Effect of the linkage chemistry on areal density and repeatability of NP attachment
Due to the stochastic nature of the assembly process, a distribution of the particle attachment density is expected for each individual experiment, as shown in Figure 3. However, minimizing the variance in NP cargo attachment density distribution across replicate experiments is crucial to determining the efficacious dosage of biohybrid systems towards robust and predictable therapeutic outcomes. Thus, we next examined the repeatability in NP attachment density outcomes for two linkage chemistries using the optimal assembly parameters of B-800-60 and E-800-60. In both NanoBEADS variants, streptavidin-coated nanoparticles were attached to the bacteria using the high affinity biotin-streptavidin interactions; however, in the ABS NanoBEADS variant, biotin was conjugated with anti-Salmonella antibody and covalently bonded to the antigen on bacteria, whereas in the Biotin-Streptavidin NanoBEADS (BS NanoBEADS) variant, biotin was adsorbed onto the bacteria via electrostatic interactions. A minimum of four independent replicate assembly experiments for each of the two NanoBEADS variants were conducted (Figure 4). For the ABS NanoBEADS, both B-800-60 and E-800-60 yielded similar results (Figures 3a, b), with E-800-60 producing a higher fraction of high NP density NanoBEADS (>20 #NPs/µm2) (Figure 4c) and a more repeatable attachment density distribution with a smaller variance (Figure 4d). Thus, we identify E-800-60 as the optimal set of assembly parameters for producing the ABS NanoBEADS. For the BS NanoBEADS, no significant difference between the B-800-60 and E-800-60 outcomes was observed (Figure 4e, f). Comparing the distribution of the attached NP areal density between the two variants (Figures 4c, g), it is evident that the ABS NanoBEADS had a significantly higher number of bacteria with greater attachment density than the BS NanoBEADS. Furthermore, only ~7% of the bacteria did not have any nanoparticle attached when the ABS linkage is used, whereas an average of ~27% did not have any NP attached when the BS linkage is used. Consistent with our observation for ABS NanoBEADS, E-800-60 produced a more repeatable attachment density distribution (Figure 4h). The viability of the bacteria was not affected by the attached NPs in either of the two variants (Figure S1).