Sensitive and Matrix-Tolerant Lateral Flow Immunoassay Based on

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A Sensitive and Matrix Tolerant Lateral Flow Immunoassay Based on Fluorescent Magnetic Nanobeads for the Detection of Clenbuterol in Swine Urine Zhen Huang, Zhijuan Xiong, Yuan Chen, Song Hu, and Weihua Lai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06449 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Journal of Agricultural and Food Chemistry

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A Sensitive and Matrix Tolerant Lateral Flow Immunoassay Based on

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Fluorescent Magnetic Nanobeads for the Detection of Clenbuterol in Swine

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Urine

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Zhen Huang*, Zhijuan Xiong*, Yuan Chen*, Song Hu*, Weihua Lai*1

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

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Nanchang 330047, China

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1Corresponding

author:

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Weihua Lai, Ph.D., Professor

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State Key Laboratory of Food Science and Technology, Nanchang University

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Address: No.235 Nanjing East Road, Nanchang 330047, China

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Tel: +86-138 7917 8802;

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The lack of sensitivity and poor of matrix tolerance is the main bottleneck of lateral

18

flow immunoassay (LFIA). Here, a sensitive and matrix tolerant method that integrated

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immunomagnetic separation and fluorescent LFIA (IMS-FLFIA) based on fluorescent

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magnetic nanobeads was developed to detect clenbuterol (CLE) residue in swine urine.

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The limit of detection (LOD) of IMS-FLFIA is 4 times lower than that of traditional

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colloidal gold LFIA. This method, which exhibits similar LOD and linearity range in

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both PBS and urine swine, is highly correlated with the LC-MS/MS for the detection

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of real swine urine samples. The result indicated that IMS-FLFIA has a universal

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resistance to swine urine matrix. The merits of this assay, high sensitivity, matrix

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tolerance, accuracy, and specificity, ensure a promising future in detection of veterinary

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drug residue.

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Key Words: Fluorescent Magnetic Nanobeads, Immunomagnetic Separation,

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Fluorescent Lateral Flow Immunoassay, Swine Urine, Clenbuterol

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1. INTRODUCTION

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Veterinary drugs are widely used to treat and promote growth in animal

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husbandry.1-3 This practice has remarkably advanced the development of this industry.

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The market size of veterinary drugs up to tens of billions dollars proves the success of

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application of these drugs in the animal husbandry.1, 4 However, uncontrolled use of

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veterinary drugs have also resulted in many environmental and public health

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problems.5-7 For example, β-adrenergic agonists have been abused to promote animal

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growth and enhance the lean meat-to-fat ratio.8-10 However, long-term or high dose

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ingestion of residual β-adrenergic agonists through meat products can cause several

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deleterious severe physiological side-effects, such as muscular pain, dizziness,

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tachycardia, nervousness, and even death.11-12 Unfortunately, although many countries

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and regions have prohibited the use of β-adrenergic agonists as growth-promoting

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agents,13-14 various β-adrenergic agonists continue to be illegally added to feed for

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economic interests. For instance, the clenbuterol (CLE) events in the middle of March

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2011 in China has led to serious discussions about β-adrenergic agonists for food

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safety.15 Thus, a simple, inexpensive, and efficient detection method is urgently needed

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to further strengthen the regulation of β-adrenergic agonists abuse to ensure food safety.

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The available instrumental analyses, such as high-performance liquid

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chromatography (HPLC),16 liquid chromatography-mass spectrometry (LC-MS),7 LC-

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MS/MS,17 and gas chromatography-MS,18 are the primary analytical methods for

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determining veterinary drugs residue, as well as β-adrenergic agonists. However, these 3



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methods are costly, complex, and time-consuming, and these characteristics hamper

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on-site determination. Therefore, lateral flow immunoassay (LFIA), own the unique

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advantages of rapid, easy-to-use, portable, and low-cost,9, 19-23 has been extended for

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the detection of veterinary drugs residue.

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The limitation of LFIA, however, is the lack of sensitivity and poor of matrix

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tolerance.23-24 Zhang et al.25 reported the significant matrix effect of swine urine on both

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time-resolved fluorescent nanobeads LFIA (TRFN-LFIA) and colloidal gold LFIA

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(CG-LFIA) for detection of CLE. The matrix effect of the urine samples causes

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significant difference between the detected concentration and the actual spiked

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concentration of CLE. To resolve these problems, immunomagnetic separation (IMS)

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based on superparamagnetic nanoparticles, usually used as a sample pretreatment

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technology for sample purification and enrichment, was introduced in the LFIA.26-27

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The IMS-based LFIA exhibits enhanced sensitivity and matrix tolerance,22, 28-29 because

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the target is enriched, and the complex matrix is removed during the IMS step. However,

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the IMS-based LFIA is too complex that including IMS, elution and LFIA steps. The

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elution step is carried out by most researchers to avoid the interference of the

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superparamagnetic

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Nevertheless, the elution step leads to the loss of the target resulting in unsatisfactory

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sensitivity. Moreover, this strategy needs two immuno-probes for the complete

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detection process, antibody modified magnetic beads for the IMS step, and antibody

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modified labels for the LFIA step. Which is expensive that cost more antibodies and

nanoparticles

on

subsequent

4


fluorescence

detection.28-30

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nanomaterials.

