optimization of the volume of Au@PDA-10-mAb Figure 4D showed the effect of the volume of Au@PDA-10-mAb on T0 and T1. With an increase in the volume of Au@PDA-10-mAb from 1.5 μL to 2.5 μL, T0 increased from 552.1 to 890.9, T1 gradually increased from 140.7 to 232.5, and ∆T gradually increased from 411.4 to 658.4. With further increasing the volume of Au@PDA-10-mAb to 3.5 μL, T0 slowly increased to 981.5, T1 increased to 328.7, whereas ∆T remained stable (652.8). Thus, 2.5 μL was considered the optimal volume of Au@PDA-10-mAb. 3.4.5 Optimization of the capping agent To avoid nonspecific absorption, five capping agents (1% casein, 10% Gly, 10% γ-PGA, 10% gelatin, and 10% BSA) were selected to block the surface of Au@PDA-mAb. As shown in Figure S6, obvious nonspecific adsorption emerged with the four other capping agents except 1% casein. Therefore, 1% casein was the optimal capping agent in this work. 3.5. Immunological kinetics analysis The immunological kinetics analysis of Au@PDA-LFIA was performed by recording the intensity of the T line of samples spiked with ZEN at 0, 0.1, and 1 ng/mL every 1 min for 40 min. As shown in Figure 5A, the intensity of the T line on samples spiked with ZEN at 0, 0.1, and 1 ng/mL reached a plateau at 30 min. Consequently, 30 min was regarded as the optimal immunoreaction time of LFIA.


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Figure 5. Performance and evaluation of LFIA. (A) Immunological kinetics analysis of Au@PDA-LFIA. Standard curve of intensity of T line versus ZEN concentrations (0, 0.01, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng/mL) in (B) Au@PDA-LFIA and (D) AuNPs-LFIA. (C) Cross-reaction of ZEN (1 ng/mL) with six other mycotoxins (FB1, AFB1, OTA, PAT, CIT, and DON all at 10 ng/mL) in Au@PDA-LFIA.

3.6. Standard calibration curve Figures 5B and 5D showed the standard curves of Au@PDA-LFIA and AuNPs-LFIA. The linear relationship of Au@PDA-LFIA was y = -260.89 lgx + 237.93 with LOD of 7.4 pg/mL, respectively (Figure 4B). The linear relationship of AuNPs-LFIA was y = -386.69 lgx + 608.55 with LOD of 76.1 pg/mL, respectively (Figure 5D). The LOD of Au@PDA-LFIA was 10-fold lower than that of 23 ACS Paragon Plus Environment


AuNPs-LFIA, indicating that Au@PDA as a signal-amplification label improved the sensitivity of LFIA. Figures S7A and S7B showed the images of test strips in Au@PDA-LFIA and AuNPs-LFIA, respectively. The color intensity of Au@PDA was the major contributor to the improved sensitivity of LFIA for ZEN detection. Table S1 compared the linear range and LOD of this method with those of the other three. The results indicated that the method had a superior LOD of detect ZEN. 3.7. Specificity experiment Figure 5C showed the cross-reaction of ZEN with six other mycotoxins (FB1, AFB1, OTA, PAT, CIT, and DON) in Au@PDA-LFIA. The intensities of the T line of samples spiked with FB1, AFB1, OTA, PAT, CIT, DON, and ZEN were 875.7, 891.5, 867.5, 919.3, 889.0, 872.5 and 211.0, indicating that no cross-reaction of ZEN with six other mycotoxins occurred. 3.8. Recovery experiment Table 1 showed the intra-assay and inter-assay recoveries of samples spiked with ZEN at 0.05, 0.1, 0.5, and 1 ng/mL by using Au@PDA-LFIA. The average recoveries for the intra-assay ranged from 93.80% to 111.82%, with a coefficient of variation (CV) ranging from 2.25% to 9.04%, whereas their inter-assay recoveries ranged from 95.05% to 102.44%, with a CV ranging from 1.08% to 7.84%. These results indicated that this method had high recovery.


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Table 1. Intra-assay and inter-assay recoveries of Au@PDA-LFIA. Spiked

Spiked concentration

Detected concentration

Recoveryc

CV

assay

(ng/mL)

(ng/mL)

(%)

(%)

0.05

0.0469±0.002

93.80

4.99

0.1

0.1068±0.002

106.80

2.25

0.5

0.5591±0.051

111.82

9.04

1

0.9421±0.062

94.21

6.55

0.05

0.0504±0.001

100.80

2.01

0.1

0.0980±0.001

98.00

1.08

0.5

0.5122±0.020

102.44

3.95

1

0.9505±0.075

95.05

7.84

Intra-assaya

Inter-assayb

a

Intra-assay: The recovery experiment was completed within one week in triplicate at

each concentration. b

Inter-assay: The recovery experiment was completed for three consecutive weeks in

triplicate at each concentration. c

Recovery = (detected concentration/spiked concentration) × 100%.

3.9. Detection of real maize samples Table 2 showed the detection results of 20 real maize samples using Au@PDA-LFIA and HPLC. Among these samples, three samples (Nos.7, 12, and 18) detected by Au@PDA-LFIA were 0.24±0.015, 4.62±0.134 and 1.13±0.048 ng/mL, respectively, whereas those detected by HPLC were 0.30±0.013, 5.18±0.096, and 1.01±0.038 ng/mL, respectively. Seventeen other samples were not detected by either method. The detection results of Au@PDA-LFIA were in agreement with those of HPLC. 25 ACS Paragon Plus Environment



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Table 2. Detection results of real maize samples using this method and HPLC. Sample number

This method (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

NDa ND ND ND ND ND 0.24±0.015 ND ND ND ND 4.62±0.134 ND ND ND ND ND 1.13±0.048 ND ND

a

CV (%)

6.08

2.91

4.24

HPLC (ng/mL) ND ND ND ND ND ND 0.30±0.013 ND ND ND ND 5.18±0.096 ND ND ND ND ND 1.01±0.038 ND ND

CV (%)

4.33

1.86

3.79

ND: Not detected.

