Evaluation of different competitive immunoassays for aflatoxin M1 determination in milk samples by means of inductively coupled plasma mass spectrometry

Abstract

12Haptens (i.e. biomolecules which molecular weight is lower than 10 kDa) 13 determination by inductively coupled plasma mass spectrometry (ICP-MS) is 14 usually performed by means of competitive immunoassays. In these 15 immunoassays, analyte quantification is indirectly carried out using two different 16 tracer species (i.e. antibodies or antigen-protein conjugates). However, the 17 benefits (and drawbacks) derived from using a given tracer species have not 18 been systematically investigated so far. The goal of this work is to evaluate the 19 influence of the tracer species employed in competitive immunoassays on the 20 analytical figures of merit for aflatoxin M 1 (AFM 1 ) determination in milk samples. 21To this end, three different strategies have been developed and evaluated, 22 namely: (i) antibody binding inhibition assay (ABIA); (ii) capture inhibition assay 23 (CIA); and (iii) capture bridge inhibition assay (CBIA). Experimental results 24show that the use of the antibody as tracer species (as in the ABIA approach) 25 42 Keywords: hapten, competitive immunoassay, nanoparticle, inductively 43 coupled plasma mass spectrometry, aflatoxin M 1 . 44 45 [1]. The use of ICP-MS in this field offers several attractive features regarding 49 other established approaches: (i) high sensitivity; (ii) specificity; (iii) compound-50 independent detection sensitivity; (iv) multi-element capabilities; (v) robustness; 51 and (vi) easy coupling to separation techniques. Initially, the analysis was 52 limited to those species containing a heteroatom detectable by ICP-MS. 53 However, analytical figures of merit were severely compromised since the 54 heteroatoms, naturally present in biomolecules (e.g. P, S, Se, As, etc.), suffer 55 from low sensitivity and (spectral and non-spectral) interferences due to matrix 56 components in biological samples (e.g. carbon, chloride, etc.) [1,2]. To improve 57 the analytical figures of merit, as well as to address with the determination of 58 non-containing heteroatom biomolecules, different strategies have been 59proposed in the literature. First, the biomolecule can be derivatized through a 60 chemical reaction with a heteroatom or an organometallic compound [3,4]. The 61 main drawback of this approach is the low selectivity of the labelling procedure. 62Alternatively, the analyte of interest can be labelled by means of an 63 immunoreaction using a heteroatom-labelled antibody [5,6]. Because of the 64 great specificity of the antigen-antibody reaction, biomolecules can be 65 successfully determined in complex mixtures. Heteroatoms labels used in 66 immunoassay includes: (i) transition metals [7,8]; (ii) lanthanides [9-11]; (iii) 67 metal nanoparticles [12][13][14]; and (iv) Quantum-Dots [15]. A priori, the use of 68 metal nanoparticles is the most advantageous approach to improve analytical 69 figures of merit (e.g. sensitivity, LoD, etc.) due to the high number of atoms 70 present in each nanoparticle. 71Non-competitive immunoassays have been traditionally employed for the 72 analysis ...

Immunoassays based on inductively coupled plasma mass spectrometry detection: So far so good, so what?

“…A large variety of NPs have been proposed (AgNPs [52][53][54][55], PtNPs [53,56], PbSNPs [57], metal/dye-doped silica NPs [58], Cs-doped magnetic NPs [58,59], UCNPs [30,31], EuPO 4 /LuPO 4 NPs [32], Quantum Dots (QDs) [60][61][62][63][64][65], etc.) but AuNPs [34,[53][54][55]63,[66][67][68][69][70][71][72][73][74][75][76] are usually preferred due to their lower cost and greater commercial availability. When compared to REE-based chelates, the use of inorganic NPs affords some attractive and unique features.…”

“…First, the high number of detectable atoms present in each NP tag is beneficial to improve both sensitivity and LoDs [75]. Pérez et al [54] investigated the influence of Ag and AuNPs of different size on the LoDs for aflatoxin M1 (AFM1) determination by means a competitive immunoassay using the antibody as a tracer specie. It was observed that, when increasing AuNPs size from 40 nm to 80 nm, LoDs in ICP-MS decreased 50-fold (LoD AuNPs 80nm 0.4 ng Kg − 1 ).…”

“…In general, NPs have been mainly employed for single analyte determinations [31,54,56,57,59,60,62,[64][65][66][67][69][70][71][74][75][76]. Unlike REEbased chelates, and despite the vast number of described NPs proposed in the literature for immunoassays, the use of NPs for multiplexed applications has been scarcely exploited [32,53,55,58,63].…”

“…Carboxyl groups are not usually employed for element tag conjugation but for immobilization purposes after an alkaline activation step (e. g. with carbonate-bicarbonate buffer) [16,31,32,48,51,61,65,66,69,74]. On this regard, it is worth to mention that most of the conjugation above-described are not exclusively employed for direct elementtagging but for antibody immobilization on solid supports [33,35,36,49,58,62,63], as well as for the introduction of biotin to make use of the well-known biotin-streptavidin system [16,32,34,48,51,54,61,66,74].…”

“…in human serum samples and cells (Table 2). Nevertheless, they have also been employed for studying manganesedependent protein dynamics of photosystem II in barley [39] and AFM1 levels in milk [54,67]. As regards the analysis of solid samples (Table 3), because traditional workflows for solid-phase immunoassays (e.g.…”