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Hence, to overcome these drawbacks to further increase the sensitivity of the LFIA,

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we developed a novel method (Figure 1) integrated IMS with fluorescent lateral flow

75

immunoassay (IMS-FLFIA) based on fluorescent magnetic nanobeads (FMNBs).

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FMNBs exhibit remarkable magnetic, fluorescence, and biological modification

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property that have been widely applied in the biomedical field,31-33 and food safety

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area.34 However, the application of FMNBs in the competitive LFIA platform has not

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been reported yet. For the design of IMS-FLFIA, we take full advantage of the dual

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functionality of FMNBs. The FMNBs-based probes worked as a carrier for the IMS

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and also as a fluorescent label for the FLFIA. Thus, only one probe was needed for the

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IMS-FLFIA, and the elution step that causes the loss of the target was omitted. To

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ensure the application of the method in actual testing, CLE in swine urine was detected

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as a model target in this study. We explored the effect of the urine matrix on the IMS-

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FLFIA and verified the results of the method by LC-MS/MS. The results indicated that

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the as-established IMS-FLFIA exhibited excellent performance of sensitivity, matrix-

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tolerance, and accuracy, that in our perception, the method may also be extended for

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the detection of other β-adrenergic agonists residue.

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2. EXPERIMENTAL

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2.1 Chemicals and Materials

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Carboxylate-modified CdSSe/ZnS quantum dots (QDs, 1.0 mg/mL (m/v), d = 10 5



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nm) were acquired from the Hangzhou Najing Technology Co., Ltd. (Hangzhou, China).

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Anti-CLE monoclonality antibody (CLE-mAb), goat anti-mouse IgG, and complete

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antigen (CLE-BSA) were provided by Jiangxi Zodolabs Biotech. Co., Ltd. (Jiangxi,

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China). The nitrocellulose (NC) membrane, absorbent pad, sample pad, and conjugate

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pad were purchased from Shanghai Jinbiao Tech. Co., Ltd. (Shanghai, China). Ferric

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chloride (FeCl3·6 H2O), ethylene glycol, anhydrous sodium acetate (NaAc), trisodium

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citrate dehydrate (SC), hydrolysis of tetraethyl orthosilicate (TEOS), 3-aminopropyl

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triethoxysilane (APTES), ammonia solution (30%wt), N-(3-dimethylaminopropyl)-N′-

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ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide (NHSS)

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were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). CLE,

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tulobutezol (TUL), brombuterol (BRO), terbutaline (TER), clorprenaline (CLO),

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ractopamine (RAC), penbutolol (PEN), salbutamol (SAL), and bambuterol (BAM)

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were obtained from Dr. Ehrenstorfer, GmbH (Augsburg, Germany). Swine urine

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samples were provided by Jiangxi Institute of Veterinary Drug and Feedstuff Control.

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Other chemicals were of analytical reagent grade and purchased from Sinopharm

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Chemical Co., Ltd. (Shanghai, China).

108

2.2 Preparation of FMNBs

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Layer-by-layer assembly35 and co-precipitation method36 have been widely used to

110

synthesis FMNBs. However, in this study, the FMNBs with a typical

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core@shell@satellite (Fe3O4@SiO2@QDs) structure were synthesized by combined

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solvothermal, sol–gel, and assembling methods.34, 37 Typically, the Fe3O4 magnetic core 6


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was first prepared by the solvothermal reduction method.38 A total of 0.819 g of

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FeCl3·6H2O was dissolved in a mixture that contained ethylene glycol (30 mL), water

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(1.1 mL), NaAc (1.8 g), and SC (0.318 g) with vigorous stirring for 2 h. Then, the

116

mixture was heated to and maintained at 200 °C for 10 h. Finally, the black products

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(Fe3O4 magnetic core) were washed several times and resuspended into ethanol for the

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further Stöber process.39

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To coat with silica shell, as-prepared Fe3O4 magnetic core (60 mg) and 15 mL of

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ultrapure water were dispersed in 100 mL of ethanol. Under continuous mechanical

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stirring, 3 mL of ammonia solution and 0.5 mL of TEOS were successively added to

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the reaction mixture. After stirring for 20 h, Fe3O4@SiO2 were obtained. For the surface

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amination of Fe3O4@SiO2, 6.7 mL of ammonia solution and 1 mL of APTES were

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added to the above mixture. After reacting for 12 h, the Fe3O4@SiO2@NH2 was washed

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several times and resuspended in water.

