1

Ultrasensitive and rapid detection of ochratoxin A in agro-products by a 1

nanobody-mediated FRET-based immunosensor 2

Zongwen Tanga,1, Xing Liua,1,*, Benchao Sua,1, Qi Chena, Hongmei Caoa, Yonghuan Yuna, Yang 3

Xub, Bruce D. Hammockc 4

aCollege of Food Science and Engineering, Hainan University, 58 Renmin Avenue, Haikou 5

570228, PR China 6

bState Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East 7

Road, Nanchang, 330047, PR China 8

cDepartment of Entomology and Nematology and UCD Comprehensive Cancer Center, 9

University of California, Davis, CA, 95616, United States 10

*Corresponding Author: Tel./Fax: +86-898-66193581, E-mail: xliu@hainanu.edu.cn (X.L.) 11

1These authors contributed equally to this work 12

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2

Abstract 14

Ochratoxin A (OTA) is a major concern for public health and the rapid detection of trace 15

OTA in food is always a challenge. To minimize OTA exposure to consumers, a nanobody (Nb)-16

mediated förster resonance energy transfer (FRET)-based immunosensor using quantum dots 17

(Nb-FRET immunosensor) was proposed for ultrasensitive, single-step and competitive detection 18

of OTA in agro-products at present work. QDs of two sizes were covalently labeled with OTA 19

and Nb, acting as the energy donor and acceptor, respectively. The free OTA competed with the 20

donor to bind to acceptor, thus the FRET efficiency increased with the decrease of OTA 21

concentration. The single-step assay could be finished in 5 min with a limit of detection of 5 22

pg/mL, which was attributed to the small size of Nb for shortening the effective FRET distance 23

and improving the FRET efficiency. The Nb-FRET immunosensor exhibited high selectivity for 24

OTA. Moreover, acceptable accuracy and precision were obtained in the analysis of cereals and 25

confirmed by the liquid chromatography-tandem mass spectrometry. Thus the developed Nb-26

FRET immunosensor was demonstrated to be an efficient tool for ultrasensitive and rapid 27

detection of OTA in cereals and provides a detection model for other toxic small molecules in 28

food and environment. 29

Keywords 30

Nanobody; quantum dots; immunosensor; förster resonance energy transfer; mycotoxin 31

32

3

1. Introduction 33

Ochratoxin A (OTA) is a mycotoxin mainly produced by Aspergillus and Penicillium 34

species through secondary metabolism (Binder, 2007). OTA has been confirmed to be 35

nephrotoxic, hepatotoxic, carcinogenic and teratogenic for animals (Damiano et al., 2018; Shin et 36

al., 2019; Stoev, 2010; Woo et al., 2012). The International Agency for Research on Cancer 37

cataloged OTA in group 2B as a human possible carcinogen (IARC, 1993). OTA is a major 38

concern around the world not only for its severe threat on human health but also for the 39

economic losses from contaminated agro-products. The occurrence of OTA is widespread in 40

coffee, beans, meats, milk, eggs, grapes, cereals, and cereal products, which could be harmful to 41

consumers for its toxic effects (Heussner and Bingle, 2015). The maximum permitted levels of 42

OTA in food have been defined by nations and organizations to ensure public health and safety. 43

Typically, the European Union has set the maximum residue limit of OTA as 10 μg/kg in soluble 44

coffee, 2 μg/kg in wine, 0.5 μg/kg in baby food, 5 μg/kg in cereal, and 3 μg/kg in cereal products, 45

respectively (Commission of the European Communities, 2006). Therefore, it is essential to 46

develop highly sensitive methods for assisting regulation and minimizing exposure to OTA. 47

Chromatography methods are routinely used for the detection of OTA because of high 48

sensitivity and reliability, such as high-performance liquid chromatography, liquid 49

chromatography-mass spectrometry, gas chromatography-mass spectrometry, and liquid 50

chromatography-tandem mass spectrometry (LC-MS/MS) (Öncü et al., 2019; Rodríguez-Cabo et 51

al., 2016; Wei et al., 2018; Zhang et al., 2019). These methods are adopted as the standard 52

protocols but not suitable for rapid and on-site analysis of OTA. Immunoassays are selected as 53

the alternative methods to detect OTA for ease of operation, good selectivity, and high-54

throughput screening. Various kinds of immunoassays have been developed for OTA and other 55

4

analytes, such as enzyme-linked immunosorbent assay, immunosensor, fluorescence 56

immunoassay, chemiluminescence immunoassay and bioluminescence immunoassay (Dong et 57

al., 2019; Liu et al., 2017; Pagkali et al., 2018; Pagkali et al., 2017; Ren et al., 2019; Sun et al., 58

2018; Zangheri et al., 2015). Immunosensor integrates advantages of both immunoassay and 59

biosensor, including speediness, high sensitivity and precision, making it a promising tool for 60

food and environment analysis. Heterogeneous immunosensors require repetitive incubation, 61

separation and washing steps, which are error-prone and cause weak binding reactions along 62

with the decrease of sensitivity (Takkinen and Žvirblienė, 2019). In contrast, the homogeneous 63

methods simplify the procedures into mixing of the sample and immune reagents followed by 64

detection, which could shorten assay time, eliminate signal variations from multiple separation 65

and washing steps, and improve detection sensitivity (Ullman, 2013). Various immunosensors 66

have been developed for homogeneous detection of large and small molecules, such as 67

electrochemical immunosensor, luminescent immunosensor, and fluorescent immunosensor 68

(Bhatnagar et al., 2016; Jo et al., 2016; Qian et al., 2017). Förster resonance energy transfer 69

(FRET), referring to the transmission of photoexcitation energy from a donor molecule to a 70

nearby acceptor molecule, is a widely used strategy for homogeneous immunodetection (Masters, 71

