In this thesis I will discuss the work I have done to develop whole cell yeast biosensors for detecting pharmaceuticals in low- and middle- income (LMICs). Yeast biosensors are a low cost and user-friendly alternative to the lab-based spectroscopic and chromatography-based technologies currently used to detect pharmaceuticals in many areas. Although powerful, these lab-based devices are expensive and require trained personnel for operation, causing them to be out of reach for many areas of the world, including many LMICs. LMICs are in particular need for ways to monitor pharmaceuticals for applications including monitoring the quality of pharmaceutical dosage forms and testing environmental samples for contamination with pharmaceuticals.1,2Whole cell biosensors are an attractive alternative to current spectroscopic technology because they can be very affordable and user-friendly while providing biologically relevant information about analytes.3,4 Whole cell yeast biosensors in particular would be useful in LMICs because they are hardy and there are well-tested methods for drying yeast for long term storage. Yeast biosensors can be made to detect an analyte of interest using genetic engineering to express genetic circuits containing a detection modality (usually an analyte sensitive receptor capable of inducing transcription) and a reporter (e.g. fluorescence, color, or luminescence). The most notable use of yeast biosensors thus far has been to detect endocrine disrupting compounds (EDCs), however, the repertoire of yeast biosensors has been expanded to include detection of DNA damage and other pathogenic fungi, to name a few analytes.5-7 While potentially powerful, few of these systems have been adapted for use in the field. To address the need for portable analytical technologies, our lab endeavored to make a portable yeast-based biosensor.We developed the first portable whole cell yeast biosensor, named the biological paper analytical device (bioPAD), which detects the antibiotic doxycycline.8 While sensitive, inexpensive, and viable after long term storage, this device was not completely field friendly due to the requirements for multi-step incubation, use of liquid nitrogen to lyse cells, and expensive and non-stable color development reagents. My research was focused on developing new techniques to make the bioPAD more field-friendly and expanding this technology to include new analytes relevant for detection in LMICs. The first question I addressed with my research was whether yeast biosensors could be made to be more field friendly through use of a different reporter system. The original bioPAD used a beta-galactosidase reporter which was hindered by the drawbacks mentioned above. Luminescent reporters also require expensive and non-shelf stable reagents for the development of signal, and most colorimetric reporters take multiple days for significant color production if reagents are not used.9-11 An alternative to these reporters in fluorescence, which does not require reagents for signal development and has shown measurable signal in little as 6 hours.12,13 I developed fluorescent bioPAD technology which proved successful at detecting doxycycline starting at 0.3 μg/mL and showed activity in biological matrices. This biosensor could be useful for monitoring drug regiment adherence through testing of biological fluids because of its increased sensitivity and robust signal in complex matrices. Because the fluorescent bioPAD showed high levels of fluorescence without the need for any additional reagents other than a small amount of yeast media, I developed a streamlined version of the bioPAD to reduce production and development costs. I also constructed a portable fluorescence detection device with the help of our tech consultant, Galen Brown. This fluorescence detector costs approximately $130 to construct, can be assembled on site, and has the potential for long-term use. I have also worked to develop a scent-based reporter for yeast based-biosensors. A scent-based yeast biosensor, or scentsor, has the potential to be used as a screening tool which needs no additional equipment for use when detecting an analyte of interest. While unlikely to be semi-quantitative like the fluorescent biosensor, the scentsor could be used to alert the user when a threshold of analyte has been reached and alert users which samples should be sent out for further analysis, thus reducing the amount of samples needed to be analyzed in detail and saving resources. I demonstrated the principle of using a scent-based reporter in yeast through construction of a galactose-responsive banana scent producing scentsor. This reporter from this scentsor was analyzed with gas chromatography and then tested with human noses. Ninety three percent of the panelists chose the treated scentsor and so I developed a scentsor that could detect a more relevant analyte. I constructed an estrogen scentsor with the aim of expanding our portable yeast biosensors in order to detect the active ingredients in hormone-based pharmaceuticals, a category of drug found to be substandard in LMICs.1,14 Panelists were able to detect banana scent at the estimated threshold of 15 nM E2, which is low enough for monitoring pharmaceutical quality and possibly identifying highly contaminated environmental samples. This work has shown that scent as a reporter modality has great potential for use in analytical technologies to be used in a field settingIn