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: [email protected] (X.L.) 11
1These authors contributed equally to this work 12
13
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
7
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
11
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: [email protected]
(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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