2014). Generally, both antibody and antigen molecules are labeled with fluorescence materials 72

serving as the energy donor and acceptor in FRET-based immunosensor, such as organic dyes 73

and gold nanoparticles. In most recent years, quantum dots (QDs) have been attractive as an 74

efficient fluorescent label in biosensors for exceptional brightness, high photostability, ease of 75

modification, size-adjustable spectrum, wide excitation spectrum, and large surface area to 76

volume ratio (Nsibande and Forbes, 2016; Xu et al., 2016; Zhou et al., 2018). QDs have been 77

demonstrated to be simultaneously effective as the donor and acceptor in FRET, and the FRET-78

5

based immunosensors using QDs of two sizes show robust performance on highly sensitive 79

detection of both large and small molecules with an ideal energy transfer efficiency (Chen et al., 80

2006; Vinayaka and Thakur, 2013). Föster’s theory indicates that the energy transfer efficiency 81

inversely correlates to the sixth power of the distance between the donor and acceptor, namely 82

effective FRET distance (Lakowicz, 1999). The monoclonal antibodies (mAbs) are widely 83

adopted in the reported FRET-based immunosensors using QDs for small molecules (Bhatnagar 84

et al., 2016; Xu et al., 2014). Therefore, the substitution of the mAb with a miniaturized antibody 85

could contribute to shorten the effective FRET distance and improve the energy transfer 86

efficiency for higher detection sensitivity. 87

Miniaturized antibodies are generated by reserving the antigen recognition sites of intact 88

antibodies through genetic engineering technology and digestion with papain and pepsin (Nelson, 89

2010). Numerous kinds of miniaturized antibodies have been developed, such as the single-chain 90

variable fragment, antigen-binding fragment (Fab), F(ab )́2 fragment, single-domain antibodies 91

(sdAbs). Among those miniaturized antibodies, the nanobody (Nb), an sdAb from the heavy 92

chain-only antibody (HCAb) devoid of light chains that occurs naturally in Camelidae, has 93

attracted widespread attention (Muyldermans, 2013). The Nb is also known as VHH, which 94

refers to the variable domain of the heavy chain of HCAbs. Compared to the rest mniaturized 95

antibodies, Nb shows characteristics of the nanoscale cylindrical shape (2.5 nm diameter and 4 96

nm height), ease of production by prokaryotic expression system, high tolerance to harsh 97

environment, good water solubility, and ease of gene manipulation (Harmsen and De Haard, 98

2007; Liu et al., 2017; Vincke and Muyldermans, 2012). Numerous Nbs have been produced 99

against various small and large molecules, including mycotoxins, pestcides, foodborne pathogens 100

and other contaminants in food and environment (He et al., 2018; Liu et al., 2014; Qiu et al., 101

6

2018; Tu et al., 2016; Wang et al., 2014; Wang et al., 2015; Wang et al., 2019a; Wang et al., 102

2019b). As the reported smallest antigen-binding domain, the Nb could be an efficient tool for 103

shortening the effective FRET distance (Qiu et al., 2016; Tang et al., 2019). Nevertheless, a 104

FRET-based immunosensor using an Nb for competitive homogeneous detection of OTA and 105

other small molecules has not yet been reported. 106

HCAbs against OTA have been raised in an alpaca and the Nbs have been cloned and 107

identified in our previous work (Liu et al., 2014). Among those Nbs, the Nb28 shows the best 108

performance in sensitivity and affinity and has been applied to various formats of immunoassays 109

(Liu et al., 2017; Sun et al., 2019; Sun et al., 2018; Sun et al., 2017; Tang et al., 2019; Tang et al., 110

2018). In this work, we set about the construction of a FRET-based immunosensor using Nb28 111

and QDs of two sizes for homogeneous and competitive detection of OTA. The optimization of 112

experiment parameters was described in detail. Based on the optimal conditions, the performance 113

of Nb- and mAb-mediated FRET-based immunosensor was compared. Moreover, the 114

constructed immunosensor was applied to the analysis of OTA in cereal samples and validated 115

by the LC-MS/MS method. 116

2. Materials and methods 117

2.1 Materials and instruments 118

N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (NHSS), N,N-119

dimethylformamide (DMF), N-(3-dimethylaminopropyl)-N -́ethylcarbodiimide hydrochloride 120

(EDC), N,N-dicyclohexylcarbodiimide (DCC), glucosamine hydrochloride, and gluconic acid 121

were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Ochratoxin A, ochratoxin 122

C (OTC), and fumonisin B1 (FB1) were obtained from Pribolab (Singapore). Aflatoxin B1 (AFB1) 123

and zearalenone (ZEN) were purchased from Fermentek (Jerusalem, Israel). Ochratoxin B (OTB) 124

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was from Bioaustralis (Smithfield, NSW, Australia). Deoxynivalenol (DON) was from Sigma-125

Aldrich (CA, USA). The 0.45 μm syringe filter was obtained from Xingya Inc. (Shanghai, 126

China). The mouse anti-OTA monoclonal antibody mAb YK232 was obtained from Yikang 127

Biotech Inc. (Haikou, China). The opaque black polystyrene 96-well microtiter plates were 128

purchased from Costar (Corning, USA). Stock solutions (8.01 μM in 50 mM borate buffer, pH 129

8.4) of the carboxylic acid-modified ZnCdSe/ZnS quantum dots with maximum emission 130

wavelength at 625 nm (RQD-COOH) and the amino group-modified ZnCdSe/ZnS quantum dots 131

with maximum emission wavelength at 525 nm (GQD-NH2) were purchased from Jiayuan Tech. 132

Co., Ltd. (Wuhan, China). All other chemicals and organic solvents were of reagent grade or 133

better. 134

Fluorescence spectra and UV–vis spectra were obtained using a spectral scanning 135

multimode reader (Infinite M200 pro, Tecan Ltd., Männedorf, Switzerland). JEM 2100 136

Transmission electron microscope (JEOL Ltd., Tokyo, Japan) was used for the morphology 137

analysis of the Nb-mediated FRET-based immunosensor. Fourier transform infrared 138

spectroscopy (FTIR) spectra were measured using a TENSOR27 TGA-IR spectrometer (Bruker 139

Ltd., Karlsruhe, Germany) within the range of 4000-400 cm-1 (resolution set as 4 cm-1). 140

