Rapid Discovery of Illuminating Peptides for Instant Detection of Opioids in Blood and Body Fluids
Authors: Yann Thillier
Ruiwu Liu
Bethany Ming-Choi Pan
Jiannan Li
Jonathan S Huynh
Shabnam Jafari
Diedra Shorty
Yousif H Ajena
Tingrui Pan
Kit S Lam
Journal: Molecules (2019)
Discipline: CHEMISTRY
Keywords: OBOC; combinatorial chemistry; drug screen; high throughput screening; molecular rotor dye
Affiliations: Department of Biochemistry & Molecular Medicine, University of California Davis, Sacramento, CA 95817, USA. ., Department of Biomedical Engineering, University of California Davis, Davis, CA 95616, USA. .

Abstract:  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. 

Retracted: No

PubPeer comments:
 In the Corrigendum to u201cTumor-targeting peptides from combinatorial librariesu201d published in Adv. Drug Del. Rew. in 2017 one can read that the split-mix strategy of Furka et al. [1] is used for the synthesis of OBOC libraries. ![file] The commented article was published 2 years later in 2019. Although the Furka et al article is cited in the commented article the Lam et al. paper [2] is again referred as the source of split-mix (split and pool) synthesis. Kit S. Lam who is corresponding author in both articles is expected to explain how is possible that the same u201cfaultu201d has been committed again. 1.tFurka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. General-Method for Rapid Synthesis of Multicomponent Peptide Mixtures. Int. J. Pept. Protein Res. 1991, 37 (6), 487u2212493. 2.tLam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. A New Type of Synthetic Peptide Library for Identifying Ligand-Binding Activity. Nature 1991, 354 (6348), 82u221284. ,