Sergio D. Aguirre

Detection of DNA using bioactive paper strips.

Paper strips containing DNA-conjugated microgels (MG) are used to achieve sensitive DNA detection in three steps: target DNA promoted ligation of a DNA primer to the MG-bound DNA, rolling circle amplification (RCA) between the primer and a circle DNA, and hybridization of the RCA products and a fluorescent DNA probe.

A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection

Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA.

Modulation of DNA-Modified Gold-Nanoparticle Stability in Salt with Concatemeric Single-Stranded DNAs for Colorimetric Bioassay Development

Gold nanoparticles (AuNPs) exhibit distinct optical properties under specific conditions. For example, spherical AuNPs of 13 nm in diameter appear red when well dispersed and blue or purple when aggregated. Two major methods used to control DNA-modified AuNP aggregation are DNA-templated assembly (also known as cross-linking aggregation) and salt-induced aggregation. The first approach modulates AuNP stability through sequence-specific hybridization and has been widely explored for biosensing applications. The second approach modulates AuNP stability by using divalent metal ions that neutralize the negative charges of DNA on AuNP surfaces, and has also been exploited to develop various colorimetric bioassays. DNA-conjugated AuNPs have also been used to make organized nanostructures with long single-stranded DNA molecules (ssDNAs) produced by rolling circle amplification (RCA). RCA is an isothermal process of amplifying DNA by using a short DNA primer, a circular ssDNA template (DNA circle) and Phi29 DNA polymerase. Because ssDNA products from RCA contain many repeating (concatemeric) units, RCA technique has been widely used to achieve sensitive detection of DNA or other biological targets. In a recent study, AuNPs and RCA have been employed together for the development of a colorimetric assay to detect single-nucleotide polymorphisms through a crosslinking strategy. Herein, we report on the novel use of RCA products to protect AuNPs against salt-induced aggregation. AuNPs modified with a short ssDNA complementary to part of an RCA product (RP) can organize onto the RP through sequence-specific hybridization. We have found that, when MgCl2 (salt) is added, the AuNP–RP assemblies form unique, globular, island-like nanostructures (Figure 1 a, lower path), which we term “NP islands (NPI)”. The color of the NPI-containing solution remains red whereas in the absence of RP, AuNPs form purple aggregates (Figure 1 a, upper path). We have also exploited this finding for the development of a colorimetric assay for ATP detection wherein the action of an ATP-dependant DNAzyme (aptazyme) is coupled to RCA and the resulted RP is used to protect AuNPs from salt-induced aggregation. AuNPs used in this study (NP1, Figure 1 b) were functionalized with a short ssDNA to a surface density of approximately 50 strands per particle. Optimization of MgCl2 concentration revealed that evident red-to-purple color transition of NP1 solution occurred at 30 mm of MgCl2 (along with 20 mm Tris-HCl, pH 7.5 at 23 8C, 100 mm NaCl); thus, this salt condition was used throughout our study. Two RCA products, RP1 (containing NP1-binding site; blue letters in Figure 1 b) and RP2 (which does not bind NP1), were prepared by using the relevant circular DNA templates and primers (for their sequences, see Figure S1a in the Supporting Information) and analyzed by agarose gel electrophoresis (Figure S1 b). Complex formation between RP1 and NP1 (but not between RP2 and NP1) was also confirmed by agarose gel electrophoresis (Figure S1 c). Before addition of MgCl2, the NP1-only, NP1–RP2, and NP1–RP1 solutions (samples 1–3, respectively) all appeared red (tube inserts in Figure 2 a, 1–3). TEM analysis revealed that gold nanoparticles in samples 1 and 2 were well dispersed (Figure 2 a, 1 and 2) whereas those in sample 3 were clustered (Figure 2 a, 3), suggesting that NP1s in this sample assembled onto RP1 by sequence-specific hybridization. With the addition of 30 mm MgCl2, samples 1 and 2 turned purple (tube inserts in Figure 2 a, 4 and 5) whereas sample 3 remained red (tube insert in Figure 2 a, 6). TEM analysis produced interesting results: huge NP aggregates were found in samples 1 and 2 (Figure 2 a, 4 and 5), howev[a] Dr. M. M. Ali, P. Kanda, S. D. Aguirre, Prof. Dr. Y. Li Department of Biochemistry and Biomedical Sciences and Department of Chemistry and Chemical Biology McMaster University, 1200 Main Street West, Hamilton (Canada) Fax: (+1) 905-522-9033 E-mail : liying@mcmaster.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201002677.

Developing Fluorogenic RNA-Cleaving DNAzymes for Biosensing Applications

Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules that have the ability to catalyze a chemical reaction. Currently, DNAzymes have to be isolated from random-sequence DNA libraries by a process known as in vitro selection (IVS) because no naturally occurring DNAzyme has been discovered. Several IVS studies have led to the isolation of many RNA-cleaving DNAzymes (RNase DNAzymes), which catalyze the transesterification of a phosphodiester linkage in an RNA substrate, resulting in its cleavage. An RNase DNAzyme and its substrate can be modified with a pair of donor and acceptor fluorophores (or a fluorophore and quencher pair) to create a fluorescence-signaling system (a signaling DNAzyme) where the RNA-cleaving activity of the DNAzyme is reported through the generation of a fluorescent signal. A signaling DNAzyme can be further coupled with an aptamer (a target-binding nucleic acid sequence) to generate a fluorogenic aptazyme in which the aptamer-target interaction confers an allosteric control of the coupled RNA-cleaving and fluorescence-signaling activity of the DNAzyme. Fluorogenic aptazymes can be exploited as valuable molecular tools for biosensing applications. In this chapter, we provide both a detailed description of methods for isolation of signaling DNAzymes by IVS and general approaches for rational engineering of fluorogenic aptazymes for target detection.

Examination of Bacterial Inhibition Using a Catalytic DNA

Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA), RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC) of antibiotics much faster than the conventional optical density (OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research.