3.10. Evaluation of the developed labeling system under conventional LFIA conditions Figure S8 exhibited the comparison between the developed labeling system and conventional LFIA conditions. The intensities of T line at ZEN concentrations of 0, 0.1, 1, and 5 ng/mL were 853, 558, 239, and 58 in the developed labeling system, and the intensities of T line at ZEN concentrations of 0, 0.1, 1, and 5 ng/mL were 832, 527.5, 202, and 76 in the conventional LFIA conditions. These results demonstrated that the sensitivity of the two methods is similar, so the developed labeling system can 27 ACS Paragon Plus Environment


also work in conventional LFIA conditions.


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4. CONCLUSIONS In this study, we synthesized a composite material (Au@PDA), and used it for the first time to detect ZEN through LFIA. AuNPs were coated with a thickness-adjustable PDA layer to enhance their color intensity, colloidal stability, and mAb coupling efficiency. The LOD of the Au@PDA-LFIA was 7.4 pg/mL, which was 10-fold lower than that of traditional AuNP-LFIA (76.1 pg/mL). The Au@PDA-LFIA also exhibited no cross reaction with six other mycotoxins and high recoveries (93.80%-111.82%). The proposed method combines a signal-amplification label (Au@PDA) with LFIA sensors to provide a promising and versatile approach for the rapid and sensitive detection of other mycotoxins at ultrasensitive levels in agricultural

products

and


foods.


AUTHOR INFORMATION Corresponding Author * Weihua Lai, E-mail: [email protected] Phone: +86 13879178802. Address: 235 Nanjing East Road, Nanchang 330047, P.R. China * Hongwei Duan. E-mail: [email protected]. Phone: +65 65141019. Address: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore.

Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by Jiangxi earmarked fund for Jiangxi agriculture research system (JXARS-03).

ASSOCIATED CONTENT Supporting Information Effect of pH and the volume of 3% H2O2 on the formation of Au@PDA; Impact of 30 ACS Paragon Plus Environment

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several structural analogues of DA on the formation of AuNPs coating; Size distribution of AuNPs and Au@PDA; UV-vis absorption spectrum of Au@PDA-10 and Au@PDA-10-mAb; Optimization of capping agents; Images of lateral flow strips; Evaluation of the developed labeling system under conventional LFIA conditions; Comparison of this method with those of previous methods.



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TOC graphic



Scheme 1. Principle of Au@PDA-LFIA. (A) Preparation procedure of Au@PDA-mAb. (B) Schematic of the proposed Au@PDA-LFIA for ZEN detection: (a) structure of immunochromatographic test strip, (b) detection mode in the presence of ZEN, and (c) detection mode in the absence of ZEN. 239x179mm (300 x 300 DPI)

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Figure 1. TEM images of AuNPs and Au@PDA. (A) AuNPs, AuNPs with the addition of (B) 5 μL of DA·HCl (Au@PDA-5), (C) 10 μL of DA·HCl (Au@PDA-10), and (D) 15 μL of DA·HCl (Au@PDA-15). All scale bars at high magnification are 10 nm.



Figure 2. Characterization and evaluation of AuNPs and Au@PDA. (A) Zeta potential of (1) AuNPs, (2) Au@PDA-5, (3) Au@PDA-10, and (4) Au@PDA-15. (B) UV-vis absorption spectrum of AuNPs, Au@PDA-5, Au@PDA-10, and Au@PDA-15 (Inset: 1, 2, 3, and 4 represent AuNPs, Au@PDA-5, Au@PDA-10, and Au@PDA-15). (C) Comparison of grayscale intensity of Au@PDA-10 and AuNPs at the same concentration on the NC membrane. (D) Images of Au@PDA-10 and AuNPs at different pH values and volumes of 10% NaCl (μL): tolerance to pH of Au@PDA-10 (a) and AuNPs (b); and tolerance to NaCl of Au@PDA-10 (c) and AuNPs (d). Absorbance of Au@PDA-10 and AuNPs at different pH values (E) and volumes of 10% NaCl (F).


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Figure 3. Evaluation of mAb coupling efficiency of Au@PDA-10 and AuNPs. (A) The standard curve of OD450 versus mAb concentration (0.005, 0.01, 0.05, 0.1, 0.5, 1, and 2.5 μg/mL) by ELISA. (B) The mAb coupling efficiency of Au@PDA-10 and AuNPs under different concentrations of mAb (1, 5, 10, 15, and 20 μg/mL)



Figure 4. Optimization of key parameters. Intensity of the T line at different (A) pH values, (B) mAb concentrations, (C) ZEN-BSA concentrations, and (D) volumes of Au@PDA-10-mAb


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Figure 5. Performance and evaluation of LFIA. (A) Immunological kinetics analysis of Au@PDA-LFIA. Standard curve of intensity of T line versus ZEN concentrations (0, 0.01, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng/mL) in (B) Au@PDA-LFIA and (D) AuNPs-LFIA. (C) Cross-reaction of ZEN (1 ng/mL) with six other mycotoxins (FB1, AFB1, OTA, PAT, CIT, and DON all at 10 ng/mL) in Au@PDA-LFIA


Lateral Flow Immunoassay Based on Polydopamine-Coated Gold

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