Patterned Biolayers of Protein Antigens for Label-Free Biosensing in Cow Milk Allergy

This paper focuses on creating one-dimensional diffractive grooved structures of antigen proteins on glass substrates for the label-free detection of antibodies to dairy allergens. In particular, the fabrication of protein structures is carried out by combining microcontact printing with physisorption, imines coupling, and thiol-ene click chemistry. The work first sets up these patterning methods and discusses and compares the main aspects involved in them (structure, biolayer thickness, functionality, stability). Homogeneous periodic submicron structures of proteins are created and characterized by diffractive measurements, AFM, FESEM, and fluorescence scanning. Then, this patterning method is applied to proteins involved in cow milk allergy, and the resulting structures are implemented as optical transducers to sense specific immunoglobulins G. In particular, gratings of bovine serum albumin, casein, and β-lactoglobulin are created and assessed, reaching limits of detection in the range of 30–45 ng·mL(−1) of unlabeled antibodies by diffractive biosensing.