## 3 Results  
In order to demonstrate multiplexed detection of each of the pathogenic bacteria simultaneously, a single sample was screened directly from stool on a single disc. A range of bacteria concentrations were tested in this multiplexed format and the resulting fluorescence measurements, displayed in relative fluorescence units (RFU), are shown in Figure 2. Each target was successfully detected in the presence of other background pathogens and complex sample matrices, conditions that would prove difficult for traditional methods without the use of extensive sample preparation. Furthermore, we characterized the sensitivity of the platform by screening a series of dilutions of each bacterial pathogen in variety of sample matrices. For each target, three discs were run and the results for samples in assay buffer are shown in Figure 3. From this we demonstrate limits of detection (LOD), defined by IUPAC standards to be three standard deviations above the noise, down to as low as 10 s of cells for the bacteria of interest. It can be seen that _E. coli_ and _Shigella_ are the most sensitively detected, following by _Salmonella_ and _Listeria_. This trend agrees with the results of the multiplexed detection experiments shown in Figure 2. The full set of LOD values for each bacteria and sample matrix is presented in Table 1.  
Figure 3: The sensitivity of our detection was demonstrated with three disc runs for a range of cell counts for all bacterial targets: (**a**) _E. coli_; (**b**) _Listeria_; (**c**) _Salmonella_; and (**d**) _Shigella_. These samples were run in assay buffer. Figure 2: Multiplexed detection of _E. coli_, _Listeria_, _Salmonella_, and _Shigella_ from a stool sample. Each target pathogen was detected in the presence of a background pool of the other non-targeted pathogen to demonstrate specificity and selectivity. As a proof-of-concept demonstration of the platform's versatility, we demonstrate the detection of _E. coli_ from a variety of clinical sample matrices such as urine, blood, and stool for a range of cell concentrations (Figure 4). Though bacteria will typically not be found in all of these matrices, the results demonstrate a robust, linear response independent of the sample matrix. It is interesting to note that the limits of detection in biological samples may be improved compared to the assay buffer in some instances. We propose that this phenomenon may be explained by the excluded volume effect, or macromolecular crowding [34; 35]. The biological sample matrices are highly complex with a large diversity and concentration of molecules. Therefore, a larger number of molecules will interact with the antibodies on the surface of the particles. Weakly binding species will be further pushed off of the beads by tighter binding partners and competition may decrease the amount of nonspecific binding to the particles prior to the wash in the density medium due to Stokes' flow. The cumulative effect may be an improvement in the limit of detection by increasing specificity. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & \multicolumn{6}{c}{**LOD (\# of Cells)**} \\ \cline{2-7}  & \multicolumn{3}{c}{_Microfluidic Singleplex_} & \multicolumn{3}{c}{_Microfluidic Multiplex_} & _Conventional Singleplex_ \\ \cline{2-7}  & **Buffer** & **Urine** & **Blood** & **Stool** & **Buffer** & **Stool** & **Buffer** \\ \hline _E. coli_ & 11 & 34 & 33 & 9 & 51 & 31 & 38 \\ _Listeria_ & 999 & 796 & 1668 & 320 & 2849 & 238 & 1745 \\ _Salmonella_ & 2416 & 703 & 1200 & 974 & 1154 & 328 & 2648 \\ _Shigella_ & 53 & 33 & 61 & 20 & 94 & 12 & 1236 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The limit of detection was determined for the full panel of bacteria in a variety of sample matrices, including assay buffer, urine, blood, and stool. Singleplex detections were performed for all matrices and multiplex detections were confined to assay buffer and stool. Conventional ELISA detections in assay buffer are also shown for comparison.