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New in Vitro Cancer Diagnostic Test Based on Protease Activity Profiling Using Nanoelectrode Arrays

詳細技術說明

Description: Kansas State University researchers have developed a new and effective invitro diagnostic method to simultaneously measure the activity of multipleproteases link to cancers.  By using anew electrochemical detection method based on novel nanoelectrode arrays, thisinnovation provides numerous potential advantages over the existingfluorescence detection commonly used today, including portability, multiplicity,sample size, accuracy and cost.Advantages:  Increased portability:The technique can be developed into a disposable chip in a portable electronic systemfor point-of-care or clinical applications, eliminating theneed for a centralizedlab.Multiplicity: The activity of up to 9(potentially to 16) proteases can be simultaneously detected.Very small sample size:A single drop of blood, serum, urine or saliva is needed to detect multipleproteases.Increased accuracy:The accuracy of the detection is significantly increased because the analysis isbased on multiple proteases as disease biomarkers.Lower cost: The cost of using mass produced chips would be lower than the existing fluorescencetechnique.Applications:  In vitro diagnostic testing of cancers or otherdiseases related to enhanced protease activitiesFrequent and easy monitoring of treatment effectswhen patients are given anticancer medicine or other drugs.Multiple analyses to improve accuracyScreening for effective protease inhibitors as drugcandidatesPatent Status:  U.S. Patent No. 14/320,215 filed in June 2014.Fig. 1 The layout of a representative multiplex nanoelectrode arrays (NEAs). Opticalimages of (a) 30 dies on a 4” SiO2-covered Si wafer and (b)individually addressed 3x3 microelectrodes on a single die. Scanning electronmicroscopy images of (c) the active sites of the 3x3 microelectrodes and (d)the NEA with exposed carbon nanofiber (CNF) tips at each microelectrode. TheCNFs are embedded in SiO2 matrix and connected to the underneathmicroelectrode pads. Different peptide substrates are functionalized atdifferent NEA pads. The proteases catalyze specific reactions at the specificNEA pads. Scale bar: (b) 2 mm, (c) 200 mm, (d) 1µm.Fig. 2 Principles of protease activity detection at a NEA. (a) VACNFs at amicroelectrode pad are embedded in SiO2 matrix leaving only the tipexposed. (b) Fc-tagged peptides attached at the CNF tips providing theelectrochemical signal. The proteolytic kinetics is recorded by measuring theelectrochemical signal vs. time and analyzed with a heterogeneousMichaelis-Menten model.Fig. 3 Illustrationof the integrated chip for protease profiling. (a) top- and (b) perspectiveviews of the packed cartridge for multiplex protease profiling, including a3x3  nanoelectrode array chip as theworking electrodes, and shared Pt ring counterelectrode and Ag/AgCl quasi-reference electrode which are packed in a polymerfluidic channel. Schemes of using the 3x3 chip for (c) selecting three bestpeptides for each protease and (d) using these 9 peptides to profile the activitiesof three protease biomarkers in a complex sample.

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美國