1. Introduction
Milk is essential for humans and perhaps the most valuable food for infants and elderly people, because of its nutritional and health benefits. That is why the safe consumption of milk is of the utmost importance for the European Union [
1]. In this regard, one of the undesirable metabolites that can be found in milk are mycotoxins. These metabolites are produced by filamentous fungi that can contaminate animal feed, reaching milk produced for human consumption. Previous studies have confirmed that low levels of aflatoxins in the human diet is a risk for health; moreover, between 4.6% to 28.2% of reported hepatocellular carcinoma cases are due to aflatoxins exposure [
2].
Aflatoxins are secondary metabolites produced by the
Aspergillus flavus,
Aspergillus parasiticus and
Aspergillus nomius fungal species [
3]. They are mainly found in cereals and crops which are included in dairy animals’ feed. Aflatoxin B1 (AFB1) is the most prevalent and most toxic of aflatoxins. It is considered the most potent naturally occurring carcinogen with a Group 1 human carcinogen designation by the International Agency on Research on Cancer (IARC) [
3,
4]. In ruminants, after ingesting feed contaminated with AFB1, the AFB1 is transformed in a hydroxylated metabolite in the liver, the Aflatoxin M1 (AFM1), which is excreted in urine and secreted in milk. The AFM1 is a heat stable metabolite and cannot be eliminated or degraded by conventional milk processing procedures (i.e., pasteurization).
AFM1 causes carcinogenicity, mutagenesis, teratogenesis, genotoxicity and immunosuppression, specially targeting the liver and lungs [
4,
5]. Since milk is a daily consumed product in many countries, the risk of exposure to AFM1 is higher than that for AFB1, particularly in children. This public concern has motivated the enforcement of strict regulations by several countries in order to minimize the content of aflatoxins in food. On this matter, the FDA has set a maximum allowed level of 0.5 µg L
−1 of AFM1 in milk [
6], but the European Commission has gone further by establishing a limit of 0.050 µg L
−1 in raw and processed milk and 0.025 µg L
−1 of AFM1 in milk intended for lactating infants [
1]. Consequently, high throughput routine controls for AFM1 in milk become essential.
High-performance liquid chromatography coupled to fluorescence detection (HPLC-FD) [
7,
8,
9] or coupled to mass spectrometry (HPLC-MS) [
10,
11,
12] are currently used as standard methods for aflatoxin quantification. Although these methods allow the multiple quantification of aflatoxins in one experiment, they require extensive sample preparation to eliminate interferences, high-cost equipment and expert operators [
4]. Additionally, the enzyme-linked immunosorbent assay (ELISA) is another method for AFM1 analysis which contributes to several commercial kits. Most ELISAs are specific, rapid and easy to use. However, these kits have limitations including cross-reactivity, lack of good recoverability and matrix interferences [
4].
One of the greatest challenges facing the dairy industry is reducing the high-cost and time-consuming detection of AFM1. In this context, the combination of the high specificity of immunosensors with the growing field of nanotechnology offers great perspectives for detection of AFM1 at trace levels, overcoming some limitations of the abovementioned techniques. In this regard, photoluminiscent semiconductor nanocrystals, known as quantum dots (QDs), have replaced conventional fluorophores as tags in immunoassays [
13,
14,
15] due to their high quantum yield, low photobleaching, high photochemical stability, size tunable emission, broad excitation spectra and easy surface modification [
16,
17]. The important advantages of fluorescence signal detection for bioassays, including a high sensitivity, low detection limits and short determination times are well recognized. However, fluorescence has some drawbacks when used in bioanalytical applications, mainly due to the scattering light and the autofluorescence from the biological media. Additionally, conventional QDs typically used as fluorophores in bioassays may exhibit an eventual toxicity derived from the nanoparticle core elemental composition (e.g., CdSe/CdTe). As an alternative, QDs exhibiting a long-lived photoluminescence (e.g., phosphorescence) and cores with low toxic elements become interesting as photoluminescence labels in immunoassays development. In this context, the use of phosphorescence-type emission as analytical signal allows the elimination of short-lived scattering light and background noise from the biological media, which would result in improved sensitivity and wider dynamic ranges of the nanoparticle-based immunoassay.