2.2 Preparation of OTA-labeled GQD conjugates 141

The amino group-modified QDs were covalently coupled to the carboxyl group on OTA by 142

the DCC/NHS method (Xu et al., 2014). Briefly, 100 μL of DMF solution containing 6.4 nmol 143

DCC and 3.2 nmol NHS was mixed with the standard solutions of 0.8, 1.6 and 3.2 nmol OTA, 144

respectively. The mixture was shaken at 30 °C for 120 min to prepare the active esters of OTA. 145

The final solution containing ester-activated OTA was dropwise added to 500 μL of GQD-NH2 146

solution (0.4 μM in 0.2 M pH 7.4 borate buffer). The mixture was vigorously shaken at room 147

8

temperature for 90 min, followed by adding 0.2 μmol ester-activated gluconic acid prepared as 148

described above to block the excess amino groups of GQD for 60 min. The OTA-labeled GQD 149

conjugates (OTA-GQDs) were obtained after twice centrifugation (27000 ×g, 30 min) at 4 °C, 150

and the residual OTA in the supernatant was quantitatively determined by an indirect 151

competitive ELISA as described previously (Liu et al., 2017). The OTA-GQDs were 152

resuspended with 500 μL of borate buffer (0.2 M, pH 7.4) and stored at 4 °C until use. The OTA-153

GQDs and free GQD-NH2 were characterized by UV–vis absorption, fluorescence spectra, TEM, 154

and FTIR. 155

2.3 Preparation of Nb-labeled RQD conjugates 156

The nanobody Nb28 (10 mg/mL) was expressed and purified as described previously (Tang 157

et al., 2019). The Nb28-labeled RQD was prepared using the EDC/NHSS method (Xu et al., 158

2014). Briefly, 2 nmol EDC, 5 nmol NHSS, and 0.2 nmol RQD-COOH were added to 500 μL of 159

borate buffer (0.05 M, pH 5.0) and gently mixed at 30 °C for 30 min. The Nb28 solution was 160

added to the ester-activated RQD solution with pH adjusted to 7.4 and incubated at room 161

temperature for 30 min. After blocking with 0.2 μmol glucosamine hydrochloride by stirring for 162

60 min, the Nb28-labeled RQD conjugates (Nb-RQDs) were separated as described for OTA-163

GQDs. The residual content of Nb28 in the supernatant was determined by a NanoDrop 1000. 164

The collected Nb-RQDs were dissolved in borate buffer (0.2 M, pH 7.4) and stored at 4 °C prior 165

to use. The Nb-RQDs were characterized by UV–vis absorption, fluorescence spectra, TEM, and 166

FTIR by comparison with free RQD-COOH. 167

2.4 Procedure of Nb-mediated FRET-based immunosensor 168

The Nb-mediated FRET-based immunosensor (Nb-FRET immunosensor) was performed as 169

follows. Briefly, 10 μL of OTA-GQDs (donor), 40 μL of Nb-RQDs (acceptor), and 130 μL of 170

9

borate buffer (0.06 M, pH 7.8) containing 20 mM sodium chloride were added into the wells of a 171

black microtiter plate. Then 20 μL of OTA standard solution with various concentrations (0, 172

0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0 ng/mL in borate buffer containing 50% methanol) 173

was added into the wells. After incubation at 37 °C for 10 min, the fluorescence intensity (FI) at 174

530 nm of the mixture caused by FRET between OTA-GQDs and Nb-RQDs was recorded (λex: 175

425 nm). The quantitative analysis of OTA was performed using the standard curve that was 176

constructed by plotting the energy transfer efficiency (E) against the logarithm of OTA 177

concentrations. The E value was calculated according to the equation: E = (FI0-FI)/FI0, where FI0 178

and FI are the fluorescence intensity at 530 nm at the absence and presence of acceptor. 179

2.5 Selectivity of the Nb-mediated FRET-based immunosensor 180

In order to evaluate the selectivity of Nb-FRET immunosensor, cross-reactivities (CRs) of 181

six substitutes of OTA (OTB, OTC, AFB1, ZEN, FB1, and DON) with two concentrations (0.1 182

and 1 ng/mL) were tested. The CR equals to the value of (IC50 of OTA/IC50 of the tested 183

substitutes)] × 100, where the IC50 is defined as the analyte concentration that yields 50% 184

inhibition of QD binding (Guo et al., 2018). 185

2.6 Sample preparation and analysis 186

Four cereal samples (rice, oats, barley, and wheat) were prepared for the Nb-FRET 187

immunosensor as shown below. Briefly, 1.0 g of ground sample was transferred to a 15 mL 188

centrifuge tube containing 5 mL of methanol-borate buffer (1:1, v/v). After ultrasonic extraction 189

for 15 min and centrifugation (8000 g, 4 °C) for 15 min, the separated supernatant was filtered 190

through a 0.45 μm syringe filter. The filtrate was diluted with borate buffer (0.06 M, pH 7.8) 191

containing 20 mM NaCl to yield sample solutions in 5% methanol before the analysis of OTA. 192

To further assess the effectiveness of the developed method, four OTA-contaminated samples 193

10

and one negative wheat sample were detected by the Nb-FRET immunosensor, and the results 194

were validated by the LC-MS/MS method listed in the supplementary material (Liu et al., 2015). 195

3. Results and discussion 196

3.1 Characterization of OTA-GQDs and Nb-RQDs 197

Using the DCC/NHS method, OTA was covalently coupled with GQD-NH2 for the 198

preparation of OTA-GQDs. Under various reactant mole ratios of OTA to GQD-NH2 (5:1, 10:1, 199

15:1, and 20:1), the binding rates of OTA were calculated to be 79.73%, 71.24%, 63.31% and 200

66.16%, which are equal to 3.98, 7.12, 9.49 and 13.23 molecules of OTA labeled on per GQD-201

NH2 on average (Table S1). As seen in Fig. 1A, a redshift of the excitonic emission peak of 202

OTA-GQDs was observed from 525 nm to 532 nm by comparison with that of GQD-NH2. 203

