domain: chim phys sde sdv

text:
Hui-Jun Fu

Lin Luo

Yu Wang

Cheng-Long Wang

Hong Wang

Yu-Dong Shen

Hong-Tao Lei

Niko Hildebrandt
email: niko.hildebrandt@univ-rouen.fr

Zhen-Lin Xu

Enzyme-Induced Silver Deposition on Gold Nanorods for Naked-Eye and Smartphone Detection of Acrylamide in Food

Keywords: acrylamide, multicolor immunoassay, nanoparticles, semi-quantitative

Acrylamide is a probable human carcinogen with wide occurrence in commonly consumed food. Owing to the lack of specific antibodies against acrylamide, immunoassays usually measure acrylamide derivates, which require long derivatization times at elevated temperatures. One exception is xanthyl acrylamide (XAA), which can be derived from acrylamide at room temperature within 30 min. Here, we present the development of a monoclonal antibody against XAA and a competitive enzyme-linked immunosorbent assay (ELISA) based on multicolor detection of gold nanorods (AuNRs). The biotinylated monoclonal antibodies bound streptavidin alkaline phosphatase, which induced XAA concentration-dependent silver deposition on the AuNRs. The resulting blue-shift of the AuNR surface plasmon resonance peak translated into a strong gray-to-orange color change that could be conveniently distinguished by the naked eye or smartphone color detection. The assay showed a broad linear range (0.3 to 17.2 ng/mL), a low limit of detection (0.06 ng/mL), and a 3.6-fold sensitivity improvement compared to conventional tetramethylbenzidine-based ELISA. The analysis of several different food products showed excellent acrylamide concentration recoveries (84% to 102%) and confirmed the applicability as an effective tool for routine monitoring of acrylamide in food. Rapid and simple color analysis demonstrated the strong potential for implementation into direct consumer product testing.

INTRODUCTION

Acrylamide is a chemical compound with 71Da molecular weight and easily generated from baked and fried food during high-temperature treatment (above ~120°C) due to the Maillard reaction. [START_REF] Stadler | Acrylamide from Maillard reaction products[END_REF] Many studies have shown that acrylamide has genotoxicity, [START_REF] Salimi | Mitochondrial, lysosomal and DNA damages induced by acrylamide attenuate by ellagic acid in human lymphocyte[END_REF][START_REF] Paulsson | Induction of micronuclei in mouse and rat by glycidamide, genotoxic metabolite of acrylamide[END_REF] carcinogenicity, [START_REF] Klaunig | Acrylamide Carcinogenicity[END_REF][START_REF] Besaratinia | A review of mechanisms of acrylamide carcinogenicity[END_REF] and neurotoxicity. [START_REF] Yang | Acrylamide Induces Abnormal mtDNA Expression by Causing Mitochondrial ROS Accumulation, Biogenesis, and Dynamics Disorders[END_REF][START_REF] Lopachin | The changing view of acrylamide neurotoxicity[END_REF] In 1994, acrylamide was classified as probable human carcinogen in group 2A by the International Agency for Research on Cancer (IARC). 8 In 2002, the Swedish national food administration and the University of Stockholm announced the wide presence of acrylamide in foods under high-temperature preparation. 9 In 2016, the U.S. Food and Drug Administration (FDA) issued guidelines for acrylamide mitigation, [START_REF]Guidance for Industry Acrylamide in Foods[END_REF] followed by the European Regulation (EU) 2017/2158 to establish both mitigation measures and benchmark levels in food products, [START_REF] Commission | Commission Regulation (EU) 2017/ 2158 of 20 November 2017 establishing mitigation measures and benchmark levels for the reduction of the presence of acrylamide in food[END_REF] such as baby food (< 150 ng/g), coffee (400-800 ng/g), biscuits (300-800 ng/g), and French fries/slices (500-750 ng/g). Although currently no maximum residue level (MRL) for acrylamide in food products has been set, the knowledge of acrylamide levels is essential for determining the effectiveness of acrylamide reduction techniques in order to mitigate the daily exposure.

Therefore, economic, rapid, and portable methods for determining acrylamide contents in foods are highly desirable.

To date, analytical techniques for the quantification of acrylamide in foods are mainly based on chromatography, e.g., high-performance liquid chromatography (HPLC), gas chromatography (GC), or GC/LC coupled with mass spectrometry (GC-MS, LC-MS). [START_REF] Milkovska-Stamenova | GC-MS Method for the Quantitation of Carbohydrate Intermediates in Glycation Systems[END_REF][START_REF] Elahi | Acrylamide in Cookie Samples: Analysis Using an Efficient Co-Derivatization Coupled with Sensitive Microextraction Method Followed by Gas Chromatography-Mass Spectrometry[END_REF] Such methods, are sensitive and selective but also time-consuming, expensive, and require highly trained personnel. Thus, it is difficult to accomplish frequent monitoring of a large number of samples. In contrast, immunoassays are simple, rapid, sensitive, and costeffective techniques, which were also proposed by the FDA to monitor acrylamide in food samples. [START_REF]Guidance for Industry Acrylamide in Foods[END_REF] Studies based on specific antibodies for detecting acrylamide have been reported. [START_REF] Assaat | Production of a polyclonal antibody against acrylamide for immunochromatographic detection of acrylamide using strip tests[END_REF][START_REF] Zhu | An indirect competitive enzyme-linked immunosorbent assay for acrylamide detection based on a monoclonal antibody[END_REF] However, only few antibodies can directly recognize acrylamide and the insufficient epitope of the low molecular weight acrylamide strongly limits a reproducible production of specific antibodies and their application for reproducible immunoassays. [START_REF] Wu | Hapten Synthesis and Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Acrylamide in Food Samples[END_REF][START_REF] Zhou | Antigen synthetic strategy and immunoassay development for detection of acrylamide in foods[END_REF][START_REF] Quan | Development of an Enhanced Chemiluminescence ELISA for the Rapid Detection of Acrylamide in Food Products[END_REF] As a consequence, acrylamide immunoassays are mainly based on antibodies against acrylamide derivatives. However, common derivatization reactions, which use 3mercaptobenzoic acid [START_REF] Preston | Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis[END_REF] or 4-mercaptobenzoic acid [START_REF] Zhu | An indirect competitive enzyme-linked immunosorbent assay for acrylamide detection based on a monoclonal antibody[END_REF] result in low efficiency and relatively long reaction times (over 1h) with the necessity of heating (over ~50°C). One exception is xanthyl acrylamide (XAA), which can be derived from acrylamide within only 30 min at room temperature, using xanthydrol as derivatization reagent. Recently, our group obtained a specific polyclonal rabbit antibody against XAA to develop more rapid and simple immunoassays for acrylamide. [START_REF] Luo | Development of an inner filter effect-based fluorescence immunoassay for the detection of acrylamide using 9-xanthydrol derivatization[END_REF] Unfortunately, polyclonal antibodies suffer from strong batch-to-batch differences, which limit their application in reproducible immunoassays, and the development of monoclonal antibodies is highly desirable.

Conventional immunoassays based on colorimetric or luminescence readout are most often based on laboratory operations as well as professional instruments, [START_REF] Fu | A rapid and simple fluorescence enzyme-linked immunosorbent assay for tetrabromobisphenol A in soil samples based on a bifunctional fusion protein[END_REF][START_REF] Fu | A full-automated magnetic particle-based chemiluminescence immunoassay for rapid detection of cortisol in milk[END_REF] which usually provide them with high diagnostic performance but also with shortcomings concerning onsite sample analysis. Naked-eye or smartphone-based analyses are usually less precise but may be sufficient for certain applications and come with the great advantage of simplicity, low cost, and rapidity for on-site monitoring. Gold nanomaterials play an important role in naked-eye detection platforms, [START_REF] Xiong | Highly Sensitive Naked-Eye Assay for Enterovirus 71 Detection Based on Catalytic Nanoparticle Aggregation and Immunomagnetic Amplification[END_REF][START_REF] Huang | Rapid detection of dibutyl phthalate in liquor by a semi-quantitative multicolor immunosensor with naked eyes as readout[END_REF][START_REF] Peng | Naked-eye" recognition: Emerging gold nano-family for visual sensing[END_REF][START_REF] Liu | A concentration-dependent multicolor conversion strategy for ultrasensitive colorimetric immunoassay with the naked eye[END_REF] owing to their unique properties, such as facile synthesis, shape-and size-dependent optical absorption, and high absorption cross sections.

It was shown that the localized surface plasmon resonance (LSPR) absorption of gold nanorods (AuNRs) is extremely sensitive to shape, size and shell composition (such as sliver coating). Multicolor immunoassays were developed for the visual analysis of carcinoembryonic antigen, prostate-specific antigen (PSA), [START_REF] Ma | A universal multicolor immunosensor for semiquantitative visual detection of biomarkers with the naked eyes[END_REF] and HIV-1 p24 antigen. [START_REF] Liu | Microfluidic-Integrated Multicolor Immunosensor for Visual Detection of HIV-1 p24 Antigen with the Naked Eye[END_REF] These assays were based on horseradish peroxidase (HRP)-labeled antibodies to quantitatively etch AuNRs via the generated 3,3′,5,5′-tetramethylbenzidine 2+ (TMB 2+ ).

Etching AuNRs via H2O2 combined with Fe 2+ for decomposing H2O2 to generate strong oxidative hydroxyl radicals (•OH) was applied for constructing multicolor assays for aflatoxin B1, alpha-fetoprotein, and microRNA. [START_REF] Ma | Gold Nanorods as Colorful Chromogenic Substrates for Semiquantitative Detection of Nucleic Acids, Proteins, and Small Molecules with the Naked Eye[END_REF] Enzyme-mediated Ag deposition on AuNRs is another approach for the design of multicolor immunoassays. This method was applied for visual detection of the H5N1 virus and PSA. [START_REF] Xu | Highly Uniform Gold Nanobipyramids for Ultrasensitive Colorimetric Detection of Influenza Virus[END_REF][START_REF] Gao | Enzyme-catalysed deposition of ultrathin silver shells on gold nanorods: a universal and highly efficient signal amplification strategy for translating immunoassay into a litmus-type test[END_REF] Overall, AuNRs modification by etching or deposition could result in significant color variations for eliminating background interference and improving the sensitivity of immunoassay. [START_REF] Liu | A multicolorimetric assay for rapid detection of Listeria monocytogenes based on the etching of gold nanorods[END_REF][START_REF] Fang | Gold nanorods etching-based plasmonic immunoassay for qualitative and quantitative detection of aflatoxin M1 in milk[END_REF][START_REF] Li | Semi-quantitative detection of p-Aminophenol in real samples with colorfully naked-eye assay[END_REF] Herein, with the aim to accomplish a more convenient and sensitive assay for acrylamide, we developed a monoclonal antibody (mAb) against XAA. The mAb was biotinylated (biot-mAb) and applied in a simple naked-eye or smartphone-based immunoassay. The combination of biot-mAb and the quantification via Ag deposition on AuNRs resulted in significant improvements concerning simplicity and sensitivity of acrylamide analysis. The analytical performance was evaluated and the applicability to real-life acrylamide screening was demonstrated by quantification in food samples.

MATERIALS AND METHODS

Materials and reagents.

Acrylamide, ethyl carbamate, methyl carbamate, acrylic acid, methyl acrylate, methacrylaimde, and L (+) ascorbic acid (AsAc) were purchased from Aladdin Chemical Technology Co., Ltd. (Shanghai, China). Hexadecyl trimethyl ammonium bromide (CTAB), sulfo-NHS-biotin, and sodium borohydride (NaBH4) were provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). 2-phospho-Lascorbic acid trisodium salt (AsAcP), streptavidin-alkaline phosphatase (SA-ALP), bovine serum albumin (BSA), and ovalbumin (OVA) were purchased from Sigma-Aldrich (China).

Silver nitrate (AgNO3) and gold (III) chloride trihydrate (99.9%, HAuCl4•4H2O) were provided by Sinopharm Chemical Reagent Co., Ltd. (China). All chemicals and reagents were used in analytical grade. Antibody and antigen were prepared in our lab. The dialysis membrane was provided by Yuanye Bio-Technology Co., Ltd (Shanghai, China). PES filters (0.22 μm) were purchased from Jinteng experimental equipment Co., Ltd (Tianjin, China).

Instruments. SpectraMax i3x multi-function detection platform (Molecular Devices, USA) was used for absorption spectra measurements. The sizes and morphologies of AuNRs were characterized by transmission electron microscopy (TEM, Tecnai G2 F30 S-TWIN, and FEI, USA). The concentration of proteins were quantified by NanoDrop 2000C system (thermo scientific, USA). Ultra-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS, Thermo Fisher, USA) was used for analytical comparison.

Preparation of AuNRs. AuNRs were synthesized using the seed-mediated growth method as reported previously. [START_REF] Ma | A universal multicolor immunosensor for semiquantitative visual detection of biomarkers with the naked eyes[END_REF] Briefly, freshly prepared cold NaBH4 (600 μL, 0.01 mol/L, 4°C) was rapidly added into a solution composed of 0.2 mol/L CTAB (5 mL) and 10 mmol/L HAuCl4 (250 μL). After vigorous stirring for 2 min, the color of the seed solution changed from yellow to brown. Then the mixture was finally stirred gently for 1h at room temperature. For the growth solution preparation, HAuCl 4 (2.5 mL, 0.01 mol/L) and AgNO 3 (400 μL, 0.01 mol/L) were mixed with the CTAB (50 mL, 0.1 mol/L). The AsAc (325 μL, 0.1 mol/L) was slowly dropped into the mixture resulting in the color of solution changing from yellow to colorless. Subsequently, the seed solution (50 µL) was injected into the above growth solution. After vigorously stirring for 30 s, the preparation was incubated at 30°C for 12 h.The obtained AuNRs were centrifuged for 15 min at 5342 g followed by removal of the supernatant. The obtained AuNRs were washed twice using the 50 mL of 0.06 mol/L CTAB solution under the same centrifuge condition. Finally, precipitation was suspended into 10 mL of ultra-pure water and kept at room temperature.

Production and biotinylation of anti-XAA-mAb. Details of production and purification of antigen and anti-XAA-mAb are shown in the Supporting Information. 200 μL of purified anti-XAA-mAb (200 μL, 5 mg/mL) was reacted with sulfo-NHS-biotin (1mg/mL, dissolved in 0.01mol/L PBS), stirring at 4°C for 12 h. Then the mixture was dialyzed in PBS (0.01 mol/L, pH 7.4) for 12 h. Dialysis was performed using a 8 kDa -12 kDa cutoff membrane. Finally, the prepared biot-mAb was kept at 4 °C until use.

Procedure of derivatization and multicolor immunoassay.

As illustrated in Scheme 1, competitive binding of XAA and coating antigen to biot-mAb specific for XAA can result in concentration-dependent streptavidin-alkaline phosphatase binding. AsAcP as the substrate can be hydrolyzed into AsAc, leading to the reduction and then deposition of silver (Ag) on the AuNRs. Modification with Ag on the AuNRs can result in a strong blue shift of the longitudinal LSPR peak and a concomitant color change that strongly depends on the acrylamide concentration. First, coating antigen (100 μL, 1 μg/mL) was incubated into the 96-microwell plate overnight at 37°C. The nonspecific sites were blocked by using 0.01 mol/L PBS containing 3% skimmed milk for 3 h after 2 times washing with PBST.

Derivatizing agent 9-xanthydrol (400 μL, 4 mg/mL) prepared in acetonitrile was mixed with 600 μL of a serial concentration of acrylamide standards. Then HCl (100 μL, 1.5 mol/L) was added to trigger the derivatization. After a 30-min reaction, derivatization was terminated by adding 0.1 mL of NaOH (1.5 mol/L). The mixture was subsequently diluted 5-fold with PBS, which was used as competitor for the following multicolor immunoassay analysis. The biot-mAb (1 μg/mL, 50 μL/well) and an equal volume of analyte in PBS or PBS alone were added into coated wells with 30 min incubation at 37 °C. Afterwards, the wells were washed five times with 0.05% PBST. SA-ALP (100 μL/well) was added to each well for 30 min incubation at 37 °C, followed by five times washing. TBST-containing AsAcP was added (100 μL per well) and incubated for 40-min at 37 °C for forming AsAc.

The mixture (18 μL per well) composed of as-prepared AuNRs (15 μL) and AgNO3 (3 μL) were added into the covered wells and incubated for 10 min at room temperature. The UVvis absorption spectra within the range of 300 nm to 800 nm of the solution were measured.

Sample analysis.

For sample analysis, normal food samples were obtained from a local shop in Guangzhou, China. The samples were analyzed by UPLC-Q-Orbitrap HRMS and the one with the lowest background level of acrylamide was applied for performing spiked sample experiments. For sample preparation for UPLC-Q-Orbitrap HRMS and immunoassay, briefly, crushed fully sample (10 g) was dissolved in 40 mL methanol. After thorough mixing and centrifugation at 2100 g for 5 min, filtered (by 0.22 μm PES filter) supernatant (20 mL) was dried with nitrogen at 25°C. The product was resolved in 5 mL distilled water. In order to remove influence of grease, N-hexane (5 mL) was added subsequently. Finally, take the 0.6 mL of water layer and submit to UPLC-Q-Orbitrap HRMS analysis or derivatization for further immunoassay analysis. The details of UPLC-Q-Orbitrap HRMS analysis are shown in the Supporting Information.

RESULTS AND DISCUSSION

Production and characterization of monoclonal antibodies. To reduce the derivatization time for acrylamide detection, we used xanthydrol as derivatizing agent. The antiserum of mice immunized was collected and characterized by indirect competitive ELISA (ic-ELISA) with two coating antigens using XAA (Supporting Figure S1) after the 4th immunization. While all three antisera displayed a certain affinity for XAA, #3 showed the highest affinity and the inhibition reached 69% in the presence of 100 ng/mL XAA with 295-OVA as coating antigen (Table 1). Thus, the spleen cells of #3 mice and 295-OVA were selected for hybridoma preparation and as coating antigen, respectively. To obtain a mAb with the highest sensitivity, XAA was used as a competitor and the concentration of XAA decreased for each round of subclonal screening. Ultimately, the obtained mAb was purified and identified by 12% SDS-PAGE (Supporting Figure S2). Previous studies have shown that using polyHRP to label streptavidin (SA) for reacting with biotinylated antibodies can effectively amplify the signal and improve the sensitivity of the immunoasasay. [START_REF] Li | Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?[END_REF] Inspired by this approach, the SA-ALP was applied for improving the sensitivity instead of using traditional goat anti-mouse IgG labeled ALP as secondary antibody. Moreover, the small size (244 Da) and high affinity of biotin for SA can result in efficient SA binding without influencing the biological activity of the assay. Biotinylated mAbs were prepared as described in the Materials and Methods section. Results of ciELISA using SA-HRP as tracer indicated that the biotinylation was successful. Feasibility of multicolor immunoassay using ALP. To verify silver deposition on AuNRs and the concomitant color change, we characterized as-prepared AuNRs under different conditions by UV-vis absorption spectroscopy (Figure 1) and TEM (Figure 2). The AuNRs UV-vis spectrum (red curve in Figure 1A) presented two characteristic absorption peaks at ~510 nm (transverse LSPR peak) and ~690 nm (longitudinal LSPR peak).