this thesis I will discuss the work I have done to develop whole cell yeast biosensors for detecting pharmaceuticals in low- and middle- income (LMICs). Yeast biosensors are a low cost and user-friendly alternative to the lab-based spectroscopic and chromatography-based technologies currently used to detect pharmaceuticals in many areas. Although powerful, these lab-based devices are expensive and require trained personnel for operation, causing them to be out of reach for many areas of the world, including many LMICs. LMICs are in particular need for ways to monitor pharmaceuticals for applications including monitoring the quality of pharmaceutical dosage forms and testing environmental samples for contamination with pharmaceuticals.1,2Whole cell biosensors are an attractive alternative to current spectroscopic technology because they can be very affordable and user-friendly while providing biologically relevant information about analytes.3,4 Whole cell yeast biosensors in particular would be useful in LMICs because they are hardy and there are well-tested methods for drying yeast for long term storage. Yeast biosensors can be made to detect an analyte of interest using genetic engineering to express genetic circuits containing a detection modality (usually an analyte sensitive receptor capable of inducing transcription) and a reporter (e.g. fluorescence, color, or luminescence). The most notable use of yeast biosensors thus far has been to detect endocrine disrupting compounds (EDCs), however, the repertoire of yeast biosensors has been expanded to include detection of DNA damage and other pathogenic fungi, to name a few analytes.5-7 While potentially powerful, few of these systems have been adapted for use in the field. To address the need for portable analytical technologies, our lab endeavored to make a portable yeast-based biosensor.We developed the first portable whole cell yeast biosensor, named the biological paper analytical device (bioPAD), which detects the antibiotic doxycycline.8 While sensitive, inexpensive, and viable after long term storage, this device was not completely field friendly due to the requirements for multi-step incubation, use of liquid nitrogen to lyse cells, and expensive and non-stable color development reagents. My research was focused on developing new techniques to make the bioPAD more field-friendly and expanding this technology to include new analytes relevant for detection in LMICs. The first question I addressed with my research was whether yeast biosensors could be made to be more field friendly through use of a different reporter system. The original bioPAD used a beta-galactosidase reporter which was hindered by the drawbacks mentioned above. Luminescent reporters also require expensive and non-shelf stable reagents for the development of signal, and most colorimetric reporters take multiple days for significant color production if reagents are not used.9-11 An alternative to these reporters in fluorescence, which does not require reagents for signal development and has shown measurable signal in little as 6 hours.12,13 I developed fluorescent bioPAD technology which proved successful at detecting doxycycline starting at 0.3 μg/mL and showed activity in biological matrices. This biosensor could be useful for monitoring drug regiment adherence through testing of biological fluids because of its increased sensitivity and robust signal in complex matrices. Because the fluorescent bioPAD showed high levels of fluorescence without the need for any additional reagents other than a small amount of yeast media, I developed a streamlined version of the bioPAD to reduce production and development costs. I also constructed a portable fluorescence detection device with the help of our tech consultant, Galen Brown. This fluorescence detector costs approximately $130 to construct, can be assembled on site, and has the potential for long-term use. I have also worked to develop a scent-based reporter for yeast based-biosensors. A scent-based yeast biosensor, or scentsor, has the potential to be used as a screening tool which needs no additional equipment for use when detecting an analyte of interest. While unlikely to be semi-quantitative like the fluorescent biosensor, the scentsor could be used to alert the user when a threshold of analyte has been reached and alert users which samples should be sent out for further analysis, thus reducing the amount of samples needed to be analyzed in detail and saving resources. I demonstrated the principle of using a scent-based reporter in yeast through construction of a galactose-responsive banana scent producing scentsor. This reporter from this scentsor was analyzed with gas chromatography and then tested with human noses. Ninety three percent of the panelists chose the treated scentsor and so I developed a scentsor that could detect a more relevant analyte. I constructed an estrogen scentsor with the aim of expanding our portable yeast biosensors in order to detect the active ingredients in hormone-based pharmaceuticals, a category of drug found to be substandard in LMICs.1,14 Panelists were able to detect banana scent at the estimated threshold of 15 nM E2, which is low enough for monitoring pharmaceutical quality and possibly identifying highly contaminated environmental samples. This work has shown that scent as a reporter modality has great potential for use in analytical technologies to be used in a field setting.