To investigate the amount of variability in EBC collected by the method developed, six student volunteers were recruited to exhale through the device for 1, 2, 3 and 4 min. The volume of collected EBC was measured by a calibrated pipette (Eppendorf, Hauppauge, NY). The amount of EBC per unit time collected by the device was determined using averages of EBC samples obtained by the volunteers under each of specific collection time tested. For each EBC collection, a different hydrophobic film and a different exhalation straw were used. In addition, the particle size distributions in the exhaled breath through mouth-breathing were also measured in a particle free bio-safety hood using an Optical Particle Counter (OPC) (GRIMM Co. Ltd., Ainring, Germany) at a flow rate of 1.2 L/min. To ensure air stream balance, the OPC was connected to a two-way tubing, which connects to clean air (Biological SafetyHood) and the breathing straw, respectively.