Short Communication Direct Electrochemical Monitoring of RNase Activity YongkangYe, a,c Wen Wen, a Yun Xiang, a Xiaodong Qi, b Jeffrey T. La Belle, a Julian J.L. Chen, b Joseph Wang a * a Biodesign Institute, Departments of Chemical Engineering, Chemistry and Biochemistry and Bioengineering, Arizona State University, Tempe, AZ 85287-5801, USA *e-mail: joseph.wang@asu.edu b Department of Chemistry and Biochemistry and School of Life Sciences, Arizona State University, Tempe, AZ 85287-5801, USA c School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, P.R. China Received: January 21, 2008 Accepted: February 7, 2008 Abstract Here we present a highly sensitive, rapid and simple electrochemical assay of RNase based on coupling magnetic separation of the enzymatically treated RNA with stripping potentiometric detection of the purine nucleobases. A detection limit of 1  10 8 U RNase (ca. 4 pg/mL) is obtained in connection to a 60 min enzymatic digestion. The attractive performance of this direct indicator-free electrochemical assay offers great promise for a wide range of molecular biology and water quality applications. Keywords: Ribonuclease, Electrochemistry, Guanine DOI: 10.1002/elan.200804172 Ribonucleases(RNase),thatcatalyzethehydrolysisbreak- down of RNA, play a key role in controlling cellular metabolism[1,2].Owingtotheirconsiderablephysiological activity there is substantial interest in monitoring RNase activity in connection to numerous molecular biology applications. RNase A is commonly the ribonuclease of choiceforRNase-activitytestingbecauseofitsbroadrange ofsubstrates. RNase measurements have traditionally relied on the Kunitz method, involving monitoring the shift of the UV absorbance of a RNA substrate upon enzymatic digestion [3, 4]. While offering a simple approach for RNase assays, this optical route offers a limited sensitivity (commonly 0.2mg/mlofRNaseA)andsuffersfrominterferencesfrom coexisting proteins. Alternately, one can use (Zymogram methods)thataremoresensitive(downto25 mg/mL)butare time consuming and cumbersome [5, 6]. Surprisingly, little attention was given to electrochemical methods, with the exception of a very recent publication describing an indicator-basedindirectapproachbasedonmonitoringthe decreasedvoltammetricpeakofaferroceneintercalator[7]. Electrochemicaldevicesareuniquelyqualifiedformeeting the size, cost and power requirements of decentralized RNasetesting. In this communication we report on a simple, rapid and sensitiveelectrochemicaldetectionofRNaseactivitybased ontheintrinsicelectroactivityofRNA[8].Thenewprotocol relies on direct stripping chronopotentiometric monitoring of the purine nucleobases cleaved from RNA during the RNase enzymatic digestion. Early work in our laboratory has illustrated that adsorptive stripping chronopotentiom- etry(AdSC)allowshighlysensitivedirectmeasurementsof nucleic acids in connection to the oxidation of the guanine and adenine nucleobases [8, 9]. This protocol couples the effectiveadsorptiveaccumulationofnucleicacidsatcarbon electrodes with an advanced background correction of the computerizedchronopotentiometricscheme.Suchsensitive electrochemical measurements have been widely used for label-freedetectionofDNAhybridization[10,11],butnot inconnectiontothemonitoringofenzymaticactivity. Fig.1. Illustration of the electrochemical RNase detection pro- tocol. A) Addition of RNase to a solution containing RNA- modified magnetic beads for enzymatic reaction. B) Magnetic separation and acid dipurinization of the cleaved RNA. C) Chronopotentiometric stripping measurements of the purine nucleobases of the cleaved RNA at a graphite pencil electrode (illustrated here for a 9.2  10 5 U RNase solution). Conditions: Incubation time of immobilized RNA with the target RNase, 60 min. After the acid dipurinization, 1 mL of acetate buffer (0.5 M, pH 5.8) containing 2 ppm Cu 2þ , was spiked into the detection cell. Preconditionining of the graphite electrode, 1 min at þ 1.4 V; accumulation, 4min at  0.05V; stripping current, þ 5 mA. 919 Electroanalysis 20, 2008,No.8,919–922 # 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim