Folding-Based Electrochemical Aptasensor for the Determination of β-Lactoglobulin on Poly-L-Lysine Modified Graphite Electrodes

Abstract

Nowadays, food allergy is a very important health issue, causing adverse reactions of the immune system when exposed to different allergens present in food. Because of this, the development of point-of-use devices using miniaturized, user-friendly, and low-cost instrumentation has become of outstanding importance. According to this, electrochemical aptasensors have been demonstrated as useful tools to quantify a broad variety of targets. In this work, we develop a simple methodology for the determination of β-lactoglobulin (β-LG) in food samples using a folding-based electrochemical aptasensor built on poly-L-lysine modified graphite screen-printed electrodes (GSPEs) and an anti-β-lactoglobulin aptamer tagged with methylene blue (MB). This aptamer changes its conformation when the sample contains β-LG, and due to this, the spacing between MB and the electrode surface (and therefore the electron transfer efficiency) also changes. The response of this biosensor was linear for concentrations of β-LG within the range 0.1-10 ng·mL −1 , with a limit of detection of 0.09 ng·mL −1 . The biosensor was satisfactorily employed for the determination of spiked β-LG in real food samples.incidence of the most popular food allergies throughout Europe [13] revealed that allergy to cow's milk is, nowadays, the most frequent food allergy, especially in early-age children, implying a percentage of around 6% in the youngest population [14,15]. Immunoglobulin E (IgE) normally mediates cow's milk allergy, and the symptoms usually occur within 2 h after milk intake [16]. In whey from cow's milk, one of the most important proteins considered to be an allergen is β-lactoglobulin (β-LG), which is classified within the lipocalins, a group of binding proteins with small ligands [17]. Its quaternary structure, identified in 1997 using X-Ray diffraction [18], is dependent on the pH. For example, at pH values between 5.2 and 7.0, it is a dimer, and its molecular weight (MW) is around 37 kDa. However, at pH values over 8.0, it exists as a monomer, and its MW is approximately 18 kDa [19]. This protein has a great potential as milk allergen because it is remarkably stable and it is not present in human milk [20], and, apart from that, it is regularly used as an additive in lots of food products [21].The development of analytical approaches used to detect and quantify food allergens has become increasingly significant [22] because labeling in food is crucial for the consumers, in order to prevent major health problems. The analysis of allergens in food samples [23] is usually carried out by immunoassay-based approaches, such as enzyme-linked immunosorbent assays (ELISAs) [11,14,24] or lateral-flow immunoassays (LFIAs) [25,26], as well as DNA-based methods, which detect the genes encoding for the proteins using amplifying methods, for instance, the polymerase chain reaction (PCR) [27,28]. Proteomic tests have been also used for the determination of allergens [29]. Although these methods are very sensitive, they are time-consuming and the instrumenta...

Recent Advances in Electrochemical Sensing Strategies for Food Allergen Detection

“…Another sensing approach always used to determine β-LB in food samples was designed by the Marrazza group [ 114 ] using a folding-based electrochemical aptasensor based on graphite screen-printed electrodes (GSPEs), modified with AuNPs/poly-L-lysine nanocomposite, and a β-lactoglobulin aptamer labeled with methylene blue (MB) as the redox probe. This aptamer changed its conformation in the presence of β-LB, and, therefore, the space between MB and the electrode surface decreased and the electron transfer was enhanced.…”

Electrochemical Biosensors in Food Safety: Challenges and Perspectives

“… Schematic representation of the sensing strategy of the aptasensor to determine b-lactoglobulin [ 212 ]. …”

“…Milk samples spiked with β-lactoglobulin were analyzed with a recovery range between 80 and 95%. Another sensing methodology always to determine β-lactoglobulin in food samples was designed by the Marrazza group [212] using a folding-based electrochemical aptasensor based on poly-L-lysine modified graphite screen-printed electrodes (GSPEs) and an anti-β-lactoglobulin aptamer tagged with methylene blue (MB). This aptamer changes its conformation when the sample contains β-LG, and this induces changes in the distance between MB and the electrode surface and consequently in the electron-transfer rate, as illustrated in Figure 13.…”

Design Strategies for Electrochemical Aptasensors for Cancer Diagnostic Devices

“…Such nanomaterials as silica and noble metal (Au, Ag, Pt, Pd) nanoparticles (NPs), graphene oxide (GO), and carbon nanotubes and their nanocomposites and nanohybrids, polymers and metal (Zn, Zr, Ce, Hf, Gd, Sn, Mn, Fe) oxides are actively used in the electrochemical aptasensor construction [ 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 ]. Electrochemical nanomaterial-based aptasensors are numerously reported in biomedical research [ 90 , 91 , 92 , 93 ] and may satisfy a huge demand for portable analytical devices with the selectivity and specificity sufficient for healthcare applications, such as POCT of biomarkers for chronic and emerging diseases: cancer, neurodegenerative disorders, cardiovascular diseases and chronic respiratory infections [ 94 ]. The unique properties of the aptamers stimulate the further development of innovative principles of such electrochemical aptasensor operation [ 95 , 96 ], and currently the electrochemical aptasensor-related articles represent ca.…”