Ricin detection by biological signal amplification in a well-in-a-well device.

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.