## 2 Methodology
While it is true that some optical techniques, e.g., laser induced fluorescence, can sometimes provide larger signal/molecule ratios than can ion-multiplication techniques, the conditions required for such sensitivity are often more difficult to achieve, they vary much more from species to species, and require more expensive gear than is generally the case with ions. It is not by accident that in many laser-based detection techniques, the laser photons are used to transform target molecules into ions. This is due to the fact that detection methods for ions are often more sensitive and general than those for photons or electronically excited molecules. By transforming airborne pathogens into charged species, we can similarly achieve many of the advantages electrospray affords with analysis of molecules, that is, high charge to mass ratio, and ability to move microbes based on external fields for examination. It is also appropriate to comment on how sensitivity is best-expressed, i.e., in what units? In chromatography, perhaps the most widely used of all analytical techniques, a specific quantity of analyte is injected into a flow of mobile phase species entering a separation column. Sensitivity is thus defined in terms of the smallest amount of injected analyte, e.g., in pico-grams or pico-moles that will give rise to a distinguishable peak in the effluent. Sensitivity defined in this manner is not very meaningful in applications where determination of concentration of a species in a gas or liquid of interest is needed or whether a particular species is present in that gas or liquid when the available quantity of that gas or liquid may be large relative to the quantity required for the analytical procedure. In such cases the appropriate measure of sensitivity is the lowest concentration of analyte in a sample liquid or gas at which detection and identification can be achieved. This minimum detectable concentration is therefore a relevant measure of sensitivity in a search for toxic chemical species by analyzing a stream of air from a region where they might be concealed. At Yale, we observed years ago that electrospray ionization, now traditionally used for mass spectrometry, has the ability to capture polar or polarizable trace species from the air [12] at levels as low as parts-per-billion without a pre-concentrator. Since the COVID-19 pandemic, there has been a flurry of development of many new methods and devices to identify potentially infectious disease transmitted by air. A key challenge in this scenario is the capture of sufficient concentrations of the target species for detection. As such, collection of more analyte from surrounding air will require a less sensitive identifier and the reverse is true. An ideal scenario is achieved if the collection device specifically captures and concentrates only the class of biomaterials of interest while ignoring the background. Indeed, Figure 3 illustrates Penicillium Spores collected from ambient air on a culture plate using electrospray and visualized using an Amscope T390B optical microscope (\(\times\)1000). Notably, greater than four times as many spores were collected using an electrospray source (ES) than from an open culture plate in 4 h without ES. The use of electrospray can be better appreciated if a known concentration of particles in an enclosed space is subjected to a charged droplet electric field. This concept is illustrated in Figure 4 where a glove box chamber is filled with 3.0-micron polystyrene microspheres to simulate bacterial human respiratory droplets and aerosolized using a collison nebulizer. The background aerosol concentration was then measured using a Met One laser particle counter in two modes, one mode where the electrospray source is turned on and the other mode where the electrospray source is turned off to illustrate electrospray collection efficiency between both modes. Figure 4, [13] clearly shows the dramatic overall background aerosol concentration drop during each cycle of electrospray operation.