Negligible absorption in the 400 nm to 800 nm range was observed for Ag + (black curve in Figure 1A) and AsAcP (magenta curve in Figure 1A). Two characteristic absorption peaks of AuNRs did not significantly change when AsAcP and Ag + were introduced separately (yellow and dark green curves in Figure 1A) or simultaneously (cyan curve in thick Ag shell around the AuNRs (Figure 2). Our measurements showed that mainly the diameter of the AuNRs increased, whereas the length was less affected. This effect may be related to less efficient growth on areas with high curvature (at the tip of the AuNRs), which was previously found to reshape the morphology of Au nanostructures. [START_REF] Rodríguez-Lorenzo | Reshaping and LSPR tuning of Au nanostars in the presence of CTAB[END_REF] Overall, the obtained results suggested that ALP could hydrolyze AsAcP into AsAc, which resulted in the reduction of Ag + and the formation of an Ag shell on the AuNRs surface. Therefore, SA-ALP should be suitable for XAA target analysis in combination with biot-mAb. inhibited the formation of the LSPR peak (Figure 3A). Therefore, an AsAcP concentration of 1 mmol/L was used for the immunoassays. The pH value of AsAcP buffer for deposition reaction also significantly impacted ALP activity. Δλ gradually increased with the pH increasing from 7.6 to 9.7 and successively decreased for a pH of 10.4 (Figure 3B). The assay performed best at pH 9.7, as expected from the effective pH value for SA-ALP of 9.7 according to the manufacturer. Higher pH (10.4) most probably resulted in the formation of AgOH, which inhibited the deposition process. Thus, we selected pH 9.7 for the immunoassays. As shown in Figures 3C/D, the optimal Ag + concentration for maximizing

Δλ was found to be 20 mmol/L, whereas the optimal SA-ALP dilution was 300-fold in TBST (5 mmol/L Tris containing 20 mmol/L Mg 2+ , and 0.05% Tween 20, pH 8.0). In summary, 1mmol/L of AsAcP, pH 9.7, 20 mmol/L of Ag + , and 3.3 µg/mL SA-ALP were determined as optimal conditions for the immunoassays. The acrylamide concentration-dependent color changes of the solutions ranged from orange to gray (Figure 4A), which was spectroscopically translated into an acrylamide concentration-dependent significant red-shift and intensity decrease of the longitudinal LSPR peak (Δλ) in the UV-vis absorption spectrum (Figure 4B), as expected from decreasing Ag deposition on the AuNRs. In contrast to AuNR size control via etching (e.g., by the commercial TMB-H2O2 system), [START_REF] Ma | A universal multicolor immunosensor for semiquantitative visual detection of biomarkers with the naked eyes[END_REF][START_REF] Liu | A multicolorimetric assay for rapid detection of Listeria monocytogenes based on the etching of gold nanorods[END_REF] which would lead to increasing intensities with increasing acrylamide concentrations, the high absorption intensities at low concentrations for our deposition approach can significantly increase the signal-to-background ratio and thereby detect lower acrylamide concentrations. [START_REF] Wei | Dual-Modal Split-Type Immunosensor for Sensitive Detection of Microcystin-LR: Enzyme-Induced Photoelectrochemistry and Colorimetry[END_REF][START_REF] Wang | A catalytic-regulated gold nanorods etching process as a receptor with multiple readouts for protein detection[END_REF][START_REF] Tian | Multicolor colorimetric detection of ochratoxin A via structure-switching aptamer and enzyme-induced metallization of gold nanorods[END_REF] For evaluating the sensitivity of the assay, the IC50 (value of acrylamide concentration leading to a 50% decrease in Δλ) of the standard curve was determined. The limit of detection (LOD) was calculated as three standard deviations of the blank control sample (no acrylamide) divided by the slope of the calibration curve. As shown in Figure 4C, the multicolor immunoassay exhibited an IC50 of 1.8 ng/mL, an LOD of 0.06 ng/mL, and a "linear" (with logarithmic concentration) signal decrease range from 0.3 to 17.2 ng/mL (y = -102.9 lgx + 110.1, R 2 = 0.991), whereas the conventional immunoassay (TMB substrate, procedure is described in the Supporting Information) had a 3.6-fold higher IC50 of 6.5 ng/mL, a 12-fold higher LOD (IC10) of 0.73 ng/mL, and a smaller linear range from 1.7 to 25.2 ng/mL.

For a more practical use of the assay (naked-eye analysis), we designed an acrylamide concentration-depended color indicator card (Figure 4D) for being used as on-site screening tool for rapid result analysis by the naked eye. For higher quantitative accuracy, which can be compromised by subjective deviations in human visual discrimination, we also analyzed the RGB color codes of the digital images taken from the different solutions (Supporting Figure S3). Although all three colors (red, green, and blue or R, G, and B)

showed acrylamide concentration-dependent intensity values, using B alone was most suitable for acrylamide quantification. Thus, we implemented the B value in the indicator card (Figure 4D) for more objective and user-friendlier (and also for weak-sighted and color-blind users) smartphone-assisted (using any RGB color value measurement application) acrylamide quantification. The proposed assay could be used to semiquantitatively analyze acrylamide concentrations with the naked eye and/or a smartphone camera by using the color indicator card.

The specificity of the acrylamide immunoassay was evaluated by analyzing the crossreactivity (CR) with five structurally related compounds, namely methyl carbamate, ethyl carbamate, acrylic acid, methyl acrylate, and methacrylaimde, at a concentration of 1 μg/mL after the same derivation reaction and with the derivatization reagent 9-xanthydrol at a concentration of 10 mg/mL. The percentage of CR was calculated using the following equation: CR (%) = [(λrelated_analogues -λ0) / (λacrylamide -λ0)] × 100, where λ0, λacrylamide, and λrelated_analogues are the wavelengths of the longitudinal LSPR peaks of blank control, acrylamide, and related analogues, respectively. As shown in Figure 5A, acrylic acid, methyl acrylate, and methacryl amide showed negligible CR. Acrylic acid and methyl acrylate are lacking the necessary amide group for derivatization with 9-xanthydrol and are therefore not recognized by the mAb. Methacryl amide has the amide group but most probably the additional methyl group resulted in critical steric hindrance that prevented binding to the mAb. 9-xanthydrol showed 4% CR but due to its high concentration of 10 mg/mL, this value can be considered as negligible influence on the analytical performance of the assay. Methyl carbamate and ethyl carbamate have very similar structures compared to acrylamide and the same derivatization strategy can be used for ethyl carbamate immunoassays. [START_REF] Luo | Development of an indirect ELISA for the determination of ethyl carbamate in Chinese rice wine[END_REF][START_REF] Fu | Development of a sensitive non-competitive immunoassay via immunocomplex binding peptide for the determination of ethyl carbamate in wine samples[END_REF] Therefore, it is not surprising that these two compounds show significant CRs of 23% (methyl carbamate) and 16% (ethyl carbamate). Despite the structural similarity, the CRs were still significantly lower compared to acrylamide, which showed the excellent specificity of the mAb for XAA binding. Moreover, methyl carbamate and ethyl carbamate are rarely found in acrylamidecontaining samples, [START_REF] Sen | Methyl carbamate and ethyl carbamate in alcoholic beverages and other fermented foods[END_REF] which means that their CR will not influence the specific determination of acrylamide in the assay. The photos of the different solutions (Figure 5B)

clearly showed that the sample colors of all CR compounds were orange and in strong contrast to the gray acrylamide sample color, which provided further evidence of the low affinity of the biot-mAb to these structurally related compounds and a concomitant strong Ag shell formation on the AuNRs.

To evaluate the reproducibility of the immunoassay, three samples with different concentrations of acrylamide were measured in triplicates by analyzing Δλ and the change of color. The standard deviations were below 7% for all samples, which confirmed the very good reproducibility of our multicolor acrylamide immunoassay (Supporting Table S2).

To highlight some pros and cons of our assay, we compared it to some recently developed acrylamide immunoassays (Table 2). Our multicolor immunoassay exhibited lower LODs than most of the other assays except one study that used polyclonal antibodies for the detection of acrylamide. [START_REF] Wu | Hapten Synthesis and Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Acrylamide in Food Samples[END_REF] However, polyclonal antibodies exhibit significant drawbacks in production, leading to reduced specificity, considerable batch-to-batch differences, and lower assay reproducibility. After careful screening, the mAb prepared in our study can overcome these drawbacks and provide high sensitivity combined with reproducible and reliable quantification of acrylamide. Most assays also require rather sophisticated instruments for acrylamide quantification. The carbon dot-based fluorescence immunoassay showed a calibration curve depending on a narrow mono-fluorescent signal variation range (0.21~6.48 ng/mL) [START_REF] Luo | Development of an inner filter effect-based fluorescence immunoassay for the detection of acrylamide using 9-xanthydrol derivatization[END_REF] and signal measurement relied on a spectrophotometer.

Electrochemical immunosensor [START_REF] Wu | Ultrasensitive immunosensor for acrylamide based on chitosan/SnO2-SiC hollow sphere nanochains/gold nanomaterial as signal amplification[END_REF][START_REF] Lau | A sample pre-treatment free electrochemical immunosensor with negative electro-pulsion for quantitative detection of acrylamide in coffee, cocoa and prune juice[END_REF] require the measurement of electrical signals and also chemiluminescence ELISAs 18 necessitate specific spectrometers for acrylamide detection.