Moreover, the fluorescence intensity (λex: 365 nm) of OTA-GQDs at 450 nm increased with the 204

reactant mole ratio of OTA to GQD-NH2, confirming the increasing amount of OTA chemically 205

coupled on GQD-NH2. However, the OTA-GQDs with the reactant mole ratio of 10:1 (OTA-206

GQDs-10) had the highest fluorescence intensity at 530 nm and the intensity decreased as the 207

mole ratio exceeded 10. This could be attributed to the surface effect caused by excessive OTA 208

immobilized on QDs, which may influence the change of its surface states from photoexcitation 209

to photoemission (Hines and Kamat, 2014). FTIR analysis was conducted to validate the 210

immobilization of OTA on the GQD-NH2 (Fig. S1A and Table S2). The FTIR spectrum of 211

GQD-NH2 showed the N─H amide II characteristic peaks at 3436.90 cm-1 which were not 212

observed in the spectrum of OTA-GQDs. In addition, the spectrum of OTA-GQDs exhibited the 213

stretching vibrations of the C─N group at 1105.13 and 1359.72 cm-1 and the bending vibrations 214

of the O─H group at 1438.79 cm-1, respectively. These results indicate the successful 215

immobilization of OTA on GQD-NH2. 216

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The anti-OTA Nb28 with high purity was confirmed by SDS-PAGE (Fig. S2) and subjected 217

to the preparation of Nb-RQDs (Fig. 1B). Compared with the unlabeled RQD-COOH, the 218

fluorescence emission spectra of Nb-RQDs with different input mole ratios of Nb28 to RQD-219

COOH (2:1, 5:1, 10:1, and 20:1) all exhibited a single peak at 630 nm when excited at 525 nm. 220

The coupling efficiencies were 71.07%, 81.69%, 66.12% and 59.06% for each group of Nb-221

RQDs (2:1, 5:1, 10:1 and 20:1). The group with the mole ratio of 5:1 (Nb-RQDs-5) showed the 222

strongest fluorescence intensity at 630 nm in the spectra. The results correspond to 1.42, 4.08, 223

6.61 and 11.81 molecules of Nb28 immobilized on the RQD-COOH (Table S1). FTIR spectra of 224

RQD-COOH and Nb-RQDs were compared (Fig. S1B and Table S3). Both RQD-COOH and 225

Nb-RQDs showed amide bond characteristic peaks at 1643.23 cm-1 (C=O amide I). The free 226

RQD-COOH also exhibited one special absorption band at 1070.42 cm-1 representing the C─O 227

primary alcohol bond. Correspondingly, three peaks were recorded at 1103.20, 1359.72 and 228

1440.72 cm-1, which stand for the stretching vibrations of the C─N group and the bending 229

vibrations of the O─H group, respectively. These results could be attributed to the modified 230

carboxyl group on RQD-COOH and thus demonstrate the successful covalent coupling between 231

Nb28 and RQD-COOH. 232

3.2 Construction of the Nb-FRET immunosensor 233

The conjugation of OTA/Nb to QDs of two sizes made it accessible to act as donor and 234

acceptor for constructing a FRET-based immunosensor. A checkerboard titration was designed 235

to select the optimal mole ratio of OTA to GQD-NH2 and of Nb28 to RQD-COOH (Table S4). 236

Due to the stronger fluorescence intensity at 630 nm of Nb-RQDs-5 (λex: 525 nm), both pairs of 237

OTA-GQDs-5@Nb-RQDs-5 and OTA-GQDs-10@Nb-RQDs-5 exhibited higher energy transfer 238

efficiency of 28.36% and 29.55% than the rest pairs. However, the efficiency of pairs of OTA-239

12

GQDs-15@Nb-RQDs-5 and OTA-GQDs-20@Nb-RQDs-5 was significantly decreased. This 240

could be ascribed to the immobilization of excess OTA molecules on GQD-NH2, which might 241

enhance the hydrophobicity of QDs and thus hinder the specific binding between QDs in FRET 242

through antigen–antibody interaction. Considering the result of titration, the pair of Nb-RQDs-5 243

(acceptor) and OTA-GQDs-10 (donor) was selected for the FRET system. The effective overlap 244

between the fluorescence emission spectrum of donor and the molar extinction spectrum of 245

acceptor is a prerequisite for FRET. As shown in Fig. 2A, the fluorescence emission spectrum of 246

Nb-RQDs-5 overlapped well with the UV-vis absorption spectrum of OTA-GQDs-10. In 247

addition, a good separation of emission spectra of Nb-RQDs-5 and OTA-GQDs-10 was observed. 248

These results ascertain the occurrence of FRET from OTA-GQDs-10 to Nb-RQDs-5. TEM test 249

was performed to confirm that the FRET was caused by the specific recognition of Nb-RQDs-5 250

to OTA-GQDs-10 (Fig. 2B). The mixture of GQD-NH2 and RQD-COOH evenly dispersed in 251

sight, whereas a number of aggregations of two, three and four QDs were observed in the 252

mixture of OTA-GQDs-10 and Nb-RQDs-5. Thus the results indicate that the interaction 253

between Nb and OTA triggered the FRET from OTA-GQDs-10 to Nb-RQDs-5. 254

3.3 Optimization of the Nb-FRET immunosensor 255

To obtain the optimal performance of the Nb-FRET immunosensor, a checkerboard titration 256

for the mole ratio of Nb-RQDs-5 to OTA-GQDs-10 was firstly performed as shown in Table S5. 257

The energy transfer efficiency increased from 23% to 34.49% with the mole ratio increasing 258

from 1:1 to 4:1. In this situation, the higher concentration of Nb-RQDs-5 could facilitate more 259

than one acceptor to react with the donor OTA-GQDs (shown in Fig. 2C) and improve the 260

overall energy transfer efficiency. However, the efficiency slightly decreased to 32.07% with the 261

ratio reaching 5:1. It could be attributed to the excess Nb-RQDs-5 that may trigger an 262

13

unpredictable fluorescence co-quenching phenomenon (Xu et al., 2014). Therefore, the mole 263

ratio of 4:1 between the acceptor and donor was selected for further research. 264

