detection-of-bacteria-using-fluorogenic-dnazymes

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

McMaster University 

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.

Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids.

The majority of bioassays utilize thermosensitive reagents (e.g., biomolecules) and laboratory conditions for analysis. The developing world, however, requires inexpensive, simple-to-perform tests that do not require refrigeration or access to highly trained technicians. To address this need, paper-based bioassays using gold nanoparticle (AuNP) colorimetric probes have been developed. In the two prototype DNase I and adenosine-sensing assays, blue (or black)-colored DNA-cross-linked AuNP aggregates were spotted on paper substrates. The addition of target DNase I (or adenosine) solution dissociated the gold aggregates into dispersed AuNPs, which generated an intense red color on paper within one minute. Both hydrophobic and (poly(vinyl alcohol)-coated) hydrophilic paper substrates were suitable for this biosensing platform; by contrast, uncoated hydrophilic paper caused "bleeding" and premature cessation of the assay due to surface drying. The assays are surprisingly thermally stable. During preparation, AuNP aggregate-coated papers can be dried at elevated temperatures (e.g., 90 degrees C) without significant loss of biosensing performance, which suggests the paper substrate protects AuNP aggregate probes from external nonspecific stimuli (e.g., heat). Moreover, the dried AuNP aggregate-coated papers can be stored for at least several weeks without loss of the biosensing function. The combination of paper substrates and AuNP colorimetric probes makes the final products inexpensive, low-volume, portable, disposable, and easy-to-use. We believe this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.

Paper-based bioassays using gold nanoparticle colorimetric probes.

Target detection by the naked eye: The action of an RNA-cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single-stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex-binding dye, the color of the dye changed from blue to purple.

Colorimetric sensing by using allosteric-DNAzyme-coupled rolling circle amplification and a peptide nucleic acid-organic dye probe.

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.

Detection of DNA using bioactive paper strips.

Modulation of DNA-modified gold-nanoparticle stability in salt with concatemeric single-stranded DNAs for colorimetric bioassay development.

Fluorogenic DNAzyme probes as bacterial indicators.

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.

Developing fluorogenic RNA-cleaving DNAzymes for biosensing applications.

Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (Î²-galactosidase (B-GAL) or Î²-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 Ã— 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-Î²-D: -glucuronide sodium salt (XG), chlorophenol red Î²-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

Article	Year
Nucleosides, nucleotides & nucleic acids\| PubMed ID: 16248023	2005
Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids. Angewandte Chemie (International ed. in English)\| PubMed ID: 18680110	2008
Paper-based bioassays using gold nanoparticle colorimetric probes. Analytical chemistry\| PubMed ID: 18847216	2008
Colorimetric sensing by using allosteric-DNAzyme-coupled rolling circle amplification and a peptide nucleic acid-organic dye probe. Angewandte Chemie (International ed. in English)\| PubMed ID: 19360817	2009
Detection of DNA using bioactive paper strips. Chemical communications (Cambridge, England)\| PubMed ID: 19865676	2009
Modulation of DNA-modified gold-nanoparticle stability in salt with concatemeric single-stranded DNAs for colorimetric bioassay development. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 21294175	2011
Fluorogenic DNAzyme probes as bacterial indicators. Angewandte Chemie (International ed. in English)\| PubMed ID: 21412961	2011
Developing fluorogenic RNA-cleaving DNAzymes for biosensing applications. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 22315083	2012
Multiplexed paper test strip for quantitative bacterial detection. Analytical and bioanalytical chemistry\| PubMed ID: 22526653	2012

M. Monsur Ali

Biography

JoVE Journal Publications

Other Publications