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Finally, FMNBs were synthesized by assembling QDs and Fe3O4@SiO2@NH2

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through bonding between amino and carboxyl.37 Briefly, 0.8 mg of QDs and 3 mg of

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as-prepared Fe3O4@SiO2@NH2 were added into 5 mL of borate saline buffer (BB, with

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0.1% Tween 20; pH 7.0) containing 10 mg of EDC and NHSS and mildly mixed at

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room temperature. With the process of reaction, QDs gradually attached to the surface

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of Fe3O4@SiO2@NH2 by covalent bond. After 12 h, the FMNBs were washed several

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times and stored in ultrapure water at the final concentration of 1 mg mL−1 and stored

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at 4 °C for further use. 7



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2.3 Construction of FMNBs-mAb Probe

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The CLE-mAb labeled FMNBs (FMNBs-mAb) probe was prepared as previously

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reported with some modifications.34 As-prepared FMNBs (1 mg) was suspended in

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1 mL of freshly made EDC and NHSS solutions (5 mg mL−1 of EDC and NHSS in 0.01

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M 2-(N-morpholino)ethane sulfonic acid buffer containing 0.1% Tween 20, pH 6.0).

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After incubation for 1 h, the EDC and NHSS solutions were removed, and the FMNBs

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were resuspended with 1 mL of mAb solution (10, 20, 30, 40, 50, or 60 μg mL−1 of

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mAb in BB) for 4 h of coupling reaction and then blocked with 1 mL of blocking buffer

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(3% BSA, 3% OVA, 3% milk, and 1 mg mL−1 glucosamine in BB) for 1 h. The FMNBs-

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mAb probe was finally collected with a magnet and resuspended in 1 mL of storage

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buffer (0.01 M BB containing 5% sucrose, 2% trehalose, 1% polyethylene glycol 20000,

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1% casein, and 0.25% Tween-20; pH 7.4) and stored at 4 °C for further use. All

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experiments were performed in triplicate.

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2.4 Fabrication of CLE Later Flow Strips

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The test strip consisted of a sample pad, a conjugate pad, an NC membrane, and an

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absorption pad. The sample pad was treated with 0.05 M BB (pH 7.4) containing 1%

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BSA, 0.5% Tween 20, and 0.05% sodium azide and then dried at 60 °C for 2 h. The

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CLE-BSA (0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40 mg mL−1) and goat anti-mouse

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IgG (0.2 mg mL−1) were spotted on the NC membrane using the BioDot XYZ platform

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(California, USA) as test line (T line) and control line (C line), respectively. The

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prepared NC membranes were dried at 37 °C for 12 h. The absorption and conjugate

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pads were used without treatment. All components of the strip were assembled as the

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strip in Figure 1. All experiments were performed in triplicate.

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2.5 Construction of the IMS-FLFIA

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The constructed IMS-FLFIA proceeded in two closely linked steps. The first step

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was the IMS for the sample purification and enrichment, and the second step was

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FLFIA for detection (Figure 1). FMNBs-mAb probes (10, 15, 20, 30, 45, and 60 μg)

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were mixed with different volumes (0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL) of the sample

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for incubation using a rotator at 15 rpm for 10, 20, 30, 40, 50, and 60 min. Then, the

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probes were separated by a magnetic separator for 5 min. The supernatant was collected

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and detected by ELISA to calculae the capture efficiency (CE) of the IMS procedure.

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The pellets were resuspended in 100 μL of PBS and added to the sample pad of FLFIA

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strips for the immunoassay. After 10 min, the photoluminescence (PL) signals of the T

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and C line were read by a portable fluorescent test strip reader (Suzhou Helmen

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Precision Instrument Co., Ltd., Suzhou, China) and recorded as ratios of the PL

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intensity of T line to that of C line (T/C) of each strip to improve the stability by

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reducing the deviation caused by the external environment.40 All experiments were

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performed in triplicate.

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2.6 Development of the ELISA and calculation of the CE

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The direct competition ELISA was performed to quantify the free CLE in the 9



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supernatant. The primary procedure was as follows: Each microplate was incubated

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overnight at 4 °C with 100 μL of CLE-BSA conjugate antigen (0.2 μg mL−1). After

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blocking, 50 μL of the supernatant or CLE standard solution (10, 5, 2.5, 1, 0.5, 0.25,

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0.1, and 0 ng mL−1) was added to each well and kept incubated with 50 μL of CLE-

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mAb at 37 °C for 2 h. The HRP–goat anti-mouse IgG (diluted 1:3000) was added to

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each well and incubated at 37 °C for 30 min. After washing, 100 μL of the 3,3′,5,5′-

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tetramethylbenzidine substrate solution was added into the wells. After 15 min at 37 °C,

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the reaction was terminated by addition of 50 μL of 0.2 M sulfuric acid and the optical

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density at 450 nm was read by a LabSystems microplate reader (Helsinki, Finland). The

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standard curve was established (Figure S1) with experimental data, and the

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concentration of free CLE in the supernatant could be calculated with the standard curve.

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Finally, the CE of IMS steps can be calculated with the following formula:

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CE(%) = 1 ―

Csup Csam

× 100%

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where the Csup and Csam represent the concentration of CLE in the supernatant and

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sample, respectively. All experiments were performed in triplicate.