Moreover, various assay conditions (reaction time, pH value, methanol concentration, ionic 265

strength, and surfactant concentration) were optimized and the energy transfer efficiency was 266

used for evaluation criterion. As shown in Fig. 3A, the energy transfer efficiency approached 267

saturation after 5 min of incubation, and no significant variation was observed after further 268

incubation. Thus, 5 min was selected as the optimal incubation time. When solution pH ranged 269

from 6.8 to 7.8, the energy transfer efficiency dramatically increased from 22.33% to a 270

maximum of 36.05%. Nevertheless, it rapidly decreased to 30.36% as the pH slightly increased 271

to 8.0 (Fig. 3B). The results indicate that immunoreaction in the developed FRET system was 272

highly sensitive to pH. Hence the recommended pH in assay buffer was 7.8. Methanol (MeOH) 273

is generally used as the cosolvent of OTA in immunoassay and could influence the antigen-274

antibody interaction. Thus the performance of the Nb-FRET immunosensor under different 275

MeOH concentrations was investigated. The FRET efficiency gradually increased from 30.07% 276

to a maximum of 36.12% as the MeOH concentration did not exceed 5%. While a precipitous 277

decline to 11.44% was observed as the MeOH concentration increased to 30% (Fig. 3C). 278

Therefore, the selected final concentration of MeOH in assay buffer was 5%. To evaluate the 279

effect of ionic strength on the Nb-FRET immunosensor, the borate buffers containing serial 280

concentrations of sodium chloride (0, 10, 20, 50, 100 and 200 mM) were tested (Fig. 3D). The 281

FRET efficiency reached a maximum of 38.28% as the NaCl concentration increased to 20 mM, 282

whereas it significantly decreased with the further enhanced ionic strength. Therefore, 20 mM 283

NaCl was selected as the optimal ionic strength in assay buffer. Furthermore, the assay buffers 284

with various contents of nonionic surfactant Tween 20 were tested to reduce the nonspecific 285

14

adsorption. But the result shown in Fig. 3E indicates that the addition of Tween 20 could 286

significantly increase the standard deviations and reduce the FRET efficiency. To ensure the 287

assay reliability and sensitivity, Tween 20 was not recommended for this proposed method. Thus, 288

the optimal working conditions for the Nb-mediated FRET-based immunosensor were 289

summarized as follows: acceptor and donor with the mole ratio of 4:1 were incubated in the 290

borate buffer (0.06 M, pH 7.8) containing 5% MeOH and 20 mM NaCl for 5 min. 291

3.4 Analytical performance of the Nb-FRET immunosensor 292

Based on the optimal assay conditions, analytical performance of the Nb-FRET 293

immunosensor was evaluated through the fluorescence spectra of the mixture of Nb-RQDs-5, 294

OTA-GQDs-10, and serial concentrations of OTA (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 295

1.0 ng/mL in borate buffer containing 50% MeOH, v/v) (Fig. 4A). The fluorescence intensity at 296

630 nm decreased as the OTA concentration increased. It confirms the competitive binding 297

between OTA-GQDs-10 and OTA to Nb-RQDs-5, which could reduce the FRET efficiency. As 298

shown in Fig. 4B, the energy transfer efficiency exhibited a good linear relationship with the 299

logarithm of OTA concentration ranging from 0.005 to 1 ng/mL (R2 = 0.996). The limit of 300

detection (LOD) of the Nb-FRET immunosensor was calculated to be 5 pg/mL, which is the 301

lowest analyte concentration required to produce a signal greater than the 3-fold standard 302

deviation of the noise level (S/N = 3). As a comparison, the mouse anti-OTA monoclonal 303

antibody (mAb 6H8) was used to prepare the mAb 6H8-labeled RQDs (mAb-RQDs) for 304

replacing the Nb-RQDs-5 (Fig. 4C). The mAb-RQDs with the input mole ratio of 2:1 between 305

mAb 6H8 and RQD-COOH (mAb-RQDs-2) was selected as the acceptor together with the donor 306

OTA-GQDs-10 for mAb-mediated FRET-based immunosensor (mAb-FRET immunosensor). 307

Under the same experimental conditions, the mAb-FRET immunosensor had a linear range of 308

15

0.05-10 ng/mL and an LOD of 50 pg/mL, which is one order of magnitude higher than that of 309

Nb-based system (Fig. 4D). Due to the tiny size of Nb, the utilization of Nb is an effective 310

approach to shorten the effective FRET distance between donor and acceptor and thus increase 311

the energy transfer efficiency and improve the detection limit of immunoassay. The selectivity of 312

the Nb-FRET immunosensor for OTA was evaluated by determining the CR with two structural 313

analogs of OTA (OTB and OTC) and four other cereal mycotoxins (AFB1, ZEN, FB1, and DON). 314

As shown in Table S6, the immunosensor cross-reacted 4.56% with OTB, 30.33% with OTC, 315

and less than 0.5% with AFB1, ZEN, FB1, and DON. These results indicate the good selectivity 316

of the developed immunosensor for OTA. Moreover, an investigation of current immunosensors 317

could provide deeper insight into the analytical performance of various biosensors and thus 318

prove the superiority of this proposed ultrasensitive single-step Nb-FRET immunosensor (Table 319

S7). 320

3.5 Detection of OTA in agro-products and validation 321

Since sample components in homogeneous methods are not removed by a wash step, the 322

sample matrix could cause nonspecific binding and decrease the sensitivity. Thus the evaluation 323

of the sample matrix effect is necessary prior to sample analysis, and the dilution method is 324

generally used to reduce the matrix effect of food in immunoassays. As shown in Fig. S3, the 325

matrix effect could be minimized after 10-, 25-, 40-, and 100-fold dilution of the extract of rice, 326

barley, wheat, and oats, respectively. Accordingly, the final LODs of the developed 327

immunosensor were 0.05 μg/kg in rice, 0.125 μg/kg in barley, 0.2 μg/kg in wheat, and 0.5 μg/kg 328

in oats, respectively. The method sensitivity well meets the maximum limits of OTA in 329

unprocessed cereal (5 μg/kg) and cereal products (3 μg/kg) set by the European Union 330

