NAGase sensing in 3% milk: FET-based specific and label-free sensing in ultra-small samples of high ionic strength and high concentration of non-specific proteins

Biosensing with biological field-effect transistors (bioFETs) is a promising technology toward specific, label-free, and multiplexed sensing in ultra-small samples. The current study employs the field-effect meta-nano-channel biosensor (MNC biosensor) for the detection of the enzyme N-acetyl-beta-D-glucosaminidase (NAGase), a biomarker for milk cow infections. The measurements are performed in a 0.5 mu L drops of 3% commercial milk spiked with NAGase concentrations in the range of 30.3 aM-3.03 mu M (Note that there is no background NAGase concentration in commercial milk). Specific and label-free sensing of NAGase is demonstrated with a limit-ofdetection of 30.3 aM, a dynamic range of 11 orders of magnitude and with excellent linearity and sensitivity. Additional two important research outcomes are reported. First, the ionic strength of the examined milk is -120 mM which implies a bulk Debye screening length <1 nm. Conventionally, a 1 nm Debye length excludes the possibility of sensing with a recognition layer composed of surface bound anti-NAGase antibodies with a size of -10 nm. This apparent contradiction is removed considering the ample literature reporting antibody adsorption in a predominantly surface tilted configuration (side-on, flat-on, etc.). Secondly, milk contains a non-specific background protein concentration of 33 mg/ml, in addition to considerable amounts of micron-size heterogeneous fat structures. The reported sensing was performed without the customarily exercised surface blocking and without washing of the non-specific signal. This suggests that the role of non-specific adsorption to the BioFET sensing signal needs to be further evaluated. Control measurements are reported.