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2.7 Detection of CLE in PBS and Urine Sample

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Three swine urine samples (referred to as Sample 1, Sample 2, and Sample 3)

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confirmed by LC-MS/MS were applied for this experiment. PBS and swine urine spiked

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with different concentrations of CLE (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025, and 0 ng

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mL−1) were detected by the IMS-FLFIA. The limit of detection (LOD) was defined as 10


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the concentration of CLE in the sample solution that caused a 10% decrease in the T/C

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ratio compared with that produced by the negative sample. All experiments were

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performed in triplicate.

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2.8 Specificity and Accuracy of the IMS-FLFIA

198

The specificity of the constructed method was evaluated using CLE and other β-

199

adrenergic agonists, namely, TUL, BRO, TER, CLO, RAC, PEN, SAL, and BAM

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spiked swine urine sample. All experiments were performed in triplicate. The cross-

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reactivity rate (CR) were calculated by comparing the half maximal inhibitory

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concentration (IC50) of CLE with those of β-adrenergic agonists in IMS-FLFIA using

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the following equation:

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CR(%) =

IC50 𝑜𝑓 𝐶𝐿𝐸 IC50 𝑜𝑓 𝑜𝑡ℎ𝑒𝑟 𝛽 ― 𝑎𝑑𝑟𝑒𝑛𝑒𝑟𝑔𝑖𝑐 𝑎𝑔𝑜𝑛𝑖𝑠𝑡𝑠

× 100%

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The accuracy of the developed method was evaluated by analyzing the recovery

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and validated with LC-MS/MS (Agilent 1260, California, USA). For recovery analysis,

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swine urine samples (Sample 1, Sample 2, and Sample 3) spiked at 0.5, 1, and 2 ng

208

mL−1 of CLE were detected by the IMS-FLFIA. Moreover, twelve real swine urine

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samples were detected with IMS-FLFIA and LC-MS/MS. The detection of CLE by LC-

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MS/MS was according to Standard Method 1063-3-2008 (Announcements of the

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Ministry of Agriculture, China) with some modifications. Briefly, 5 mL of swine urine

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sample was adjusted to pH 9.5 with 5 M NaOH. Then, 10 mL of tert-butanol/tert-butyl

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methyl ether (60:40, v/v) was added to the sample, which was adequately oscillated. 11



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After centrifugation at 7000 rpm for 10 min, the supernatant was transferred to a glass

215

test tube and concentrated using a nitrogen evaporator. The residue was dissolved in 1

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mL of 0.2% aqueous formic acid (v/v). The reconstituted solution was separated and

217

purified by SPE, and the purified solution was filtered through a 0.22 µm cellulose

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membrane for LC-MS/MS analysis. The LC system for CLE detection included a C18

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column (2.1 × 100 mm, 1.7 µm), which was kept at 40 °C for chromatographic

220

separation. The flow rate of the mobile phase was 0.20 mL/min, and the injection

221

volume of the sample solution was 5 µL. The mobile phase consisted of aqueous formic

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acid (methanol:0.2% formic = 50:50, v/v). An electrospray positive-ion source was

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used, and MS acquisition was performed in multiple-reaction monitoring mode. The

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monitoring ion pairs were CLE m/z 276.5/202.5 (quantitative ion) and 276.5/258.7

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(qualitative ion). The results of both methods were compared, and correlation analysis

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was conducted. All experiments were performed in triplicate.

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3 RESULTS AND DISCUSSION

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3.1 Characterization of FMNBs and FMNBs-mAb Probe

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The morphologies and structures of the FMNBs were characterized by scanning

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electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2A

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shows that the FMNBs were monodispersed and approximately spherical with average

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size of 250 nm, which were consistent with the results from the TEM (Figure 2B). More

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importantly, the TEM image clearly shows the core@shell@satellite structure of

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FMNBs that was displayed as a 3D structure model in Figure 2C. A single Fe3O4

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magnetic core with 100 nm in size was coated with a 75 nm thick silica shell, and

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numerous QDs were well attached to the silica shell.

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For the designed strategy, FMNBs not only served as a carrier for IMS but also as

238

a fluorescent probe for FLFIA. Thus, the magnetic and fluorescent properties of

239

FMNBs were further characterized. Figure 2D illustrates that the FMNBs had magnetic

240

saturation values of 12.50 emu g−1 and exhibited favorable superparamagnetism,

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because zero coercivity was obtained during the magnetic field cycling between

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−20,000 Oe and 20,000 Oe. During magnetic separation, the FMNBs could be

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completely recycled in 3 min, as shown in the internal drawing of Figure 2D. The

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excitation and emission spectra of the FMNBs (Figure 2E) showed that the FMNBs

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inherited excellent fluorescence properties of narrow emission spectra, broad excitation

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range, and wide Stokes’ shift of the QDs. These properties were beneficial to enhance

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the sensitivity of FLFIA. Moreover, the FMNBs displayed an optimal excitation

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wavelength at 365 nm and an optimal emission wavelength at 610 nm. Thus, the

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FMNBs exhibited a bright red color under 365 nm UV light (internal drawing of Figure

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2E).