Conventional ELISAs [START_REF] Zhu | An indirect competitive enzyme-linked immunosorbent assay for acrylamide detection based on a monoclonal antibody[END_REF][START_REF] Wu | Hapten Synthesis and Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Acrylamide in Food Samples[END_REF][START_REF] Zhou | Antigen synthetic strategy and immunoassay development for detection of acrylamide in foods[END_REF][START_REF] Preston | Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis[END_REF] using TMB 2+ show simple color change from yellow to colorless, which is more prone to subjective deviations in human visual discrimination compared to our multicolor results for naked-eye or smartphone detection. However, no method is perfect and our multicolor immunoassay also has some shortcomings. Vivid colors produced by the deposition of Ag on AuNRs was implemented by relatively complicated experimental procedures including addition of AuNRs, AsAcP, and AgNO3. In addition, a precise quantitative analysis of multicolor assay requires screening of LSPR peaks, which can be time-consuming and also requires specific instruments. Analysis of commercial food samples. To test the applicability of our immunoassay to quantify acrylamide in actual food products, we selected a food product (instant noodles) with a known acrylamide concentration of 40.8 ng/g and spiked it with acrylamide concentrations of 15, 30, 60, and 120 ng/g. It should be noted that due to pre-processing after sample extraction using a 10-fold dilution, the LOD of the actual sample analysis was increased to 0.6 ng/g and the linear range became 3 to 172 ng/g. However, the analytical performance should still be sufficient and in addition, the dilution can significantly reduce sample matrix effects, which was confirmed by the results of the recovery test. Using the same derivatization conditions as for the standard curve, the obtained recovery was in the range of 84% to 102% with coefficients of variation between 7% and 9% (Table 3).

Importantly, based on the color change of the spiked samples and using the color indicator card, the acrylamide concentration ranges could be obtained by naked-eye and smartphone analyses, which were consistent with the UV-vis analysis results. For going a step further in evaluating the applicability of the assay for food sample analysis, ten different food products were collected randomly from a local market and analyzed by UV-vis spectroscopy, color indication (using the color indicator card), and UPLC-Q-Orbitrap HRMS as standard method. As shown in Table 4, there was an excellent agreement between the different methods, which confirmed the applicability of our multicolor immunoassay for accurate acrylamide analysis in different typical food products.

Notably, even after the 10-fold dilution in our assays, samples 1, 3, 6, 8, and 9 contained acrylamide concentrations that exceeded the upper limit of quantification with UV-vis (172 ng/g). Still these samples could be identified as high acrylamide concentration samples by both UV-vis and color indicator analysis. However, additional dilution would be necessary for a more exact quantification of these highly concentrated samples. Another interesting finding was the relatively low concentration of acrylamide in samples 5 and 7. In fact, the packaging information indicated that the production of these two samples avoided hightemperature frying, such that the generation of acrylamide should also be avoided, as confirmed by our measurements. Although UPLC-Q-Orbitrap HRMS resulted in a significantly more accurate quantification compared to our semiquantitative analysis, it required expensive instrumentation and highly trained personnel. Independent sample analysis mode (8 min/sample) also leads to low efficiency for the screening of a large number of samples via UPLC-Q-Orbitrap HRMS. The proposed multicolor immunoassay with high throughput capability, simplicity, and straightforward readout is a highly complementary method to UPLC-Q-Orbitrap HRMS because it provides the possibility of simple and userfriendly monitoring of acrylamide concentrations in most fried food products with excellent analytical performance and applicability. S3.

CONCLUSIONS

In summary, we successfully designed, characterized, and applied a multicolor immunoassay for the determination of acrylamide in fried food samples. The assay principle was based on specifically developed monoclonal antibodies that recognized xanthyl acrylamide and competitive binding that was used for tuning the thickness of Ag shells on AuNRs via ALP-induced silver deposition. The strong acrylamide concentrationdependent color change resulted in excellent assay performance and a very low detection limit of 0.06 ng/g, which allowed us to use a 10-fold dilution of food samples to suppress sample matrix effects. In addition to quantification via UV-vis spectroscopy, the assay demonstrated rapid, sensitive, and specific quantification of acrylamide in various different food samples by naked-eye or smartphone color detection as verified by UPLC-Q-Orbitrap HRMS. The present assay also possessed very good stability and reproducibility, which provides a powerful and simple screening strategy for risk monitoring and exposure assessment of acrylamide in common food samples.

Table of Contents graphic

Scheme 1 .

 1 Scheme 1. Schematic principle of the multicolor immunoassay for acrylamide analysis. After acrylamide derivatization for 30 min (a), XAA and biot-mAb were incubated for 30 min in antigencoated microwell plates (b). XAA-antibody complexes were washed out (c1) and the remaining biot-mAb was bound to antigen on the plate (c2). After washing, the antigen-antibody complexes could bind with SA-ALP after 30 min of incubation (d). After washing, the wells were incubated with 2phospho-L-ascorbic acid trisodium salt for 40 min, which resulted in the formation of AsAc (e).AuNRs and AgNO3 were added and Ag + was reduced to Ag by AsAc (f), which resulted in the formation of an Ag shell on the AuNRs (g) and an acrylamide concentration-dependent color change (h) caused by the different Ag shell thicknesses.

Figure 1 .

 1 Figure 1. UV-vis spectra (A) and images (B) of solutions containing AuNRs, Ag + , AsAcP, or mixtures thereof. Curves a, d, and e and curves b and c largely overlap and are therefore not very well distinguishable. The shift of the longitudinal peak is indicated by the blue arrow. AsAcP: 1mmol/L; Ag + : 50 mmol/L; SA-ALP: 3 μg/mL; TBST buffer: 5 mmol/L Tris containing 20 mmol/L Mg 2+ , and 0.05% Tween 20, pH 8.0; reaction time: 10 min.

Figure 1A )

 1A Figure 1A) to the AuNRs solution. The color of all AuNRs containing solutions was pink

Figure 2 .

 2 Figure 2. TEM images of AuNRs (A) and AuNRs after Ag deposition (B), HAADF-STEM image (C) and corresponding EDX element mapping images of Au (D), Ag (E), and their overlay (F).

Figure 3 .

 3 Figure 3. UV-vis spectra and images (insets) of assay solutions for different optimization conditions. Effect of AsAcP concentration (A), pH of the detection solution (B), concentration of Ag + (C), and dilution factor of SA-ALP (D). The blue shift of the longitudinal LSPR peaks (Dl) is indicated by the arrows between the dotted lines.

Figure 4 .

 4 Figure 4. Calibration of multicolor immunoassay. A: Images of solutions (with acrylamide concentrations ranging from 0 to 17 ng/mL to be within the linear concentration range of the commercial ELISA) used for calibration (corresponding concentrations are shown in Supporting TableS1). B: UV-vis spectra of selected solutions from A (selected such that their absorption spectra were well distinguishable). C: Assay calibration curves of multicolor (blue) and conventional TMB (red) immunoassay. Error bars show standard deviation (n=3). D: Color indicator card including the blue value range of the RGB image and concentration ranges in ng/mL for naked-eye or smartphone semiquantitative analysis.

Figure 5 .

 5 Figure 5. Specificity evaluation of multicolor immunoassay (A) and corresponding images (B) of solutions. Concentrations of the structurally related compounds was 1 μg/mL. Concentration of 9xanthydrol was 10 mg/mL. Error bars show standard deviations (n = 3)

a

  Standard deviation; b Coefficient of variation; c Average blue (B) value of RGB image obtained from a smartphone (n=3).

Table 1 .

 1 Characterization of antiserum against XAA using homologous and heterologous coating antigens.

			#1 a		#2		#3
	Coating antigen
		Titer b	Inhibition c	Titer	Inhibition	Titer	Inhibition
	(1000 ng/mL)
		(×10 3 )	(%)	(×10 3 )	(%)	(×10 3 )	(%)
	295-OVA	16	43	32	60	64	69
	309-OVA	64	22	128	20	128	34

a #1, the antiserum is from NO.1 mice immunized. b Titer is defined as dilution factor of antiserum with absorbance at 450 nm being from 1.0 to 1.5. c Percentage inhibition was calculated as follow: Inhibition (%) = [1-(B/B0)] × 100. B0 was mean absorbance of the wells with no XAA; B was mean absorbance of the wells with 100 ng/mL XAA as competitor.

Table 2 .

 2 Comparison of available immunoassay methods for acrylamide analysis

	Antibody	Method	Signal output	Sample

Table 3 .

 3 Recovery

analysis of food samples (instant noodles) by multicolor immunoassay (n=3).

Table 4 .

 4 Comparison of the blind analysis results for acrylamide content in food samples by immunoassay (n=3) and UPLC-Q-Orbitrap HRMS. Upper limit of quantitation (172 ng/g); b Corresponding images are shown in Supporting Table

		Immunoassay
	Sample	UV-Vis analysis, Mean ± SD (ng/g)	Indicator analysis (ng/g) b	UPLC-Q-Orbitrap HRMS (ng/g)
	NO.1 (Cookies)	>172 a	>125	959.8
	NO.2 (Cookies)	126.7±1.2	>125	128.0
	NO.3 (Potato chips)	>172	>125	369.6
	NO.4 (Cookies)	72. 5±3. 7	54-125	74.8
	NO.5 (Potato chips)	45.8±1.4	20-54	45.4
	NO.6 (Cookies)	>172	>125	499.3
	NO.7 (Instant noodles)	41.2±1.1	20-54	40.8
	NO.8 (French fries)	>172	>125	406.2
	NO.9 (Cookies)	>172	>125	647.7
	NO.10 (Cookies)	83.0±1.1	54-125	84.5

a

ACKNOWLEDGEMENTS

This work was supported by the National Nature Science Foundation of China (31822039, 31972157), the Special Support Program of Guangdong Province, China (2019TX05N052), the Brain Pool program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (2021H1D3A2A0204958912), Seoul National University, Université Paris-Saclay, Université de Rouen Normandie, INSA Rouen Normandie, CNRS, European Regional Development Fund, Labex SynOrg (ANR-11-LABX-0029), Carnot Institute I2C, XL-Chem graduate school (ANR-18-EURE-0020

XL CHEM), and the Region Normandie. Hui-Jun Fu was supported by a China Scholarship Council (CSC) scholarship to visit Université de Rouen Normandie.