(Commission of the European Communities, 2006). Based on the optimal dilution factors, the 331

16

spiking and recovery experiments were performed to evaluate the effectiveness of the Nb-FRET 332

immunosensor on cereal sample analysis. The negative cereal samples (rice, barley, wheat, and 333

oats) with three spiking levels of OTA (0.02, 0.2 and 2 μg/kg) were pretreated and tested. As 334

shown in Table S8, the average recovery rate and the relative standard deviation (RSD) of intra-335

assay ranged from 81% to 111% and from 3% to 9.4%, respectively. With respect to inter-assay, 336

the average recovery rate and RSD were in the range of 80−109% and 4.5−9%, respectively. 337

These results indicate the acceptable accuracy and precision of the Nb-FRET immunosensor. To 338

further verify its reliability, nine OTA-contaminated samples (rice-1, -2, -4; oats-1, -2, -3; barley-339

1; wheat-1) and four negative samples (rice-3, oats-4, barley-2, wheat-2) were detected by the 340

immunosensor, and the results were validated by the LC-MS/MS method as shown in Table 1. 341

Thus, the Nb-FRET immunosensor is demonstrated to be applicable for rapid, ultrasensitive and 342

selective detection of OTA in agro-products. 343

4. Conclusions 344

In this work, a novel FRET-based immunosensor using Nb and QDs was developed for 345

quantitative analysis of ultra-trace OTA in cereal. The OTA and anti-OTA Nb were covalently 346

coupled with QDs of two sizes, serving as the energy donor and acceptor, respectively. 347

Compared with the traditional mAb, ease of expression of Nb in the prokaryotic system with 348

high yield could contribute to reducing the cost. Moreover, the extremely small size of Nb made 349

it more suitable for highly sensitive FRET-based immunoassay. Since the effective FRET 350

distance between two QDs could be shortened using Nb, the FRET efficiency was accordingly 351

improved for higher detection sensitivity. Due to the homogeneous detection system, the 352

proposed method was also superior to both Nb- and mAb-based heterogeneous immunoassays 353

for no-wash, single-step analysis with higher sensitivity. Thus, this study demonstrated that the 354

17

Nbs have great application potential in FRET-based biosensing systems for detecting OTA and 355

other small molecules in food and environment. To our knowledge, an Nb-mediated FRET-based 356

immunosensor using QDs of two sizes for homogeneous and competitive detection of OTA and 357

other low-molecular-weight compounds has not been reported yet. 358

Acknowledgements 359

This work was financially supported by the National Natural Science Foundation of China 360

(grant number 31760493 and 31901800), the Natural Science Foundation of Hainan Province 361

(grant number 219QN149), and the Scientific Research Foundation of Hainan University [grant 362

number KYQD1631 and KYQD(ZR)1957]. We thank the approval of Analytical and Testing 363

Center of Hainan University for the TEM and FTIR tests. 364

Declarations of interest: none. 365

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526

26

Table 1. Determination of OTA content in cereal samples. 527

Sample LC-MS/MSa

(μg/kg)

Nb-FRET immunosensor

(μg/kg)

rice-1 2.03 ± 0.09 1.45 ± 0.08

rice-2 1.80 ± 0.17 1.68 ± 0.13

rice-3 NDb ND

rice-4 1.45 ± 0.18 1.58 ± 0.15

oats-1 0.82 ± 0.10 0.89 ± 0.07

oats-2 7.53 ± 0.11 8.63 ± 0.95

oats-3 12.0 ± 0.30 13.8 ± 0.90

oats-4 ND ND

oats-5 1.22 ± 0.04 1.34 ± 0.10

barley-1 1.83 ± 0.14 1.64 ± 0.11

barley-2 ND ND

wheat-1 ND 0.006 ± 0.00006

wheat-2 ND ND

aThe LOD of the LC-MS/MS method is 0.01 ng/mL (Liu et al., 2015). 528

bNot detected. 529

530

27

Figure captions 531

Fig. 1. Fluorescence emission spectra (λex: 425 nm) of different mole ratios (5:1, 10:1, 15:1, 20:1) 532

of OTA to GQD-NH2 (A) and fluorescence emission spectra (λex: 530 nm) of different mole 533

ratios (2:1, 5:1, 10:1, 20:1) of Nb28 to RQD-COOH (B). 534

Fig. 2. (A) The fluorescence emission spectra of OTA-GQD-10 (λex: 425 nm) and Nb-RQD-5 (λex: 535

530 nm) and the UV absorption spectra of OTA-GQD-10. The TEM analysis of the mixture of 536

GQD-NH2 and RQD-COOH (B) and the mixture of OTA-GQDs-10 and Nb-RQDs-5 (C). 537

Fig. 3. The effects of immunoreaction time (A), pH value (B), methanol concentration (C), ionic 538

strength (D), and Tween-20 concentration (E) on the energy transfer efficiency of the Nb-FRET 539

immunosensor. Error bars indicate standard deviations of data from experiments performed in 540

triplicate. 541

Fig. 4. Fluorescence emission spectra of the mixture of OTA-GQD-10 and Nb-RQD-5 with 542

various concentrations of OTA (A), and the plot of the energy transfer efficiency against the 543

logarithm of OTA concentrations (B). Fluorescence emission spectra (λex: 530 nm) of different 544

mole ratios of OTA-specific mAb to RQD-COOH (C) and plot of the energy transfer efficiency 545

against the logarithm of OTA concentrations (D). The fluorescence spectra were recorded with 546

excitation at 425 nm. Inset in: the linear portion of the plot. The error bars indicate standard 547

deviations of data from experiments performed in triplicate. 548

549

28

550

551

552

Fig. 1 553

554

29

555

556

557

Fig. 2 558

559

30

560

561

562

Fig. 3 563

564

31

565

566

Fig. 4 567

1

Graphical Abstract

S-1

Supplementary Material for

Ultrasensitive and rapid detection of ochratoxin A in agro-products

by a nanobody-mediated FRET-based immunosensor

Zongwen Tanga,1, Xing Liua,1,*, Benchao Sua,1, Qi Chena, Hongmei Caoa, Yonghuan

Yuna, Yang Xub, Bruce D. Hammockc

aCollege of Food Science and Engineering, Hainan University, 58 Renmin Avenue,

Haikou 570228, PR China

bState Key Laboratory of Food Science and Technology, Nanchang University, 235

Nanjing East Road, Nanchang, 330047, PR China

cDepartment of Entomology and Nematology and UCD Comprehensive Cancer

Center, University of California, Davis, CA, 95616, United States

*Corresponding Author: Tel./Fax: +86-898-66193581; E-mail: xliu@hainanu.edu.cn

(X.L.)