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The FMNBs were modified with anti-CLE mAb to specifically recognize and

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capture CLE. The UV-visible spectroscopy analysis and hydration particle size analysis

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were performed to confirm the successful modification of mAb on the surface of

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FMNBs. Figure 3F shows a distinct characteristic absorption peak of protein at 280 nm 13



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after the FMNBs conjugated with the mAb. Hydration particle size analysis was also

256

performed to monitor changes in the particle size of FMNBs (Figure 2G). The hydration

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particle size of bare FMNBs was 322.5 nm. However, when mAb coupled to the surface

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of FMNB, the hydration layer of the particles significantly increased.Thus, larger

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hydration particle size of FMNBs-mAb (377.3 nm) was observed. The results of the

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two analyses were consistent with previous reports,41-42 indicating the successful

261

synthesis of the FMNBs-mAb probe.

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3.2 Construction and Optimization of the IMS-FLFIA

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The FMNBs not only participate in the IMS step as carriers for sample purification

264

and enrichment but also participated in the FLFIA step as the fluorescence label. Thus,

265

these two modular steps were seamlessly linked to form the method demonstrated in

266

Figure 1. The IMS was performed by adding FMNBs-mAb probes to the samples (PBS

267

or swine urine) containing spiked CLE, followed by incubation for a time to specifically

268

recognize and capture the targets. Then, the FMNBs-mAb probe and FMNBs-mAb-

269

CLE complex were separated from the sample matrix using the magnetic field and

270

resuspended in 100 μL of PBS for the next FLFIA. As the 100 μL of resuspension was

271

added to the sample pad of the strip, the FMNBs-mAb probe and FMNBs-mAb-CLE

272

complex migrated along the strip by capillary action. Subsequently, as the antigen-

273

antibody reaction proceeds, detectable PL signal appeared on the T and C line. Given

274

that the FLFIA step was presented in a competitive mode, the PL signal intensity of T

275

line decreased as the target concentration increased. To quantitatively detect CLE, the 14


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standard calibration curve was obtained by plotting the T/C value against the logarithm

277

of various CLE concentrations. Compared with previously reported IMS-based FLFIA,

278

the proposed method innovatively utilized a composite nanoparticle with excellent

279

magnetic and optical properties to integrate enrichment and detection in one step to

280

avoid the loss of the target and enhance the sensitivity.

281

Experiments were first performed to optimize the concentration of CLE-BSA on

282

the T line. PBS and 1 ng mL−1 of CLE-spiked PBS were detected as negative and

283

positive samples (Unless stated, the same sample is used for optimization experiments

284

below). We observed that higher T/C value was achieved by enhancing the

285

concentration of CLE-BSA on the T line for both positive and negative samples (Figure

286

3A). The reaction opportunities between FMNBs-mAb probe and CLE-BSA increased

287

with the concentration of CLE-BSA. Thus, more FMNBs-mAb probe may be

288

immobilized on the T line, leading to higher PL signal and T/C values. Therefore, the

289

inhibition rate was further examined. This quantity is the extent to which the T/C value

290

of the positive sample was lower than that of the negative sample. Figure 3A shows that

291

the inhibition rate was non-monotonic as the concentration of CLE-BSA increased from

292

0.10 mg mL−1 to 0.50 mg mL−1. When the concentration of CLE-BSA was 0.10 mg

293

mL−1 and 0.30 mg mL−1, high inhibition rates of 30.80% and 32.28% were obtained,

294

respectively. However, as the concentration of BSA-CLE was 0.1 mg mL−1, the large

295

error level (30.80% ± 3.73%, average ± standard deviation) indicated that the test strip

296

was unstable under this condition. Therefore, considering stability and sensitivity, 0.30 15



297

mg mL−1 of CLE-BSA was chosen for the further study.

298

To investigate the effect of the FMNBs-mAb probe on the method, probes prepared

299

with different conditions were studied. The blocking agents impacted significantly on

300

the FLFIA step (Figure S2). Unblocked and 1 mg mL−1 of glucose amine blocked

301

FMNBs-mAb probe exhibited strong non-specific binding to NC membrane, thus, the

302

inhibition rate is meager. Moreover, 3% (W/V) of OVA, BSA, and milk blocked

303

FMNBs-mAb probe well, and the BSA blocked probe exhibited the most substantial

304

inhibition rate of 45.32%. Subsequently, the FMNBs-mAb probes prepared with

305

different concentrations of mAb were evaluated from both CE and inhibition rate

306

aspects. As shown in Figure 3D, the CE of the IMS step continuously increased when

307

the mAb concentration increased from 10 μg/mL to 60 μg/mL and reached a maximum

308

value of 34.06%. However, for the inhibition rate, the trend of inhibition rate showed a