NOTES

The authors declare no conflict of interest.


domain: chim sde sdv

text:
Hui-Jun Fu
email: huijun_foo@163.com

Ruifang Su
email: ruifang.su@univ-rouen.fr

Lin Luo
email: lin.luo@scau.edu.cn

Zi-Jian Chen

Thomas Just Sørensen

Niko Hildebrandt
email: niko.hildebrandt@univ-rouen.fr

Zhen-Lin Xu

Rapid and wash-free time-gated FRET histamine assays using antibodies and aptamers

 Although the European Union, United States Food and Drug Administration (FDA), and China have set regulatory limits for HA in food ranging from 50 to 1000 mg/kg, 9,10 foodborne poisoning associated with HA ingestion has not been

ABSTRACT: Histamine (HA) is an indicator of food freshness and quality. However, high concentrations of HA can cause food poisoning. Simple, rapid, sensitive, and specific quantification can enable efficient screening of HA in food and beverages. However, conventional assays are complicated and time-consuming as they require multiple incubation, washing, and separation steps. Here, we demonstrate that time-gated Förster resonance energy transfer (TG-FRET) between terbium (Tb) complexes and organic dyes can be implemented in both immunosensors and aptasensors for simple HA quantification using a rapid, single-step, mix-and-measure assay format. Both biosensors could quantify HA at concentrations relevant in food poisoning with limits of detection of 0.19 µg/mL and 0.03 µg/mL, respectively. Excellent specificity was documented against the structurally similar food components tryptamine and L-histidine. Direct applicability of the TG-FRET assays was demonstrated by quantifying HA in spiked fish and wine samples with both excellent concentration recovery and agreement with conventional multistep enzymelinked immunosorbent assays (ELISAs). Our results show that the simplicity and rapidity of TG-FRET assays do not compromise sensitivity, specificity, and reliability and both immunosensors and aptasensors have a strong potential for their implementation in advanced food safety screening. KEYWORDS: Aptasensors, Immunoassays, Food testing, Fluorescence, Förster resonance energy transfer eradicated. [START_REF] Colombo | Histamine Food Poisonings: A Systematic Review and Meta-Analysis[END_REF] Thus, to ensure human health and evaluate the quality of food, simple and efficient monitoring of histamine in food and beverage samples is an important challenge for sensor development.

Histamine can be formed from L-histidine (essential amino acid) decarboxylation by microbial activity. Therefore, the specificity of the detection method is paramount.

Historically, the major analytical approach for HA determination in food samples was chromatographic separation, [START_REF] Zhang | Determination of 6 Biogenic Amines in Food Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry without Derivatization[END_REF][START_REF] Kamankesh | Determination of Biogenic Amines in Lighvan Cheese Using a Novel Hollow-Fibre Electromembrane-Microextraction Coupled with Gas Chromatography-Mass Spectrometry[END_REF][START_REF] Chimalakonda | Development and Validation of a Liquid-Chromatography Tandem Mass Spectrometry Method to Determine in Vitro and in Vivo Histamine Release[END_REF][START_REF] Chen | High Throughput Determination of Free Biogenic Monoamines and Their Metabolites in Urine Using Thin-Film Solid Phase Microextraction[END_REF][START_REF] Zhang | Rapid Determination of Histamine Level in Seafood Using Read-out Strips Based on High-Performance Thin Layer Chromatography Modified with Self-Visualization Nanomaterials[END_REF] which is typically connected to high instrumentation costs and the necessity of trained operators. To meet the requirements for a more rapid and lowcost detection within a large number of samples, highly sensitive and specific immunological methods, such as enzyme-linked immunosorbent assays (ELISAs), using specific HA antibodies have been used for HA sensing. [START_REF] Luo | Synthesis of Novel Haptens and Development of an Enzyme-Linked Immunosorbent Assay for Quantification of Histamine in Foods[END_REF][START_REF] Dong | Portable Amperometric Immunosensor for Histamine Detection Using Prussian Blue-Chitosan-Gold Nanoparticle Nanocomposite Films[END_REF][START_REF] Mattsson | Modified Carbon Black as Label in a Colorimetric On-Chip Immunoassay for Histamine[END_REF][START_REF] Yang | A Sensitive Sandwich ELISA Using a Modified Biotin-Streptavidin Amplified System for Histamine Detection in Fish, Prawn and Crab[END_REF][START_REF] Li | Histamine Detection in Fish Samples Based on Indirect Competitive ELISA Method Using Iron-Cobalt Co-Doped Carbon Dots Labeled Histamine Antibody[END_REF][START_REF] Luo | Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine[END_REF] Aptamers are alternative molecular recognition elements, usually composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) oligonucleotides (typically 15-80 nucleotides) that can bind their target via the formation of specific three-dimensional structures in aqueous solution. [START_REF] Xiao | Oligonucleotide Aptamers: Recent Advances in Their Screening, Molecular Conformation and Therapeutic Applications[END_REF] The specificity and affinity of aptamer-target binding can be equivalent or even stronger than for antibodies. [START_REF] Dwidar | Programmable Artificial Cells Using Histamine-Responsive Synthetic Riboswitch[END_REF] Despite the development of advanced antibodies and aptamers, most of these developed assays for HA analysis are heterogeneous, which means that they include several inevitable and time-consuming washing and separation steps. [START_REF] Ho | Generation and Screening of Histamine-Specific Aptamers for Application in a Novel Impedimetric Aptamer-Based Sensor[END_REF][START_REF] Mairal Lerga | High Affinity Aptamer for the Detection of the Biogenic Amine Histamine[END_REF] Only few simple and rapid single-step (homogeneous) assays, such as aptamer-based assays using colorimetry (gold nanoparticle aggregation or dispersion) [START_REF] Mairal Lerga | Gold Nanoparticle Aptamer Assay for the Determination of Histamine in Foodstuffs[END_REF] and fluorescence (quenching or recovery), [START_REF] Dwidar | Development of a Histamine Aptasensor for Food Safety Monitoring[END_REF] have been developed but inevitably suffered from interferences with the sample environment.

Förster resonance energy transfer (FRET) is a strongly donor-acceptor distancedependent energy transfer mechanism that has been widely used for the quantification of biomolecular distances and concentrations via fluorescence biosensing. [START_REF] Fret -Förster | Resonance Energy Transfer: From Theory to Applications[END_REF][START_REF] Algar | FRET as a Biomolecular Research Tool -Understanding Its Potential While Avoiding Pitfalls[END_REF][START_REF] Chen | Recent Advances in Fluorescence Resonance Energy Transfer-Based Probes in Nucleic Acid Diagnosis[END_REF][START_REF] Qiu | A Clinical Role for Förster Resonance Energy Transfer in Molecular Diagnostics of Disease[END_REF] FRET-based immunoassays are usually sandwich assays (binding of donor-antibody and acceptorantibody to the same target results in FRET) or competitive assays (free target competes with donor or acceptor labeled target that is bound to an acceptor or donor labeled antibody) and have the advantage of simpler and quicker wash-free procedures (the binding-related FRET signal is different from other non-specific signals) and more precise ratiometric detection (if both donor and acceptor are fluorescent). [START_REF] Algar | FRET as a Biomolecular Research Tool -Understanding Its Potential While Avoiding Pitfalls[END_REF][START_REF] Qiu | A Clinical Role for Förster Resonance Energy Transfer in Molecular Diagnostics of Disease[END_REF][START_REF]The Immunoassay Handbook[END_REF] When lanthanide-based donors with very long (up to milliseconds) excited state lifetimes are used in FRET, the use of time-gated (TG) detection can efficiently reduce sample autofluorescence and other interfering signals, resulting in highly sensitive homogeneous assay formats. [START_REF] Algar | FRET as a Biomolecular Research Tool -Understanding Its Potential While Avoiding Pitfalls[END_REF][START_REF] Zwier | Time-Gated FRET Detection for Multiplexed Biosensing[END_REF][START_REF] Qiu | Multiplexed Biosensing and Bioimaging Using Lanthanide-Based Time-Gated FRET[END_REF][START_REF] Sy | Lanthanide-Based Luminescence Biolabelling[END_REF] Using terbium (Tb) donors and dye or quantum dot acceptors, we have developed noncompetitive TG-FRET assays against various protein or nucleic acid biomarkers. [START_REF] Qiu | Multiplexed Biosensing and Bioimaging Using Lanthanide-Based Time-Gated FRET[END_REF][START_REF] Xu | Single-Measurement Multiplexed Quantification of MicroRNAs from Human Tissue Using Catalytic Hairpin Assembly and Förster Resonance Energy Transfer[END_REF][START_REF] Dekaliuk | Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer[END_REF][START_REF] Qiu | Advanced MicroRNA-Based Cancer Diagnostics Using Amplified Time-Gated FRET[END_REF][START_REF] Qiu | Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET[END_REF][START_REF] Geißler | Six-Color Time-Resolved Forster Resonance Energy Transfer for Ultrasensitive Multiplexed Biosensing[END_REF] However, implementing TG-FRET into specific, sensitive, rapid, and wash-free competitive assays for small molecules, such as histamine, remains to be demonstrated.

For an efficient translation into different sensing concepts, it would also be highly desirable, if such a homogeneous HA assay could be generically applied to different molecular recognition molecules, such as antibodies and aptamers. Moreover, direct applicability to actual food samples would be extremely important.

