Published: April 28, 2011 r2011 American Chemical Society 4273 dx.doi.org/10.1021/ac200697y | Anal. Chem. 2011, 83, 4273–4280 ARTICLE pubs.acs.org/ac Paper Bioassay Based on Ceria Nanoparticles as Colorimetric Probes Maryna Ornatska, Erica Sharpe, Daniel Andreescu, and Silvana Andreescu* Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, New York 13699-5810, United States b S Supporting Information C olorimetric sensors on patterned paper or plastic were developed as the least expensive, user-friendly alternative to conventional analytical instrumentations for ‘point-of-care’ medical diagnosis, environmental monitoring, and food quality control. 14 These low-cost platforms have been integrated with both colorimetric 5,6 and electrochemical 79 detection systems. Such sensors are miniaturized and disposable and can be used for on-site analysis. Examples of paper bioassays include patterned paper fabricated by photolithography for detection of glucose and bovine serum albumin (BSA), 1 inkjet-printed solgel bioinks for detection of neurotoxins, 10 aptamernanoparticle-based lateral flow devices for detection of DNA sequences. 11 In conventional colorimetric paper-based enzyme assays, an enzymatic reaction associated with a colorimetric process invol- ving soluble reagents is performed onto a paper or plastic platform. Typically, the enzyme is immobilized onto the sensing platform. The level of target analyte is quantified by a visible color change detectable with the naked eye. Most strategies reported previously involve the migration of soluble dyes onto hydrophilic paper with well-defined hydrophobic regions to provide control of reagents and biological liquids. This type of assay has been reported initially by the Whitesides group for simultaneous detection of glucose and BSA in urine. 5 The assay is based on the enzymatic conversion of glucose by glucose oxidase to form H 2 O 2 which reacts with horseradish peroxidase (HRP) and potassium iodide, resulting in a color change from clear to brown. Abe et al. 12 have developed a multianalyte paper-based microfluidic device by inkjet printing to quantitatively detect human serum albumin, glucose, and pH. Dungchai et al. 6 have proposed an alternative design in which multiple colorimetric indicators are used to improve the ability to visually discriminate between different analyte concentrations such as glucose, lactate, and uric acid. The assay is based on the oxidation of soluble indicators such as 4-aminoantipyrine, 3,5-dichloro-2-hydroxy- benzenesulfonic acid, o-dianisidine dihydrochloride, and potas- sium iodide by H 2 O 2 produced by analyte-specific oxidase enzymes. In another report, a biocompatible enzyme-based solgel ink was printed onto a colorimetric paper strip to create a bioactive surface. The sensor was used for the indirect detection of acetylcholinesterase (AChE) inhibitors, paraoxon, and afla- toxin B1, by measuring the residual activity of AChE on paper using the conventional Ellman’s colorimetric assay. 10 In addition to the use of soluble dyes, nanoparticle-based colorimetric bioassays have been reported, with some adapted to paper platforms. 13 Traditionally, these assays are based on gold nanoparticles and the detection principle relies on nanoparticle- induced aggregation or dispersion in the presence of the analyte. 13,14 Most colorimetric nanoparticle assays are DNA assays; few have been adapted to enzymatic reactions. An enzymatic paper “dipstick” for detection of enzyme inhibitors Received: March 18, 2011 Accepted: April 28, 2011 ABSTRACT: We report the first use of redox nanoparticles of cerium oxide as colorimetric probes in bioanalysis. The method is based on changes in the physicochemical properties of ceria nanoparticles, used here as chromogenic indicators, in response to the analyte. We show that these particles can be fully integrated in a paper-based bioassay. To construct the sensor, ceria nanoparticles and glucose oxidase were coimmobilized onto filter paper using a silanization procedure. In the presence of glucose, the enzymatically generated hydrogen peroxide induces a visual color change of the ceria nanoparticles immobilized onto the bioactive sensing paper, from white-yellowish to dark orange, in a concentration-dependent manner. A detection limit of 0.5 mM glucose with a linear range up to 100 mM and a reproducibility of 4.3% for n = 11 ceria paper strips were obtained. The assay is fully reversible and can be reused for at least 10 consecutive measurement cycles, without significant loss of activity. Another unique feature is that it does not require external reagents, as all the sensing components are fixed onto the paper platform. The bioassay can be stored for at least 79 days at room temperature while maintaining the same analytical performance. An example of analytical application was demonstrated for the detection of glucose in human serum. The results demonstrate the potential of this type of nanoparticles as novel components in the development of robust colorimetric bioassays.