309

peak shape with increase in the concentration of mAb (Figure 3C). This phenomenon

310

resulted from the blending of IMS and FLFIA steps. As we all know, for competitive

311

immunochromatographic models, increasing the antibody concentration usually results

312

in reduced inhibition rate. However, for the IMS models, increasing the antibody

313

concentration usually increases CE. Thus, as Figure 3C shown, the inhibition rate

314

increased from 11.59% to 29.75% as the concentration of mAb increased from 10 μg

315

mL−1 to 30 μg mL−1. Then, the inhibition rate gradually decreased to 2.45% as the

316

concentration of mAb increased to 60 μg mL−1. Similar phenomena have also been

317

observed when different amounts of FMNB-mAb probes were used to detect CLE 16


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(Figures 3E and F). The CE increased from 9.70% to 33.10% as the amount of FMNBs-

319

mAb probes increased from 10 μg to 35 μg. In the meantime, largest inhibition rate of

320

24.10% was obtained when 20 μg of FMNBs-mAb probes were used. In summary, 20

321

μg of FMNBs-mAb probes conjugated with 30 μg mL−1 of mAb and blocked with 3%

322

BSA were chosen as the optimal probe to improve the sensitivity of test result.

323

The effects of increasing sample volume and incubation time on IMS-FLFIA were

324

studied. Six samples with different volumes were tested to demonstrate how volumetric

325

enrichment can affect the test sensitivity. The CE of IMS step decreased with increase

326

in the sample volume (Figure 3H). With increasing sample volume, the FMNBs-mAb

327

probes in the sample were diluted, which decreased the efficiency of antigen-antibody

328

binding between probe and CLE, resulting in reduced CE. Then, the amounts of

329

captured CLE in the different sample volumes were calculated by multiplying the

330

capture rate, sample volume, and concentration. The calculated results showed that 1.60,

331

1.81, 1.56, 1.40, 0.67, and 0.74 ng of CLE were captured when the sample volumes

332

were 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL, respectively. By analyzing these data, we

333

inferred that maximum inhibition rate would be obtained when the sample volume was

334

0.3 mL. Subsequent experiments confirmed our conjecture, as shown in Figure 3G, the

335

maximum inhibition rate of 42.53% was obtained when 0.3 mL of sample was tested.

336

The effects of incubation time on IMS were also studied (Figures 3I and J). During

337

incubation, FMNBs-mAb probes specifically recognized and captured the targets by

338

the antigen–antibody reaction. Thus, increasing the incubation time from 10 min to 40 17



339

min increased the CE from 15.42% to 65.40% and the inhibition rate from 18.07% to

340

42.82%. The CE and inhibition rate remained stable with prolonged incubation time,

341

because the FMNBs-mAb probes were limited. By these study, 0.3 mL of sample was

342

tested with 40 min of incubation for this method.

343

Finally, under the optimal conditions, PBS at 0, 1, and 5 ng mL−1 of CLE were

344

detected. After 100 μL of resuspension were dropped onto the test strip, the test strip

345

was immediately read, and the T/C value was recorded every 30 s for 20 min to

346

construct the Immuno-kinetic curve (Figure 3B). As the immunochromatography

347

progressed, the T/C value gradually decreased and eventually reached equilibrium.

348

When the time reached 10 min, the T/C values of the three samples reached equilibrium,

349

which indicates that the stabilization process of immunochromatography had no

350

correlation with the content of CLE in the sample. Thus, the strips for detection of

351

samples containing any CLE could be read at 10 min.

352

3.3 Study of Sensitivity and Matrix Tolerance of the IMS-FLFIA

353

Urine, as a metabolic channel for a variety of drugs, has the advantages of easy

354

access and easy pre-treatment and can meet the requirements of liveness detection.

355

Therefore, the urine is widely selected as a sample for various tests.43 However, the

356

composition of urine is complex that can seriously affect immunochromatography,

357

leading to inaccurate detection results.25, 44-45 PBS and negative urine sample (Sample

358

1) spiked with different concentrations of CLE were tested to verify the utility of the

18


Page 18 of 42

Page 19 of 42


359

constructed method. Figure 4A shows that CLE with the same concentration in different

360

matrices resulted in similar T/C values. The correlation analysis, as shown in Figure

361

4B, showed that the T/C value of PBS sample and that of urine sample were

362

considerably correlated (R2 = 0.997, slope = 1.01), indicating that the IMS-FLFIA had

363

excellent matrix tolerance. However, the control experiment that CLE in PBS and urine

364

was detected by CG-LFIA (Supporting Information S1) showed significant matrix

365

effect (Figure S3). The t-test analysis shows the T/C value of CG-LFIA in PBS and that

366

in urine are significantly different (P < 0.05), indicating the urine affect greatly on the

367

detection results of CG-LFIA, which is consistent with the conclusions of Zhang et al.25

368

To further verify the universal resistance of IMS-FLFIA to swine urine matrix, negative

369

urine samples (Sample 1, Sample 2, and Sample 3) spiked at 1 ng mL−1 of CLE were

370

detected by CG-LFIA and IMS-FLFIA, respectively (Figure S4). The T/C values of

371

IMS-FLFIA in different urine samples are very stable and has no significant difference

372

from the T/C value in PBS, exhibited much better matrix tolerance than CG-LFIA. The

373

elimination of matrix effects of urine should be attributed to the application of IMS.