Here, we present the development and application of two different competitive homogeneous TG-FRET assays for rapid and simple HA quantification. The first assay is based on the competition between a Tb-donor conjugated antigen and HA, both binding to an antibody (immunoglobulin G, IgG) labeled with dye-acceptors. We show that different dyes can be used to tune the concentration range and sensitivity of the assay. The second assay uses the competition between Tb-cDNA-to-dye-aptamer hybridization and HA-toaptamer binding. The performance of the two assay formats was compared. Both assays showed excellent HA sensing performance and allowed us to perform HA concentration recovery experiments in spiked fish and wine samples. We also performed a comparison to conventional heterogeneous ELISA, which showed that both TG-FRET assays could simply and rapidly quantify HA with high accuracy and reliability in actual food and beverage samples. With these advantages, the homogeneous TG-FRET assays have a strong potential for their implementation into rapid food and beverage quality monitoring.

MATERIALS AND METHODS

Materials. Lumi4-Tb-NHS (Tb-NHS) complexes were provided by Lumiphore, Inc.

(Berkeley, USA) Cyanine5.5 NHS ester (Cya5.5-NHS) and sulfo-Cyanine5.5-NHS ester (sulfoCya5.5-NHS) were purchased from Lumiprobe GmbH (Hannover, Germany). ATTO-655-NHS was purchased from ATTO-TEC GmbH (Siegen, Germany). Cy5.5 (GE Healthcare) was purchased readily conjugated to the RNA aptamer from Eurogentec (Seraing, Belgium). Chemical structures of these FRET donors and acceptors can be found in Supporting Figure S1. Anti-HA monoclonal antibody and antigen (specific hapten with ovalbumin, OVA, as carrier protein) were produced by our lab as previously reported. [START_REF] Luo | Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine[END_REF] HA was purchased from Heowns Biochem Technologies Co. Ltd. (Tianjin, China). The analogues of HA (such as L-histidine and tryptamine), Tris base, phosphate buffered saline (PBS), hydrochloric acid (HCl), sodium hydroxide (NaOH), N,N-dimethylformamide (DMF), sodium bicarbonate (NaHCO3), 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), and bovine serum albumin (BSA) were purchased at Merck/Sigma-Aldrich (St. Quentin Fallavier, France). Cy5.5 conjugated aptamers and complementary chain were synthesized and purified by high-performance liquid chromatography (HPLC) from Eurogentec. Their sequences are shown in Table 1. Gel electrophoresis (Supporting Figure S2) showed only one band between 25 to 50 bp, which confirmed that the aptamer (37 nt) did not form multiple secondary structures under the reaction condition and can be efficiently used for HA binding. High-quality Milli-Q water (18.2 MΩ.cm) was used for preparing solutions. Bioconjugation. The complete antigen (hapten-labeled OVA carrier protein) was used for Tb-NHS bioconjugation because OVA provided accessible amino groups, which was not the case for the hapten. In addition, direct attachment of Tb to the hapten may interfere with antibody binding. 8 nM Lumi4-Tb-NHS was dissolved in DMF and then mixed with the antigen in 100 µL of 100 mM carbonate buffer (pH 9.0) using a 30:1 (330 µM Tb and 11 µM antigen) reaction ratio. The mixture was incubated for 4 h at 25 rpm (rotations per minute) at room temperature using an ELMI Intelli-Mixer (Riga, Latvia). For purification, the sample was washed 4 times with 100 mM Tris-HCl (pH 7.4) using Zeba Spin desalting columns (Thermo Fisher Scientific, France) with 7K molecular weight cut-off (MWCO).

For Tb-complementary chain conjugates, 20-fold molar excess of Tb-NHS was mixed with the amino-functionalized complementary oligo (200 µM Tb and 10 µM DNA) and incubated in 100 µL of 100 mM carbonate buffer (pH 9.0) overnight at 4 °C. The conjugates were purified 3 times with HEPES buffer (100 mM, pH 7.4) using Zeba Spin desalting columns with 7K MWCO. Tb concentrations were determined by absorbance measurements at 340 nm using an extinction coefficient of 26,000 M -1 cm -1 . Antigens were quantified by absorbance measurements at 280 nm using an extinction coefficient of 31,400

M -1 cm -1 (OVA). The oligo concentration was obtained by measuring the absorbance at 260 nm using an extinction coefficient of 124,800 M -1 cm -1 .

For preparation of dye-IgG conjugates, Cya5.5-NHS, ATTO655-NHS, or sulfoCya5.5-NHS were mixed with IgG in 50 µL of 100 mM carbonate buffer (pH 8.0) using a 8:1 (208 µM dye and 26 µM IgG) reaction ratio and incubated for 4h at 25 rpm at room temperature using an ELMI Intelli-Mixer. After the same purification procedure as for Tb conjugation, the conjugation ratios were determined by absorption spectroscopy, using linear combinations of the IgG and dye absorption spectra to best fit the absorption spectra of the IgG-dye conjugates. IgG concentration was determined using an extinction coefficient of 210,000 M -1 cm -1 at 280 nm. Because the dye absorption spectra significantly changed upon IgG conjugation, the original dye spectra (using the peak extinction coefficients provided by the suppliers: 198,000 M -1 cm -1 for Cya5.5; 235,000 M -1 cm -1 for SulfoCya5.5; 125,000 M -1 cm -1 for ATTO655) were corrected, such that the integrals of the original (dye in buffer) and changed (IgG-conjugated dye) spectra were the same (Supporting Figure S3). These corrected extinction coefficient spectra were used for calculating the dye concentrations in the IgG-dye conjugates.

Determination of FRET Parameters. [START_REF] Hildebrandt | How to Apply FRET: From Experimental Design to Data Analysis[END_REF] Förster distances (R0, donor-acceptor distance for which FRET efficiency is 50%) were calculated using the following equations:

R0 = 0.021 (κ 2 ΦD n -4 J) 1/6 nm (1) 𝐽 = ∫ ID (λ)εA(λ)λ 4 dλ 700 nm 475nm (2)
A dipole-dipole orientation factor (κ 2 ) of 2/3 was assumed, as justified by the random orientation of donor and acceptor during the FRET time (dynamic averaging) taking into account the long PL lifetime of the Tb donor and their unpolarized emission. The Tbcentered PL quantum yield (Φ D ) was 0.7±0.1. For aqueous solutions, the refractive index of the medium (n) was taken as 1.35. The overlap integral (J) was calculated by the spectral overlap between the area normalized (to unity) PL spectrum of Tb (the entire spectrum between 475 and 700 nm) [START_REF] Hildebrandt | Luminescent Terbium Complexes: Superior Förster Resonance Energy Transfer Donors for Flexible and Sensitive Multiplexed Biosensing[END_REF] and the molar extinction coefficient (M -1 cm -1 ) spectrum of the dyes from 475 to 700 nm (cf. Figure 1A).

Procedures for TG-FRET assays. Two different homogeneous TG-FRET assays based on IgG and aptamer recognition of HA were developed. In the IgG system, all samples were prepared in 100 mM Tris-HCl containing 0.5% (w/w) BSA buffer.

Measurement mixtures (150 µL) were composed of 50 μL IgG-dye, 50 μL antigen-Tb, and 50 μL HA solutions at different concentrations and incubated at room temperature for 2 h. For the aptamer system, all samples were prepared in HEPES buffer (50 mM HEPES, pH Assay calibration curves were recorded as the relative FRET ratio (Equation 3) against HA concentration. FRET ratios (Equation 4) were calculated from the TG intensities (0.1-0.9 ms) in the acceptor (I A ) and donor (I D ) detection channels and FRET ratio (c = 0) presents the FRET ratio of samples without HA (zero analyte control), whereas FRET ratio (c = x) presents the FRET ratio of samples with a specific concentration (x) of HA.

All samples were measured in triplicate. In cases where error bars are invisible, they were smaller than the data points.

rel. FRET ratio =

FRET ratio (c = x)

FRET ratio (c = 0) (3)
FRET ratio =

" A " D (4)
Preparation of Food Samples. It has been reported that certain seafood is rich in free histidine in the muscle, which can be transformed into HA and lead to HA contamination. [START_REF] Lehane | Histamine Fish Poisoning Revisited[END_REF][START_REF] Tao | A Survey of Histamine Content in Seafood Sold in Markets of Nine Countries[END_REF] In addition, fermented food or beverages, such as wine, can also contain high HA concentrations due to microbial activity. [START_REF] Ordóñez | Evaluation of Biogenic Amines Profile in Opened Wine Bottles: Effect of Storage Conditions[END_REF][START_REF] Esposito | Level of Biogenic Amines in Red and White Wines, Dietary Exposure, and Histamine-Mediated Symptoms upon Wine Ingestion[END_REF] Considering this, we purchased fresh salmon and wine (both supposed to have negligible HA concentrations) from a local supermarket as representative samples to assess the practicality of the developed TG-FRET immunoassays. Samples were prepared as previously reported. [START_REF] Luo | Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine[END_REF] In brief, 2 g of salmon were mixed with 2 mL PBS and homogenized. 8 mL of PBS were added into the homogenate (2 g) and shaken vigorously for 1 min. The mixture was centrifuged at 3000 g for 10 min and the water layer of supernatant was diluted 20-fold in PBS. This solution was then spiked with different concentrations of HA standard, namely 100, 200, and 400 mg per kg of fresh fish for TG-FRET assays and conventional ELISAs. For wine, 1 mL of wine was added into 9 mL PBS and then spiked with different concentrations of HA standard, namely 10, 15, and 20 mg per L of wine, for TG-FRET assays and conventional

ELISAs. Methods for isothermal titration calorimetry (ITC), nucleic acid electrophoresis, and ELISA assays are reported in the Supporting Information.

