Here, a novel EBC collection method and device was developed and evaluated in collecting EBC samples from human subjects using culturing and molecular methods. Compared to those currently available devices shown in Table S1 , our device is lightweight with simplicity, reusability, and lower cost. The developed collection device itself costs less than $10, with about $0.5 for consumables (straw and hydrophobic film) per collection. The time needed for 100 ml EBC including sample collection and removal was around 2 min. The physical collection efficiency of the device is shown in Figure 3 . The data points shown in the figure were averages of the EBC samples collected from six volunteers under each of the collection times (1, 2, 3 and 4 min) tested. In general, the amount of EBC sample collected was observed to increase with increasing collection time were observed among subjects. As also observed in Figure 3 , the method has a good reproducibility (small variations). ANOVA analysis indicated that the collection time had a statistically significant effect on the amount of EBC sample collected per unit of time (p-val- Table S2 ; F and M indicate Female and Male, respectively, 1-7 indicate the subject ID corresponding to those listed in Table  S2 ; EBC collection time was 3 min; data points represent averages and standard deviations from at least three replicates. doi:10.1371/journal.pone.0041137.g005 ue = 0.0026). For the 4 min collection, the volume of collected EBC (168.7 mL) was 1.8 times of that (60.0 mL in average) by 1 min. In our study, when no EBC was collected about 1 mL of liquid was obtained from the hydrophobic surface in an environment with a temperature of 17.9-19.3uC and a relative humidity level of 46-52%. In addition, during the breath sample collection, the collection device had a higher air pressure due to the exhaling, thus it is less likely that environmental air would come into the device. This suggests that environmental water vapor had limited impact on the collection method given the total amount of EBC collected. A recent study indicated that the minimum required volume of EBC was 50 mL for follow-up biological and chemical analysis, such as multiplexed cytokine analysis [35] . This on the other hand implies that the EBC device developed in this study can provide adequate amount of EBC sample for rapid analysis. Here, only one type of hydrophobic surface (parafilm) was tested, and in the future different Table S2 ; DI water was used as the negative control. Table S2 ; Bacillus subtilis species was used as the positive control and DI water was used as the negative control; the curves shown here include two duplicates for each EBC sample. doi:10.1371/journal.pone.0041137.g007 hydrophobic materials should be also explored to improve the overall efficiency.