According to this, the intended addition of transition metal impurities in semiconductor quantum dots (doped quantum dots) constitutes an interesting approach for tuning quantum dots photoluminescence emission. As previously reported, doping ZnS quantum dots with Mn results in exceptional photoluminescence properties typical of phosphorescent emission, as a longer Stokes shift between excitation and emission and longer luminescent lifetimes in the order of ms [
18,
19]. Longer dopant emission lifetime provides the opportunity to eliminate background fluorescence through time-resolved measurements. In this context, recent advances in controlling the synthesis and capping of such QDs have allowed well characterized, highly luminescent and aqueous-stable Mn-doped ZnS phosphorescent QDs to be obtained [
18,
20,
21,
22]. In fact, several analytical applications have been already successfully developed using Mn-doped QDs as luminescent labels [
23,
24,
25,
26,
27].
Engineered nanostructures are highly valuable for multifunctional purposes such as signal transduction and amplification or molecular recognition. Unfortunately, one of the main limitations of nanoparticle-based techniques is the lack of reproducibility on the preparation of the bioconjugated materials. The morphology of nanomaterials may change from batch to batch and deviations are accumulated during multistep preparation procedures. The assurance of reproducibility requires an exhaustive control of the nanoparticle synthesis and functionalization process at several stages, ensured by using complementary analytical techniques that provide information. The nanoparticle concentration can be obtained by combining Inductively Coupled Plasma Mass Spectrometry (ICP-MS) with X-Ray Diffraction (XRD), the confirmation of bioconjugation effectiveness can be evaluated by using an Asymmetrical Flow Field-Flow Fractionation (AF4), the hydrodynamic radius by Dynamic Light Scattering (DLS) and the morphology of the QD and the antibody QD conjugate can be obtained by High-Resolution Transmission Electron Microscopy (HR-TEM).
In this work, two different immunoassay schemes using Mn-doped QDs (d-QDs) were designed and critically compared for sensitive detection and quantification of AFM1 in bovine milk. One scheme was based on the conjugation of phosphorescent dihydrolipoic acid-capped Mn:ZnS QDs to a derivative of the antigen (AFM1–BSA conjugate). In addition, a direct immunoassay scheme, in which d-QDs were bioconjugated with anti-IgG antibodies, was also designed and investigated. The approach showing the best analytical performance was then implemented in the development of an indirect competitive phosphorescence immunosensor for the quantification of AFM1 in milk samples.
2. Materials and Methods
2.1. Reagents, Solutions and Materials
All experiments were carried out with analytical-grade chemical reagents used as received without further purification. Deionized ultrapure water (18.2 MU/cm) was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). Zinc sulfate heptahydrate, manganese chloride tetrahydrate, l-cysteine hydrochloride monohydrate, and standard solutions of 1000 mg L−1 of Mn, Zn and S were obtained from MERCK (Darmstadt, Germany). Sodium sulfide nonahydrate, sodium hydroxide, lipoic acid, potassium tert-butoxide, N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), TWEEN 20, bovine serum albumin (BSA), aflatoxin M1 BSA conjugate, free aflatoxin M1 and methanol HPLC gradient grade were purchased from Sigma-Aldrich (Schnelldorf, Germany). Rat monoclonal anti-aflatoxin and goat polyclonal anti-rat antibody were obtained from Abcam (Cambridge, UK).
2.2. Instrumentation
QDs photoluminescent spectra were performed with the Varian Cary Eclipse Fluorescence Spectrometer (Agilent, Santa Clara, CA, USA) equipped with a xenon discharge lamp (peak power equivalent to 75 kW), a Czerny–Turner monochromator and photomultiplier tube detector (Model R-298). The emission spectra (1 nm data interval), which presented a maximum emission wavelength at 590 nm, were recorded upon excitation at 290 nm, with a delay time of 0.2 ms and a gate time of 5 ms. Excitation and emission slits were set at 10/10 nm respectively, and the averaging time selected to perform the experiments was 5 ms. A microplate reader accessory was used for phosphorescent immunoassay measurements.
QDs characterization was carried out by simultaneous detection and quantification of the elements constituting the QD core (S, Zn and Mn) using an ICP-MS/MS system (Agilent 8800 ICPQQQ, Tokyo, Japan). For separation and characterization of nanoparticles and their bioconjugates the ICP-MS/MS was coupled on-line to the AF4 system (AF2000, Postnova Analytics, Landsberg, Germany). Separation conditions are summarized in
Table S1 (Electronic Supplementary Material, ESM). Dynamic light scattering spectra were measured by using a NanoZS90 instrument from Malvern Instruments, Houston, TX, USA.
2.3. Procedures
2.3.1. Synthesis of DHLA Capped Mn-Doped ZnS QDs
Mn-doped ZnS QDs were synthesized as previously reported using a Mn:Zn precursors molar ratio of 3% [
18] to ensure optimum luminescent properties. To ensure a proper colloidal stability of the nanoparticles in aqueous and biological media, the surface of the synthesized QDs was modified with dihydrolipoic acid (DHLA), providing not only aqueous stabilization of the nanocrystals but also the capacity to be further bioconjugated to antibodies or biomolecules using conventional chemistry bioconjugation reactions.
The successful formation of the QDs was confirmed by Dynamic light scattering (DLS). A DLS spectrum of the product from the synthesis is provided in
Figure S1 in the ESM. Results showed that product of the synthesis displayed a narrow nanoparticle-size distribution with a hydrodynamic diameter of 11 nm.
2.3.2. Bioconjugation Protocols and Purification
One of the major drawbacks to achieve a routine use of inorganic nanoparticles as photoluminescence tags in immunosensing is, perhaps, the absence of “one bioconjugation reaction fits all”, whereby one can link QDs to any biomolecule by following a reproducible scheme. Here, taking advantage of the surface modification of the QDs with DHLA ligands, a carbodiimide chemistry has been used for creating QDs bioconjugates based on the attachment the carboxyl groups of the QDs surface to amine groups of the antibodies. The bioconjugation reaction was carried out at room temperature with constant stirring for 2 h in 10 mM pH 7.4 phosphate-buffered saline (PBS) containing 0.05% Tween 20. Reaction conditions include the use of 66 nM of the antibodies Ab, 2 µM QDs (to ensure a QDs:Ab molar ratio of 30:1) and × 1500 and × 3000 molar excess EDC and NHS, respectively (the concentration of EDC and NHS were 3 mM and 6 mM respectively). During the first 10 min of stirring, QDs were mixed only with EDC and NHS in order to activate the carboxylic groups. After this initial time, the antibody was added until the completion of the reaction time. Finally, a purification step of the bioconjugate was performed by removing the unconjugated QDs and other excess of reagents by ultracentrifugation using a 100 kDa cut-off centrifugal device (AMICON®, Millipore, Madrid, Spain).
Similarly, a derivative of the AFM1 (AFM1-BSA from Sigma-Aldrich) was used here to facilitate the bioconjugation of the phosphorescent d-QDs to the analyte analog using the carbodiimide-mediated conjugation reaction. The reaction took place ensuring the same experimental conditions as those used for the conjugation of the antibody with the QDs; however, here different concentrations of the reactants were used: 110 nM AFM1-BSA, 3.3 µM QDs and × 1500 and × 3000 molar excess of EDC and NHS (5 mM and 10 mM, respectively). Considering the differences on the molecular weight of the AFM1-BSA and of the AFM1-BSA-QD, the unbound AFM1-BSA and other excess of reagents were removed by ultrafiltration (UHF) using a 100 KDa cut-off membrane filter.
2.3.3. Phosphorescent Immunoassay Formats Assayed
A schematic representation of the different competitive immunoassays here developed and critically compared for AFM1 quantification is presented in
Figure 1.
The phosphorescent QDs-based immunoassay formats assayed for AFM1 determination consisted of a competitive format, because AFM1 is a small molecule, having a single binding site with the antibody. Two different approaches have been investigated: an indirect competitive immunoassay, based on the phosphorescent QDs as tags of a derivative of the antigen (AFM1–BSA conjugate) and a direct competitive immunoassay, in which anti-IgG antibodies were labelled with the d-QDs.
In the indirect competitive assay, the competition was established via the binding between the target molecule (i.e., AFM1 from the sample) and the AFM1-BSA–QDs bioconjugate for the limited binding sites of the anti-AFM1 antibody previously immobilized in a microtiter plate. Here, the phosphorescent signal is inversely proportional to the concentration of AFM1 in the sample (
Figure 1A). In this approach, the wells of the microtiter plates were coated with the anti-AFM1 antibodies (100 µL of 2.5 µg mL
−1 in 10 mM PBS pH 7.4). The antibody solution was incubated at 37 °C for 6 h and then overnight for 10 h at 4 °C. This solution was removed and the plate was blocked with 150 µL of BSA (1%
w/
v). After blocking step, three washings of the wells were performed (3 × 150 µL washing buffer 10 mM PBS of pH = 7.4 containing 0.05% Tween 20).
The competitive assay was then based on an incubation of such microtiter plates with standards or samples containing AFM1 and labelled antigen. In a typical assay 50 µL of the sample containing the AFM1 were mixed with the QD labelled BSA-AFM1 (50 µL of 1 µg mL−1 QD-BSA-AFM1) and the mixture was transferred to a well of the microtiter plate coated with the anti-AFM1 antibody and incubated for 2 h at 37 °C. Then, three washing steps were carried out before the room temperature phosphorescence (RTP) of each well was recorded.