RESULTS AND DISCUSSION

Characterization of the FRET systems. Acceptor dyes were selected, such that their absorption would significantly overlap with the Tb donor emission and that their PL could be measured beyond the Tb emission spectra (wavelengths larger than 700 nm). In principle, also other lanthanide complexes or nanoparticles (containing trivalent ions of, e.g., Eu, Sm, Dy, or Er) emitting in the visible spectrum could be selected as appropriate donors. [START_REF] Sy | Lanthanide-Based Luminescence Biolabelling[END_REF] However, combining Tb donors with very red (emission beyond 700 nm) dye acceptors was shown to result in very low Tb background PL and strong dye-sensitization. [START_REF] Geißler | Six-Color Time-Resolved Forster Resonance Energy Transfer for Ultrasensitive Multiplexed Biosensing[END_REF] The selection criteria also allowed us to compare different types of organic fluorophores and the variations in the bioconjugates. Four similar dyes Cyanine5.5 (Cya5.5), Sulfo-Cyanine5.5 (sulfoCya5.5), ATTO 655 (ATTO655), and Cy5.5 were used in the Tb-dye donor-acceptor pairs. As shown in Figure 1A, the area-normalized Tb emission spectra (gray) and absorption spectra of the different dye acceptors exhibited good spectral overlap, which resulted in similar overlap integrals (from 475 nm to 700 nm) of J(Tb- Supporting Figure S3). These different dye-bioconjugate conditions led to different environments that caused non-specific adsorption or aggregation of some of the dyes, which, in turn, resulted in spectral broadening and redshift as well as an increase of the lower-wavelength absorption peak compared to the higher-wavelength peak. [START_REF] Chen | FRET-Modulated Multihybrid Nanoparticles for Brightness-Equalized Single-Wavelength Barcoding[END_REF][START_REF] West | The Effects of Solvents and of Solid Substrates on the Visible Molecular Absorption Spectrum of Cyanine Dyes[END_REF][START_REF] Levitus | Cyanine Dyes in Biophysical Research: The Photophysics of Polymethine Fluorescent Dyes in Biomolecular Environments[END_REF] This behavior is very well illustrated by the different Cya5.5 absorption spectra, for which sulfoCya5.5 (green spectrum in Figure 1A) shows less aggregation compared to Cya5.5

Cya5.5) = 2.4×10 15 M -1 cm -1 nm 4 , J(Tb-sulfoCya5.5) = 3.4×10 15 M -1 cm -1 nm 4 , J(Tb- ATTO655) = 2.7×10 15 M -1 cm -1 nm 4 ,
(magenta spectrum in Figure 1A). The emission spectra (Figure 1B) also show differences in the spectral positions of their peaks. However, all of them provided significant PL emission beyond 700 nm to avoid overlap with Tb PL. Ratiometric TG PL detection was performed in two detection channels, namely 494±10 nm for Tb and 716±20 nm for the dyes (bandpass filter transmission spectra are shown in gray in Figure 1B).

TG-FRET immunosensor and aptasensor detection principles. Although TG-

FRET detection is the same for both assay types, the mechanisms of HA quantification using antibodies (Figure 2A) or aptamers (Figure 2B) are significantly different. The immunosensor is used in a competitive immunoassay, in which a Tb-labeled antigen and HA compete for binding sites on dye-labeled IgGs. The aptasensor, which is composed of a dye-labeled aptamer and a Tb-labeled complementary DNA (cDNA), is separated via the specific HA-aptamer interaction, which displaces the cDNA from the aptamer. Both competitive assays result in decreasing FRET (less FRET pairs) with increasing HA concentration because FRET is disrupted via HA-IgG and HA-aptamer binding. Both TG-FRET assays were ratiometric, which means that TG dye acceptor and donor PL intensities

were measured simultaneously and their ratio (rel. FRET ratio, Equation 3) was used for HA quantification. Influence of the type of dye-IgG bioconjugate on immunosensor performance. TG-FRET immunoassays were used to investigate the influence of the specific nature of the antibody-dye conjugate on the assay performance. All dyes were labeled (using the same conditions) via their functional NHS ester groups to free amines on the anti-HA IgG. Bioconjugation of the dyes to the IgG resulted in partial dye adsorption and aggregation as witnessed by the redshifted and broadened absorption spectra (vide supra). SulfoCya5.5 was least affected by IgG-conjugation (no broadening and higher intensity ratio of long-wavelength to short-wavelength absorption peaks -cf. Figure 1A), which indicated that the negatively charged sulfo groups provided a better solubility of the IgG-conjugated dye (and therefore less adsorption and aggregation) in the carbonate buffer used for bioconjugation. Despite the differences, all three IgG-dye conjugates were functional. A comparative assay (Supporting Figure S4 and Table 2), performed at the same IgG concentrations (12.4 nM), antigen concentrations (27.4 nM), and HA concentration range (0 -400 μg/mL), showed that assays with SulfoCya5.5-IgG had a significantly broader detection and concentration range. On the other hand, Cya5.5-IgG provided a lower limit of detection (LOD), which may be related to the lower amount of dyes per IgG or the better overlap of the PL emission with the wavelength range of the detection filter (cf. Figure 2B). Although a more detailed bioconjugation study would be necessary to understand how the different dyes can be used to adjust the assay performance, the different types of dyes clearly resulted in different assay calibration curves, which showed that the choice of acceptor dye and bioconjugation procedure plays an important role in TG-FRET sensor development. In the case of HA quantification, where relatively high concentrations need to be distinguished, the SulfoCya5.5-IgG conjugates with the broadest detection and concentration range are most probably the best choice. Comparison of TG-FRET HA immunosensors and aptasensors. Bioconjugation of the RNA aptamer and c-DNA was quite different from the random labeling of IgG with several dyes because the nucleic acids were functionalized only on their two termini (3' and/or 5'). Normally, this results in significantly less adsorption or aggregation issues and makes donor-acceptor distance control much easier. However, the 1:1 donor-acceptor ratio can also result in less overall FRET signal changes upon target binding, thus, leading to a higher background signal at high target concentrations. This assumption was confirmed by a direct comparison of the HA assays in a 0.03 to 400 µg/mL concentration range using both immunosensors (SulfoCya5.5-IgG with 3.2±1.0 dyes plus Tb-antigen with 1.6±0.3 Tb) and aptasensors (Cy5.5-aptamer with 1 dye and Tb-cDNA with 1 Tb). Both homogeneous TG-FRET assays showed excellent analytical performance over the entire concentration range (Figure 3). The signal change of both TG Tb PL increase and TG dye PL intensity decrease (and therefore also the rel. FRET ratio decrease) over the complete concentration range was significantly stronger for the IgG-based assay, thereby providing a larger detection range and better distinction of higher HA concentrations. At lower concentrations, the aptamer-based assay provided a better FRET ratio distinction and thus, also a lower LOD. To verify if this lower LOD was not caused by differences in HA binding affinity, we determined the dissociation constant of the IgG by isothermal titration calorimetry (ITC) and found a value of KD = 0.53 µM (Supporting Figure S8), which was very close to the value reported for the RNA aptamer used in our study (0.4 µM). [START_REF] Dwidar | Programmable Artificial Cells Using Histamine-Responsive Synthetic Riboswitch[END_REF] Therefore, we assume that the larger size of the antibody-antigen complex, composed of IgG (~150 kDa and 6 nm × 9.5 nm × 15 nm size) [START_REF] Guddat | Three-Dimensional Structure of a Human Immunoglobulin with a Hinge Deletion[END_REF] and OVA (45 kDa, 7 nm × 4.5 nm × 5 nm), 52 requires higher HA concentrations for a significant change in FRET ratio compared to the relatively small aptamer-cDNA complex with a donor-acceptor distance of 13 base pairs, for which already low HA concentrations could result in significant FRET changes. The PL intensities of the PL decay curves (Figure 3) were quite weak (due to the relatively low IgG and aptamer concentrations in the assays) and did not allow for a reliable PL lifetime analysis, which could have been used for measuring the donor-acceptor distances via the FRET spectroscopic ruler. [START_REF] Guo | Conformational Details of Quantum Dot-DNA Resolved by Forster Resonance Energy Transfer Lifetime Nanoruler[END_REF][START_REF] Léger | Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbium-to-Quantum Dot Förster Resonance Energy Transfer[END_REF] However, the shapes of the PL decay curves clearly show a Histamine selectivity of TG-FRET immunosensors and aptasensors. In addition to good sensitivity (target-concentration dependent signal change) in a suitable target concentration range, the specificity of an assay is extremely important. This is especially true when similar components can be found in the sample. In order to evaluate the specificity of both TG-FRET assay for HA, we used tryptamine, as a representative biogenic amine, and histidine, as precursor of histamine, as possibly interfering components. In particular, L-histidine exists at high concentration levels in related foods and its structure is closely related to the one of HA. Using the same conditions for HA, tryptamine, and L-histidine analysis, we found negligible cross-reactivity to L-histidine, and tryptamine (Figure 4), which demonstrated the excellent specificity of both IgG and aptamer based HA assays. Concentration recovery in spiked food samples. Based on their favorable analytical performance and to go a significant step further toward actual food analysis, we challenged our homogeneous TG-FRET assays with HA spiking and concentration recovery experiments in fish and wine samples. Solutions extracted from fresh fish (200fold diluted) and wine samples (10-fold diluted) were spiked with increasing levels of the HA standard (100, 200, and 400 mg per kg of fresh fish and 10, 15, and 20 mg per L of wine). The different samples were then analyzed by both TG-FRET assays and a conventional indirect competitive ELISA for comparison (calibration curves see Figure S9). As shown in Figure 5, all assays could very well quantify the HA concentrations. In the fish samples, IgG-based assays had a mean recovery rate of 103 to 107% and aptamerbased assays of 99 to 103%, with corresponding coefficients of variance (CVs) of 3 to 11% and 7 to 16%, respectively. In wine samples, recovery rates were 95 to 105% for IgG-based assays and 90 to 98% for the aptamer-based tests, with CVs of 6 to 11% and 3 to 12%, respectively. The excellent agreement with the results of the conventional heterogeneous ELISA assay clearly showed that the simplicity and rapidity of our homogeneous TG-FRET assays are combined with high accuracy and reproducibility. The recovery ranges were also in very good agreement to or better than for other IgG or aptamer based assays from previous studies (Table 3). S1.