1These authors contributed equally to this work

S-2

Contents

Table S1………………………………………………………………………….S-3

FTIR analysis…………………………………………………………………….S-4

Figure S1…………………………………………………………………………S-5

Table S2………………………………………………………………………….S-6

Table S3………………………………………………………………………….S-7

Table S4………………………………………………………………………….S-8

Table S5………………………………………………………………………….S-9

Table S6………………………………………………………………………….S-10

Table S7………………………………………………………………………….S-11

Table S8………………………………………………………………………….S-12

Figure S2…………………………………………………………………………S-13

Matrix effect……………………………………………………………………. .S-14

Figure S3…………………………………………………………………………S-15

Procedures of the LC-MS/MS method for OTA………………………………....S-16

References………………………………………………………………………..S-17

S-3

Table S1. Evaluation of binding rate and QDs-conjugates coupling rate by

determination of the supernatant for each sample. Combined with the results of the

QD recovery rates, the average QDs-conjugates coupling ratio was calculated with the

molar concentrations of both components.

Nb-RQDs OTA-GQDs

Reactant ratio (1:x)

Nb binding rate (%)

Coupling ratio (1:x) Reactant

ratio (1:x) OTA binding

rate (%) Coupling ratio (1:x)

2 71.07 1.42 5 79.73 3.98

5 81.69 4.08 10 71.24 7.12

10 66.12 6.61 15 63.31 9.49

20 59.06 11.81 20 66.16 13.23

S-4

FTIR analysis

FTIR spectra were measured using a TENSOR27 TGA-IR spectrometer (Bruker,

Germany) within the range of 4000-400 cm-1 where the resolution was set as 4 cm-1.

The FTIR spectra of free RQD-COOH and Nb-RQDs (Figure S1A) both show amide

bond characteristic peaks at 1643.23 cm-1 (C=O amide I). The free RQD-COOH also

exhibits one special absorption bands at 1070.42 cm-1 stands for the C─O primary

alcohol bond. Correspondingly, three peaks were recorded at 1103.20 cm-1, 1359.72

cm-1 and 1440.72 cm-1, which represent the stretching vibrations of C─N group and

the bending vibrations of O─H group, respectively. The results can be attributed to

the modified carboxyl group and demonstrates the successful covalent coupling

between Nb28 and RQD-COOH.

The FTIR spectrum of free GQD-NH2 shows N─H secondary amide

characteristic peaks at 3436.90 cm-1 which were not observed in the spectrum of

OTA-GQDs. Moreover, the latter spectrum (Figure S1B) exhibits the stretching

vibrations of C─N group and the bending vibrations of O─H group at 1105.13 cm-1,

1359.72 cm-1 and 1438.79 cm-1, respectively. The above observations indicate the

successful immobilization of OTA with the GQD-NH2.

S-5

Figure S1. FTIR spectra of free RQD-COOH and Nb-RQDs (A) and free GQD-NH2

and OTA-GQDs (B).

S-6

Table S2. Identification of notable infrared peaks and bands in FTIR spectrum of

GQD-NH2 before and after bioconjugation.

free GQD-NH2 OTA-GQDs

Bands and peaks (cm-1)

Characteristic groups Bands and peaks

(cm-1) Characteristic

groups

- - 698.18-925.76 C-N bending

1641.30 C=O, amide I 1641.30 C=O, amide I

- - 1105.13 C─N stretching

1359.72 C─N stretching

1438.79 O─H bending

3436.90 N─H, amide II - -

- - 3222.82 N─H stretching

3448.47 N─H stretching

S-7

Table S3. Identification of notable infrared peaks and bands in FTIR spectrum of

RQD-COOH before and after bioconjugation.

free RQD-COOH Nb-RQDs

Bands and peaks (cm-1)

Characteristic groups Bands and peaks

(cm-1) Characteristic

groups

- - 783.04-925.76 C-N bending

1643.23 C=O, amide I 1643.23 C=O, amide I

1070.42 C─O primary alcohol

- -

- - 1103.20 C─N stretching

1359.72 C─N stretching

1440.72 O─H bending

3436.90 N─H secondary amide

- -

- - 3218.96 N─H stretching

3442.69 N─H stretching

S-8

Table S4. Checkerboard titration of OTA-GQDs and Nb-RQDs with various labeling

ratios.

OTA-GQDs Quenching efficiency of combined Nb-RQDs (%)a 2:1 5:1 10:1 20:1

5:1 10.08 ± 0.35 28.36 ± 0.33 26.48 ± 0.35 22.92 ± 0.28 10:1 20.76 ± 0.32 29.55 ± 0.52 24.00 ± 0.24 25.32 ± 0.45 15:1 22.32 ± 0.43 23.48 ± 0.41 25.01 ± 0.37 23.72 ± 0.47 20:1 24.23 ± 0.32 21.77 ± 0.59 21.00 ± 0.48 27.64 ± 0.20

aEach assay was performed in three replicates on the same day.

S-9

Table S5. Quenching efficiency for different reactant ratios between donor and

acceptor.

Mole ratio of acceptor:donor Quenching efficiency (%)a 1:1 23.00 ± 0.38 2:1 27.92 ± 0.55 3:1 31.15 ± 0.35 4:1 34.49 ± 0.52 5:1 32.07 ± 0.27

aEach assay was performed in three replicates on the same day.

