Rapid Discovery of Illuminating Peptides for Instant Detection of Opioids in Blood and Body Fluids

The United States is currently experiencing an opioid crisis, with more than 47,000 deaths in 2017 due to opioid overdoses. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS), which require expensive instrumentation and extensive sample preparation. Our aim was to develop a portable point-of-care device that can be used for the instant detection of opioids in body fluids. Here, we reported the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in the blood using a homogeneous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified using a high throughput one-bead one-compound (OBOC) combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), that are coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescent activation under a confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify the peptide-beads that fluoresce in the presence of an anti-morphine antibody, but lost fluorescence when the free morphine was present. After the positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were then synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for the detection of morphine in plasma. We demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for the detection of illicit drugs and cancer biomarkers in body fluids.