Figure 8(a) depicts the fluorescence intensity of conventional glass slide substrate, GLAD MEF substrate, and AgNMPA MEF substrate. Both GLAD and AgNMPA MEF substrates were fabricated using a same GLAD process with the deposition angle of 89°. The fluorescence intensity and background signal of GLAD MEF substrate were ~ 63 and ~ 17.5 times higher than those of glass substrate. The enhancement of background signal was mainly due to the enhanced electromagnetic field effects because of the effective blocking process, however, the higher enhancement of fluorescence intensity was due to not only the enhanced electromagnetic fields and also the strong binding of antibody on the Ag nanorods. The fluorescence intensity and background signal of AgNMPA MEF substrate were ~ 71 and ~ 2.8 times higher than those of glass substrate. The signal enhancement factor was similar to the GLAD MEF substrate, however, the dramatic reduction of background signal enhancement was achieved in the AgNMPA MEF substrate. It clearly shows the AgNMPA MEF substrate provide similar fluorescence signal enhancement to the GLAD MEF substrate with more than 6 times lower background signal. Figure 8(b) compares the SBR of conventional glass slide substrate, GLAD MEF substrate, and AgNMPA MEF; the values were 1.6, 5.9, and 41.4, respectively. The comparison plot indicates ~ 7× enhancement in SNR on AgNMPA MEF substrate than on GLAD MEF substrate.