title: Ricin detection by biological signal amplification in a well-in-a-well device.
abstract: This paper presents a ricin detection method based on ricin's inhibitory effects on protein synthesis. Biological synthesis (expression) of a protein includes the steps of gene transcription (DNA --> RNA) and protein translation (RNA --> proteins); these reactions can be coupled into a one-step operation and carried out in a cell-free medium. Ricin is known to inhibit protein synthesis by interacting with 28S ribosome RNA; the inhibitory effect is exploited as the sensing mechanism in this work. For each copy of DNA, thousands of copies of proteins can be produced. As a result, the inhibitory effects of ricin are amplified, leading to a significantly enhanced detection signal (the difference between the positive control and samples). An array of protein expression units is developed to accommodate positive/negative controls and multiple samples. The array device contains a solution without any reagent captured on a solid surface, offering flexibility without comprising the activities of biomolecules. The miniaturized well-in-a-well design possesses a mechanism to supply nutrients continuously and remove byproducts, leading to higher protein expression yields and thus larger detection signals (lower detection limit) when ricin is present. We demonstrate the production of green fluorescent protein and luciferase in the device. A calibration curve has been obtained between the luciferase expression yield and the ricin concentration, showing a detection limit of 0.01 nM (0.3 ng/mL) ricin. The nested-well device is also used for measuring the toxicity level of ricin after physical or chemical treatment.
authors.affiliation: Department of Mechanical and Aerospace Engineering, University of Florida, P.O. Box 116250, Gainesville, Florida 32611, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Mei
authors.forename: Carl K
authors.initials: CK
authors.lastname: Fredrickson
authors.forename: Wei
authors.initials: W
authors.lastname: Lian
authors.forename: Shouguang
authors.initials: S
authors.lastname: Jin
authors.forename: Z Hugh
authors.initials: ZH
authors.lastname: Fan
mesh_terms: D002138:Calibration; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012339:RNA, Ribosomal, 28S / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015203:Reproducibility of Results; D012276:Ricin / Q000032:analysis* / Q000494:pharmacology / Q000633:toxicity; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D011500:Protein Synthesis Inhibitors; D012339:RNA, Ribosomal, 28S; D012276:Ricin; D008156:Luciferases