The direct competitive immunoassay was performed by tagging secondary anti-IgG antibodies with the QDs. Here, the wells of the microtiter plates were coated with the derivative of the antigen (BSA-AFM1) and the sample containing AFM1 and anti-AFM1 antibodies (in excess) are added to each of the wells. A competition to bind the antibodies is then established between the free AFM1 from the sample or the immobilized BSA-AFM1. After a washing step to remove the immunocomplex that is not immobilized, the anti-IgG QD-labelled antibody will bind the anti-AFM1 antibody bound to the immobilized BSA-AFM1. The analytical signal will be inversely proportional to the initial AFM1 concentration in the sample (
Figure 1B). Here, wells of a microtiter plate were coated with the BSA-AFM1 conjugate (i.e., 100 µL of 1 µg mL
−1 in 10 mM PBS pH 7.4). The BSA-AFM1 solution was incubated at 37 °C for 6 h and then overnight for 10 h at 4 °C. The solution was removed and the plate was blocked with BSA (150 µL 1%
w/
v) for 1 h at 37 °C. After blocking, three washing steps of the wells were performed before the addition of follow-up solutions (3 × 150 µL washing buffer 10 mM PBS, pH = 7.4 containing 0.05% Tween 20). Then, the sample to be analysed was incubated with anti-AFM1 rat antibody for 2 h at 37 °C (50 µL of sample; 50 µL 2 µg mL
−1 anti-AFM1 rat antibody). A total of 100 µL of this solution was added into each well and incubated for 1 h at 37 °C. A further washing step was performed before adding 100 µL of bioconjugate solution (3 µg mL
−1, in terms of antibody conjugation) which was incubated for 1 h at 37 °C). Finally, after the corresponding washing steps, the room temperature phosphorescence (RTP) of each well was recorded as analytical signal.
Finally, the applicability of the developed immunoassay to real sample analysis was investigated by analyzing a commercial skimmed cow milk spiked with different levels of AFM1 in order to assess the analyte recoveries. Such commercial cow milk was selected due to the high similitude of its matrix to the real bovine milk and the absence of detectable amounts of AFM1.
It should be noted that it was not necessary to perform any sample pretreatment to the milk samples, except for a 10-fold milk dilution with PBS 10 mM, prior to their analysis. For calibration, AFM1 standards diluted in PBS 10 mM were used.
4. Conclusions
A competitive immunosensor for sensitive quantification of food toxins which makes use of the advantages of the phosphorescence detection provided by Mn-doped QDs used as antibodies tags is here proposed.
The comparison of the analytical parameters derived from the doses–response curves obtained for the two immunoassays configurations assayed (direct and indirect formats) revealed a better detection limit for the format based on the conjugation of the water-stabilized phosphorescent QDs to a secondary anti-IgG antibody. Such direct competitive immunoassay was successfully developed and exhibited good analytical features for the sensitive quantification of AFM1 in milk, being a highly valuable alternative to conventional techniques for the quantification of aflatoxin M1 in milk.
The proposed immunoassay is an environmentally friendly, robust, quick and low-cost methodology. The calibration statistics and validation of this procedure with skimmed milk samples have demonstrated its compliance with the EU legislation limits for the maximum allowed AFM1 concentration in milk products. Moreover, the simplicity of the sample pre-treatment of the raw milk allows the proposed procedure to be carried out on-site by using portable luminescence instruments for collecting the final RTP signal.
In brief, we have demonstrated the feasibility of the use of Mn:ZnS QDs as highly valuable phosphorescent labels in the development of a quantitative immunoassay for sensitive AFM1 detection in milk samples (a demand of high interest in food quality control). In addition, it is important to point out that it was not necessary to perform any complex sample pre-treatment and an aqueous sample dilution is only required for the success of the quantification.
It is envisaged that the competitive phosphorescent immunoassays format developed here have wide potential applicability in different areas such as bioscience, food analysis, clinical settings, etc., just by selecting an adequate selective receptor specific of the desired analyte, making it possible to expand the applicability to other relevant problems. Although in the present study AFM1 was used as a target analyte, the approach here developed (based on the labelling of a secondary antibody with the phosphorescent QDs) can be directly translated for detection or quantification of any other environmental, clinical or food relevant small molecules by just selecting an appropriate capture primary antibody.