S-10

Table S6. Cross-reactivity of the Nb-FRET immunosensor with mycotoxins.

Analyte Chemical structure IC50 (ng/mL) CR (%)

OTA

0.5 100

OTB

12 4.2

OTC

2 25

AFB1

>1200 <0.05

FB1

>1200 <0.05

ZEN

>1200 <0.05

DON

>1200 <0.05

S-11

Table S7. Analytical performance of various biosensors for the detection of OTA.

Protocol LOD Assay time References

Electrochemical immunosensor 39 fg/mL >60 min (Zhang et al., 2018) Colorimetric immunosensor 28 fg/mL 24 h (Ren et al., 2018)

Thermodynamical photoluminescence immunosensor

10 pg/mL 25 min (Viter et al., 2018)

Photoluminescent immunosensor 4.4 pg/mL 30 min (Myndrul et al., 2018) Photoelectrochemical

immunosensor 0.02 pg/mL 6 h (Qileng et al., 2018)

Fluorescent aptasensor 1 ng/mL >12 h (Lu et al., 2017) Fluorescent aptasensor 2.5 pg/mL 60 min (Tian et al., 2018)

Terbium chelated nanoparticle sensor

20 ng/mL Not shown

(Altunbas et al., 2020)

Monolithically integrated optoelectronic biosensor

2 ng/mL 50 min (Pagkali et al., 2017)

Nb-FRET immunosensor 5 pg/mL 5 min This work

S-12

Table S8. Recoveries of OTA from the spiked cereal samples tested by the Nb-FRET

immunosensor.

aEach assay was performed in triplicate on the same day.

bEach assay was performed in triplicate on three days.

Sample Spiking

level (μg/kg)

Intra-assaya Inter-assayb

Recovery ± SD (%)

RSD (%)

Recovery ± SD (%)

RSD (%)

Rice 0.02 111 ± 9.3 8.4 109 ± 7 6.4

0.2 90 ± 2.7 3 92 ± 4.2 4.5

2 89 ± 3.7 4.2 84 ± 6 7.1

Oats 0.02 97 ± 3.1 3.2 94 ± 8 8.5

0.2 89 ± 6.5 7.3 86 ± 5 5.8

2 85 ± 4.8 5.6 83 ± 6 7.2

Barley 0.02 99 ± 7.5 7.6 96 ± 6.3 6.5

0.2 97 ± 5.2 6.9 106 ± 7.4 7

2 86 ± 7.2 8.4 89 ± 6.4 7.2

Wheat 0.02 85 ± 5 5.9 84 ± 6.2 7.4

0.2 87 ± 8.2 9.4 90 ± 7 7.8

2 81 ± 6.4 7.9 80 ± 7.2 9

S-13

Figure S2. SDS-PAGE analysis of the expression and purification of Nb28. M:

Prestained protein ladder; Lane 1: total cellular protein before induction; Lane 2: total

cellular protein after induction; Lane 3: Nb28 purified by nickel-nitilotriacetic acid

sepharose; Lane 4: Nb28 concentrated by ultrafiltration centrifuge tube. Gels were

stained with 0.1% (w/v) coomassie brilliant blue-R250 staining solution

(isopropanol-glacial acetic acid-deionized water, 25:10:65, v/v/v) at room temperature

for 1 h. Destaining were then performed with ultrapure water by microwave heating

for 10 min.

S-14

Matrix effect

The matrix effect is one of the most critical influences on accuracy and sensitivity

of immunoassays for food analysis. The food matrix effect can be eliminated by many

methods, such as dilution method, solid-phase extraction, immune-affinity

purification, and BSA shielding effect. Dilution is the most commonly used method

for minimizing food matrix effect because of the advantages of low cost and ease

operation. An appropriate dilution factor is important to decrease matrix effect. To

evaluate the matrix effect, standard calibration curves using the cereal sample extracts

with different dilution factors were set up and compared with the one using the

optimal reaction buffer. As shown in Figure S3, the cereal matrix effects were

minimized as the cereal extract was diluted 10 times for rice (Figure S3A), 25 times

for barley (Figure S3B), 40 times for wheat (Figure S3C) and 100 times for oats

(Figure S3D), and the standard inhibition curves corresponded well with the one

performed in optimal buffer.

S-15

Figure S3. Plot of the energy transfer efficiency of rice (A), oats (B), barley (C) and

wheat (D) extracts with different dilution factors against the logarithm of OTA

concentrations. The error bars indicate standard deviations of data from experiments

performed in triplicate.

S-16

Procedures of the LC-MS/MS method for OTA

The LC-MS/MS method for the detection of OTA was performed as described

previously (Liu et al., 2015). Briefly, 1 g of ground sample and 4 mL of

acetonitrile-water-acetic acid buffer (v/v/v, 79/20/1) were mixed by vigorous

vortexing and ultrasonication for 20 min. The precipitate was isolated by

centrifugation at 10000×g for 10 min. The supernatant was mixed with equal volume

of acetonitrile-water-acetic acid buffer (v/v/v, 20/79/1). The solution was filtered

through a 0.22 μm filter prior to LC-MS/MS analysis. Briefly, LC analysis was

performed using an LC-MS system equipped with a binary solvent pump, an

autosampler, and a mass detector coupled with an analytical workstation. The

separation was performed on a UPLC BEH C18 column (2.1×100 mm, 1.7 μm,

Waters Corporation, MA, USA). The mobile phase was acetonitrile-acetic acid-water

buffer (v/v/v, 10/0.1/89.9) with a flow rate of 0.25 mL/min. The sample injection

volume was 10 μL with 10 min of running time. The Quattro Premier tandem mass

spectrometer (Waters Corporation, MA, USA) was operated in a positive electrospray

interface (ESI) mode with the below working conditions: cone gas flow, 25 L/h;

desolvation gas flow, 700 L/h; source temperature, 120 °C; desolvation temperature,

350 °C; and capillary, 3.00 kV.

S-17

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