Superhydrophobic Paper-Based Microfluidic Field-Effect Transistor Biosensor Functionalized with Semiconducting Single-Walled Carbon Nanotube and DNAzyme for Hypocalcemia Diagnosis

Hypocalcemia is caused by a sharp decline in blood calcium concentration after dairy cow calving, which can lead to various diseases or even death. It is necessary to develop an inexpensive, easy-to-operate, reliable sensor to diagnose hypocalcemia. The cellulose-paper-based microfluidic field-effect biosensor is promising for point-of-care, but it has poor mechanical strength and a short service life after exposure to an aqueous solution. Octadecyltrichlorosilane (OTS), as a popular organosilane derivative, can improve the hydrophobicity of cellulose paper to overcome the shortage of cellulose paper. In this work, OTS was used to produce the superhydrophobic cellulose paper that enhances the mechanical strength and short service life of MFB, and a microfluidic field-effect biosensor (MFB) with semiconducting single-walled carbon nanotubes (SWNTs) and DNAzyme was then developed for the Ca2+ determination. Pyrene carboxylic acid (PCA) attached to SWNTs through a non-covalent π-π stacking interaction provided a carboxyl group that can bond with an amino group of DNAzyme. Two DNAzymes with different sensitivities were designed by changing the sequence length and cleavage site, which were functionalized with SPFET/SWNTs-PCA to form Dual-MFB, decreasing the interference of impurities in cow blood. After optimizing the detecting parameters, Dual-MFB could determine the Ca2+ concentration in the range of 25 μM to 5 mM, with a detection limit of 10.7 μM. The proposed Dual-MFB was applied to measure Ca2+ concentration in cow blood, which provided a new method to diagnose hypocalcemia after dairy cow calving.