CONCLUSIONS

In conclusion, we successfully developed and tested two TG-FRET assays for simple and rapid HA quantification using IgGs or aptamers for HA binding. We first investigated the influence of different acceptor dyes on the immunoassay performance and found that sulfonated dyes provided a better solubility and less adsorption and aggregation when conjugated to IgG. Although the SufloCya5.5-IgG immunosensors provided the broadest detection and concentration range, ATTO655-IgG and Cya5.5-IgG, which showed partial adsorption and aggregation when conjugated to IgG, were also functional in the HA assays, and the Cya5.5-IgG based assay even provided the lowest LOD of 0.040±0.005 µg/mL.

Owing to the overall best performance, the SulfoCya5.5-IgG based assay with an LOD of 0.19±0.03 μg/mL was selected for comparison with the Cy5.5-aptamer based assay. The latter provided a less broad detection and concentration range but a lower LOD of 0.030±0.004 μg/mL, most probably due to the very different donor and acceptor conjugates.

Various donors and acceptors in the IgG-antigen system resulted in a donor-acceptor distance distribution, whereas the single donor-acceptor pair in the aptamer-cDNA complex led to a well-defined donor-acceptor distance. Considering that both LODs were well below the regulatory limits for HA in food (50 to 1000 mg/kg), direct applicability to

a

  photoluminescence (PL) emission spectrum of Tb was recorded on FluoTime 300

  7.0, 250 mM NaCl, and 0.1 mM MgCl2, 0.01%Tween 20). Measurement mixtures (150 µL) were composed of 50 μL of Tb-cDNA, 50 µL of dye-aptamer, and 50 µL of HA, heated to 55°C for 20 min, and then incubated at 22°C for 1h. TG-FRET assay and PL decay measurements (performed in black 96-well KRYPTOR microplates -Thermo Fisher Scientific) were performed using 140 μL of the measurement mixtures. TG-FRET assays take 5 s per sample (100 pulses with a repetition rate of 20 Hz). Both IgG and aptamer based assays are single-step assays (no separation or washing steps) that only require a certain incubation time. The total assay times were 120 min for the IgG-based assays and 80 min for the aptamer-based assays.

  and J(Tb-Cy5.5) = 3.4×10 15 M -1 cm -1 nm 4 and Förster distances of R0(Tb-Cya5.5) = 5.5±0.4 nm, R0(Tb-sulfoCya5.5) = 5.9±0.4 nm, R0(Tb-ATTO655) = 5.7±0.4 nm, and R0(Tb-Cy5.5) = 5.9±0.2 nm. The different shapes of the dye absorption spectra resulted from the slightly different types of dyes as well as from the different bioconjugates (the dye spectra shown in Figure 1 correspond to the IgG and aptamer conjugated dyes -comparison with the dye spectra in solution are shown in

Figure 1 .

 1 Figure 1. Absorption and emission spectra of the FRET donor and acceptors. (A) Absorption spectra of Tb (black), Cya5.5 (magenta), sulfoCya5.5 (green), ATTO655 (red), and Cy5.5 (blue). The area-normalized Tb emission spectrum (gray) is shown for visualization of donor-acceptor spectral overlap. (B) PL emission spectra of Tb (black), Cya5.5 (magenta), sulfoCya5.5 (green), ATTO655 (red), and Cy5.5 (blue). Excitation wavelengths were 340±2.5 nm for Tb and 630±2.5 nm for the dyes. Bandpass filter transmission spectra for Tb donor and dye acceptor detection channels

Figure 2 .

 2 Figure 2. TG-FRET assay principles based on competition of Tb-antigen (Tb-AG) and HA for dye-

  multiexponential decay for the IgG-based assays, which confirmed the donor-acceptor distance distribution caused by the multiple and randomly labeled dyes per IgG. The almost monoexponential decay for the aptamer-based assays confirmed the single donor-acceptor pair within the relatively rigid double stranded DNA-RNA hybrid.

Figure 3 . 5 .

 35 Figure 3. Selected PL decay curves of Tb donors and dye acceptors (green: no HA; magenta: 0.39 µg/mL HA; blue: 3.13 µg/mL HA; red: 25 µg/mL HA; black: 400 µg/mL HA) and HA assay calibration curves (showing all measured HA concentrations) of TG-FRET assays based on IgGs and aptamers. Dynamic range minimum and maximum values have an error of 20%. Determination of IC50 and IC10 values is shown in Supporting Figure 5. Donor and acceptor PL decay curves and TG PL intensities of all measured concentrations are shown in Supporting Figures S6 (IgG assays)

  assays) and S7 (aptamer assays).

Figure 4 .

 4 Figure 4. Specificity analysis of related analogues (L-histidine and tryptamine) of TG-FRET assays based on IgGs (A) and aptamers (B). Cross-reactivity (IC50(HA) / IC50(cross-reactive compound)) was below 0.1%. C: Chemical structures of the different compounds.

Figure 5 .

 5 Figure 5. HA concentration recovery results in spiked fish (A) and wine (B) samples using TG-FRET-IgG assays (red), TG-FRET-aptamer assays (green), and conventional ELISA (blue). Gray columns present spiked concentrations. Full data is shown in Supporting TableS1.

  HA quantification in actual food samples was demonstrated by concentration recovery experiments in spiked fish and wine samples. The excellent recovery rates of TG-FRET were in very good agreement with results from conventional heterogeneous ELISA assays but with a much simpler and faster assay format. Whereas the ELISA required i) overnight antigen incubation, ii) two washing steps, iii) blocking buffer incubation for 1h, iv) HA and IgG incubation for 40 min, v) five washing steps, vi) secondary antibody incubation for 40 min, vii) five washing steps, viii) TMB (tetramethylbenzidine) solution incubation for 10 min, and ix) H2SO4 addition to stop the reaction, TG-FRET assays were performed in a single-step format by simply mixing all components, incubating for 120 min (IgG-based assays) or 80 min (aptamer-based assays), and measuring for 5 s. Taking into account the solution-phase format of TG-FRET assays, reduction of the incubation times should be possible to further reduce the overall assay time. In conclusion, our results showed that the important TG-FRET assay advantages of simplicity and rapidity go hand-in-hand with accuracy and reproducibility and that TG-FRET is suitability for the quantification of small contaminants in food with a strong potential for the development of rapid test kits for food and beverage analysis.ASSOCIATED CONTENTSupporting InformationChemical structures of FRET donor and acceptors, gel electrophoresis analysis of aptamers, absorption spectra of pure and IgG-conjugated dyes, HA assay calibration curves for IgG assays with different dyes, PL decay curves and TG PL intensities of Tb donors and dye acceptors for IgG and aptamer based assays, IgG-HA affinity determination by ITC, ELISA

Table 1 .

 1 Sequences of aptamer and complementary chain.

	Name	Sequence (5′→ 3′) a

Table 2 .

 2 Comparison of assay performance using different IgG-dye conjugates.

	Dye	Conjugation ratio (dye/IgG)	Detection range a	Linear range (μg/mL) b	IC50 μg/mL	in	LOD (IC10) in μg/mL
	Cya5.5	2.4±1.2	~0.4	0.10 -1.97	0.45±0.05	0.040±0.005
	ATTO655	4.9±2.0	~0.5	0.39 -5.87	1.52±0.18	0.18±0.03
	SulfoCya5.5	3.2±1.0	~0.8	0.53 -18.2	3.11±0.36	0.19±0.03

a Difference between the highest and lowest relative FRET-ratios in the calibration curves. b Both minimum and maximum values have 20% relative errors.

Table 3 .

 3 Mean recovery rates of different assays for HA analysis in fish and wine.

	Recognition molecule	Assay format	Sample	Recovery	Reference
			fish	83% -95.7%
					17
			red wine	89.1% -110.1%
			fish	97.25% -105%	18
			saury	84.1% -96%
		heterogeneous	red wine	92.5% -106.8%	22
	IgG		fish	98.28% -98.68%	55
			rice wine	91.52% -91.58%
			fish	91.07% -116.5%	21
			fish, prawn and crab	80.19% -108.3%	20
			fish	103.7% -107.2%
		homogeneous			this work
			red wine	94.5% -105.3%
			fish	80% -113%	24
			fish	86.34% -123%	56
	Aptamer	homogeneous
			fish	99.5% -103.1%
					this work
			red wine	89.9% -98.3%

Acknowledgments

We thank Lumiphore, Inc. for the gift of Lumi4 reagents. This work has been supported by China Scholarship Council (CSC), National Nature Science Foundation of China (31822039), National Research Foundation of Korea, Seoul National University, University of Copenhagen, Villum Fonden (grant#14922), Université Paris-Saclay, Université de Rouen Normandie, INSA Rouen, CNRS, Normandie Université, European Regional Development Fund, Labex SynOrg (ANR-11-LABX-0029), Carnot Institute I2C, XL-Chem graduate school (ANR-18-EURE-0020 XL CHEM), and Region Normandie.

HA assay calibration curve, full data of concentration recovery results, and supporting methods.

Conflict of interest

The authors declare no conflict of interest.