Easy-to-use dipstick tests for detection of lead in paints using non-cross-linked gold nanoparticle–DNAzyme conjugatesw

Designing easy-to-use biosensors for trace metal ions in the environment is of considerable importance as these metal ions are large in number, small in quantity and high in toxicity. Metal-specific DNAzymes, functional DNA molecules that can catalyze a reaction in the presence of a particular metal ion, have emerged as a new class of metal-ion sensors because DNAzymes with desired metal specificity and affinity can be obtained by a combinatorial biology technique called in vitro selection. When combined with gold nanoparticles (AuNPs), the DNAzymes have been transformed into highly sensitive and selective colorimetric biosensors, producing color changes between red (dispersed AuNPs) to blue (aggregated AuNPs) in response to a target recognized by DNA. Using this method, we have demonstrated colorimetric biosensors for metal ions such as Pb, UO2 2+ and Cu. While these colorimetric biosensors have taken an important step towards real-time sensing as the signal is detectable by the naked eye, without the need for expensive instrumentation, they still require laboratory type operations, such as precise transfer and mixing of multiple solutions. In addition, although the sensitivity is high when the absorbance is recorded using a UV-Vis spectrophotometer, it is often difficult to distinguish the red color of dispersed nanoparticles against a blue background from the aggregates, particularly at low metal-ion concentrations. Furthermore, AuNPs are not very stable in the solution state; they are vulnerable to aggregation under a variety of conditions thereby making it difficult to store the sensors for a long period of time. Lateral flow devices are an ideal platform for making dipstick type tests to further improve the performance of DNAzyme–AuNP colorimetric sensors. In addition to eliminating precise solution transfer and allowing separation of AuNPs to make it easier to distinguish colors, the reagents can be prepared in a dry or nearly dry state, making the device stable at ambient conditions for a long period of time. The home pregnancy test is the most commonly used application of lateral flow devices that use antibodies for detection. Being more stable to denaturation, DNA is an attactive molecule to replace antibodies in the dipstick tests. Despite the promise, DNA-based dipstick tests are not common. Glynou et al. reported a lateral flow device for the detection of DNA. To expand on the range of analytes detected, we previously reported dipstick tests for the detection of adenosine and cocaine using AuNP conjugated to aptamers. A paper based bioassay using aptamers and the protein enzyme DNAase I which also involved the disassembly of nanoparticle aggregates that were dried onto paper substrates was reported by Yingfu Li and coworkers. Even though our previously reported methodology can be applied to almost any target for which aptamers can be obtained, because aptamers with high affinity for metal ions have been difficult to select, this methodology has not been applied to dipstick tests for metal ions. To extend the applicability of this test for metal ions, metal-specific DNAzymes are an excellent choice. However, it is not trivial to adopt the aptamer-based dipstick methodology by simply replacing aptamers with DNAzymes because DNAzymes undergo not only binding as aptamers do, but also catalytic activity and product release, making the design more complicated. Herein, we report a method to convert DNAzyme– AuNPs into dipstick tests for metal ions, specifically for Pb. In the process, we showed that the cross-link based method used in the previous aptamer–AuNP system did not work for the DNAzyme–AuNP system. Instead, we succeeded in developing a non-cross-linked DNAzyme–AuNP system for detection. Furthermore, we have demonstrated the dipstick tests to be ideal for detection of lead in household paints, in accordance with the EPA defined threshold of 1 mg cm 2 Pb for paint to be classified as a lead-based paint. We chose the 8–17 DNAzyme to construct the dipstick tests for Pb because of its very high activity as shown by a fast cleavage rate (estimated kobs B50 min 1 at pH 7.0). Fig. 1a shows its reaction scheme. In the presence of Pb, the enzyme strand (called 17E) catalyzes the cleavage of the substrate (called 17S) at the single ribo-adenosine base (shown in red). Based on this reaction scheme, the 8–17 DNAzyme has been previously converted into fluorescent, colorimetric and electrochemical sensors for Pb. Unlike aptamer-based colorimetric tests, the presence of target does not cause immediate disassembly of the aggregates in the DNAzyme-based colorimetric tests. Disassembly in the case of DNAzymes has been shown to require heating or the use of invasive DNA strands to release the cleaved product trapped in the nanoparticle aggregates. Either of these methods will lead to added complexity and they are not feasible for lateral flow devices. Therefore, we decided to use an alternative approach that does not involve formation of nanoparticle aggregates Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL, USA. E-mail: [email protected]; Fax: +1-217-244-3186; Tel: +1-217-333-2619 w Electronic supplementary information (ESI) available: Experimental section; optimization of DNAzyme construct; specificity data; details for detection of lead in paint. See DOI: 10.1039/b917772h