374

Subsequently, standard curves of IMS-FLFIA in different matrices were established

375

(Figure 4C). For PBS, a linear relationship was observed in the range of 0.25 ng mL−1

376

to 5 ng mL−1 with the LOD of 0.16 ng mL−1. The LOD of IMS-FLFIA is 4 times lower

377

than that of traditional CG-LFIA (0.62 ng mL−1, Figure S5). And for swine urine, a

378

linear relationship was also observed in the range of 0.25 ng mL−1to 5 ng mL−1 with

379

LOD of 0.22 ng mL−1. This result again indicated that the swine urine matrix had little 19



380

effect on the sensitivity and linear range of the method. Thus, we think that the FMNBs-

381

based strategy actually improved the sensitivity and matrix tolerance of LFIA although

382

the IMS-FLFIA may be limited by antibody performance (Table S1).

383

3.4 Specificity and Accuracy Study of the IMS-FLFIA

384

As shown in Figure 5A, the negative urine, 1 ng mL−1 of CLE and10 ng mL−1 of

385

other eight β-adrenergic agonists spiked urines were detected by IMS-FLFIA for the

386

specificity test, only CLE spiked sample caused a significant decrease in the T/C value.

387

The data of CR (Table S2) further verified the specificity of the IMS-FLFIA. The CRs

388

of other eight β-adrenergic agonists are not more than 0.137%, indicating the method

389

can detect CLE specifically. This high specificity of the method relies on the high

390

specificity of the conjugated antibody to CLE. Then, recovery experiments were

391

conducted to evaluate the accuracy and precision of the method. As shown in Table 1,

392

the average recovery of this method was 79.1%–108.9% with coefficients of variation

393

of 3.4%–10.3%, which are the acceptable levels for CLE quantitative analysis. To

394

ensure the accuracy of the method, twelve real swine urine samples were detected with

395

IMS-FLFIA and LC-MS/MS, respectively, as shown in (Figure 5B). The test results of

396

IMS-FLFIA and LC-MS/MS for the twelve real samples showed significant correlation

397

(R2=0.979, slope=1.03). The result indicated that the IMS-FLFIA has good accuracy

398

with LC-MS/MS in multiple urine samples. Therefore, the established method has a

399

universal resistance to swine urine matrix and can be applied to the detection of actual

400

samples. 20


Page 20 of 42

Page 21 of 42


401

In conclusion, we first proposed the IMS-FLFIA based on FMNBs for the detection

402

of CLE with only one probe and without elution steps. The feasibility of this approach

403

for practical applications was demonstrated with the determination of CLE in swine

404

urine. The IMS-FLFIA could quantitatively detect CLE in the range of 0.25 ng mL−1 to

405

5 ng mL−1 with the LOD of 0.22 in urine, and 0.16 ng mL−1 in PBS. By comparing with

406

traditional CG-LFIA, this method is much more matrix tolerant and 4 times higher

407

sensitivity. IMS-FLFIA also exhibited great specificity when it distinguished CLE from

408

eight other β-adrenergic agonists with high concentration. Moreover, the proposed

409

method resulted in significant recovery of 79.1%–108.9% with coefficients of variation

410

of 3.4%–10.3%. The IMS-FLFIA was highly correlated with the LC-MS/MS (R2=0.979,

411

slope=1.03) when six real urine samples were detected. Therefore, the method can not

412

only meet the detection of CLE but may also be widely applied to detect other β-

413

adrenergic agonists residues.

414

ACKNOWLEDGMENTS

415

This work was partially supported by the National Natural Science Foundation of

416

China (no. 31772066), the free explore issue of State Key Laboratory of Food Science

417

and Technology of Nanchang University (SKLF-ZZB-201719), the Jiangxi Special

418

Fund for Agro-scientific Research in the Collaborative Innovation (JXXTCX201703-

419

1), and the Earmarked Fung for the Jiangxi Agriculture Research System (JXARS-03).

21



420

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29



573

APPENDIX

574

TOC

575

30


Page 30 of 42

Page 31 of 42


576

Table 1. The recovery of IMS-FLFIA in CLE spiked swine urine samples. (n=3)

577 578

Spiked CLE Recovery SDb -1 (ng mL ) (%) a Sample 1 0.5 79.1 2.7 1.0 88.9 9.2 2.0 101.3 5.6 Sample 2 0.5 81.9 7.0 1.0 100.1 9.1 2.0 97.5 7.5 Sample 3 0.5 88.0 4.3 1.0 94.2 8.6 2.0 108.9 5.5 a Recovery = (detected concentration/spiked concentration) ×100%. b SD = Standard Deviation; CV = Coefficient of Variation. Sample

31


CV (%) b 3.4 10.3 5.5 8.5 9.1 7.7 4.9 9.1 5.1


579 580

Figure 1. Schematic illustration of the IMS-FLFIA. The IMS-FLFIA process is

581

performed in two modular steps, namely, IMS for sample purification and enrichment

582

and FLFIA for detection.

