The nonspecific binding observed with ELISA of Autiopronin to anti-HA was not observed with the QCM. QCM naturally prohibits nonspecific binding through the acceleration of adhered particles. This acceleration is generated by the oscillation of the quartz and can help remove weakly bound or nonspecifically bound molecules [55, 56] . Similar binding of anti-FLAG to FLAG-Au and HA-Au-FLAG but not HA-Au was measured with the QCM (Fig. 3b) . Thus, orthogonal and normalized binding at the QCM is observed consistent with immunological results obtained from ELISA, but is observable in a much shorter time interval (10 min compared to the 4 h it took to complete the ELISAs) when using the developed immunoassay and the QCM. The demonstrated binding between bi-functionalized nanoparticles and their respective antibodies makes evident the practicality of their use as a simulant for microorganisms, while lacking the difficulties associated with use of an attenuated or killed pathogen. When a change in Fig. 4 Calibration of HA protein binding to anti-HA. a The average change in mass of H5N1 HA protein binding was determined at varying concentrations. Briefly, (black line) 40 μg/mL (n=2), (red line) 20 μg/mL (n=5), (blue line) 10 μg/mL (n=3), (teal line) 5 μg/mL (n=3), and (pink line) 1 μg/mL (n=4). Also shown for comparison is the binding of HA-Au (green line) and HA-FLAG-Au (orange line). b A linear representation of the Langmuir isotherm produced by this average binding is shown with a linear relationship of y ¼ 0:035 AE 0:004 ð Þ x þ 4:90 AE 15:5 ð Þ Â 10 À10 and R 2 =0.96. c Calibration curve for an assay time of 1.5 min where the black squares are HA proteins, and the red and blue dots are HA-FLAG-Au and HA-FLAG, respectively b mass is observed upon introduction of the functionalized MPC to the immunosensor, the immunological response is shown to be above the limit of detection (∼3 ng, calculated by three times the average noise). Results show the average change in mass for protein G to be 128±48 ng, for antibodies to be 276±75 ng, and for the nanoparticle simulants to be 37±8 ng (Table 1 ). While the time was held constant for each step, the binding varied slightly, which could be due to slight variations in surface roughness and the surface coverage of prior adsorption steps. Even with a standard deviation of 75 ng for antibody adsorption, a deviation of only 8 ng was measured for the final detection step. The binding of the nanoparticles demonstrates saturation behavior (Fig. 3) . Based on the shape of the QCM curves and the rapid increase in mass with time, it can be assumed that the kinetics would occur quickly and with presumably large equilibrium association constants. To test this theory, a calibration of anti-HA to HA binding was determined, and the binding of the functionalized nanoparticles was compared. The HA protein from H5N1 was exposed to anti-HA antibodies at concentrations ranging from 1 to 40 μg/mL (Fig. 4a, b) . This binding can be compared at any time point to generate a calibration curve (Fig. 4c , example shown is at 1.5 min). Based upon the desired separation of the lower data points, the binding can continue for several more minutes. Also, the amount of nanoparticle exposed to the surface can be lowered to prevent overloading of the sensor. Measuring the maximum binding that occurs, as opposed to lower time points, can be used to determine the equilibrium association constant (K a ) and increase our understanding of the affinity of our sensor. This constant was determined by fitting to a linear rearrangement of the Langmuir adsorption isotherm, where C was plotted versus C/Δm (Eq. 3, Fig. 4b ) [30] :