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.

Turning tryptophanase into odor-generating biosensors.

An odor-based sensor system that exploits the metabolic enzyme tryptophanase (TPase) as the key component is reported. This enzyme is able to convert an odorless substrate like S-methyl-L-cysteine or L-tryptophan into the odorous products methyl mercaptan or indole. To make a biosensor, TPase was biotinylated so that it could be coupled with a molecular recognition element, such as an antibody, to develop an ELISA-like assay. This method was used for the detection of an antibody present in nM concentrations by the human nose. TPase can also be combined with the enzyme pyridoxal kinase (PKase) for use in a coupled assay to detect adenosine 5'-triphosphate (ATP). When ATP is present in the low µM concentration range, the coupled enzymatic system generates an odor that is easily detectable by the human nose. Biotinylated TPase can be combined with various biotin-labeled molecular recognition elements, thereby enabling a broad range of applications for this odor-based reporting system.

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.

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 each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.(1) PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.(2-4) Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.(5) DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.(6-8) These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 10(16) individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).(9-16) These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.(6-8) Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.(17-29) RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity. More recently, we developed a method of isolating RFDs for bacterial detection.(5) These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets. Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).(5) This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3). Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

Multiplexed paper test strip for quantitative bacterial detection.

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 (ß-galactosidase (B-GAL) or ß-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-ß-D: -glucuronide sodium salt (XG), chlorophenol red ß-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.

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.

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.

Paper-based bioassays using gold nanoparticle colorimetric probes.

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.