32


Page 32 of 42

Page 33 of 42


583 584

Figure 2. Characterization of FMNBs and FMNBs-mAb probe. (A) SEM image, (B)

585

TEM image, (C) 3D structural model, (D) room-temperature hysteresis loops, and (E)

586

PL spectra of FMNBs. (F) UV-visible spectroscopy and (G) hydration particle size

587

analyses of the FMNBs and FMNBs-mAb probe.

33



588 589 590 591 592 593

Figure 3. Optimization of the IMS-FLFIA. Effects of (A) concentration of CLE-BSA on the T Line, (C) concentration of mAb for FMNBs-mAb probe, (E) amount of FMNBs-mAb probe, (G) sample volume, and (I) incubation time on IMS-FLFIA; Effects of (D) concentration of mAb for FMNB-mAb probe, (F) amount of FMNB-mAb probe, (H) sample volume, and (J) incubation time on the CE of IMS. (B) Immunological kinetic curve of the FLFIA step. The PBS and 1 ng mL-1 CLE spiked PBS were detected as negative and positive sample, respectively. In (A), (C), (E), (G), and (I), bar and line charts represent T/C Value and Inhibition rate, respectively. (n=3)

34


Page 34 of 42

Page 35 of 42


594 595

Figure 4. Detected CLE in PBS (black) and swine urine (red) by IMS-FLFIA. (A) The

596

T/C value of different concentrations of CLE (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025,

597

and 0 ng mL−1) spiked negative urine sample. (B) Correlation between the T/C value

598

of spiked PBS and urine samples. (C) Standard calibration curve for CLE in PBS and

599

urine samples were obtained by plotting the T/C value against the logarithm of various

600

CLE

concentrations.

35


(n=3)


601 602

Figure 5. Specificity and methodological comparison of the IMS-FLFIA. (A)

603

Specificity analysis of IMS-FLFIA. The concentration of CLE is 1 ng mL−1, and other

604

β-adrenergic agonists are at a concentration of 10 ng mL−1. (B) Correlation between the

605

detection results of LC-MS/MS and IMS-FLFIA of twelve unknown real samples. (n=3)

36


Page 36 of 42

Page 37 of 42


TOC 84x47mm (300 x 300 DPI)



Figure 1. Schematic illustration of the IMS-FLFIA. The IMS-FLFIA process is performed in two modular steps, namely, IMS for sample purification and enrichment and FLFIA for detection. 235x151mm (300 x 300 DPI)


Page 38 of 42

Page 39 of 42


Figure 2. Characterization of FMNBs and FMNBs-mAb probe. (A) SEM image, (B) TEM image, (C) 3D structural model, (D) room-temperature hysteresis loops, and (E) PL spectra of FMNBs. (F) UV-visible spectroscopy and (G) hydration particle size analyses of the FMNBs and FMNBs-mAb probe.



Figure 3. Optimization of the IMS-FLFIA. Effects of (A) concentration of CLE-BSA on the T Line, (C) concentration of mAb for FMNBs-mAb probe, (E) amount of FMNBs-mAb probe, (G) sample volume, and (I) incubation time on IMS-FLFIA; Effects of (D) concentration of mAb for FMNB-mAb probe, (F) amount of FMNB-mAb probe, (H) sample volume, and (J) incubation time on the CE of IMS. (B) Immunological kinetic curve of the FLFIA step. The PBS and 1 ng mL-1 CLE spiked PBS were detected as negative and positive sample, respectively. In (A), (C), (E), (G), and (I), bar and line charts represent T/C Value and Inhibition rate, respectively. (n=3)


Page 40 of 42

Page 41 of 42


Figure 4. Detected CLE in PBS (black) and swine urine (red) by IMS-FLFIA. (A) The T/C value of different concentrations of CLE (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025, and 0 ng mL−1) spiked negative urine sample. (B) Correlation between the T/C value of spiked PBS and urine samples. (C) Standard calibration curve for CLE in PBS and urine samples were obtained by plotting the T/C value against the logarithm of various CLE concentrations. (n=3)



Figure 5. Specificity and methodological comparison of the IMS-FLFIA. (A) Specificity analysis of IMS-FLFIA. The concentration of CLE is 1 ng mL−1, and other β-adrenergic agonists are at a concentration of 10 ng mL−1. (B) Correlation between the detection results of LC-MS/MS and IMS-FLFIA of twelve unknown real samples. (n=3)


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Sensitive and Matrix-Tolerant Lateral Flow Immunoassay Based on

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