This work is supported by K-INBRE P20GM103418, Kansas Soybean Commission (KSC/PSU 1663), ACS PRF 56629-UNI7 and PSU polymer chemistry startup fund, all to SS. We thank the university videographer, Mr. Jacob Anselmi, for his outstanding work with the video. We also thank Mr. Roger Heckert and Mrs. Katha Heckert for their generous support for research.

1.Synthesis and Functionalization of Multi-Parametric Magneto-Fluorescent Nanosensors (MFnS).
<ol>
	<li>Synthesis of superparamagnetic iron oxide nanoparticles (IONPs)
	<ol>
		<li>To prepare for IONP synthesis, prepare the following 3 solutions: Solution 1: FeCl3 (0.70 g) and FeCl2 in H2O (2 mL), Solution 2: NH4OH (2.0 mL, 13.4 M) in H2O (15 mL), and Solution 3: polyacrylic acid (0.855 g) in H2O (5 mL).</li>
		<li>Add 90 &#181;L of 2 M Hydrochloric acid (HCl) to Solution 1 and then immediately mix with Solution 2 while vortexing at 875 rpm. Then add Solution 3. Continue vortexing for 60 min.</li>
		<li>Centrifuge for 20 min at 1,620 x g. Centrifuge the supernatant for 20 min at 2,880 x g.</li>
		<li>Purify the final supernatant via dialysis. Add the supernatant to a dialysis bag (MWCO 6&#8722;8 K) and place it in a beaker containing phosphate buffered saline (PBS) (pH = 7.4) and spin bar. Let it soak for 12 h, replacing with fresh buffer every 2-3 h.</li>
	</ol>
	</li>
	<li>Conjugation of target-specific antibodies to surface of IONPs
	<ol>
		<li>Prepare the following four solutions: Solution A: 5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in 250 &#181;L MES [2-(N-morpholino)ethanesulfonic acid] buffer (0.1 M, pH = 6.8), Solution B: 3 mg of N-Hydroxysuccinimide (NHS) in 250 &#181;L of MES buffer (0.1 M, pH = 6.8), Solution C: 5 &#181;g of IgG1, the E. coli mAb, in 225 &#181;L of phosphate-buffered saline (PBS) (pH 7.4), and Solution D: 4 mL of IONP (5.0 mmol) in 1 mL of PBS (pH 7.4).</li>
		<li>Add 2-(N-Morpholino)ethanesulfonic acid (MES) buffer to Solution A, then immediately add Solution A to Solution D in small increments within 15 s, inverting the solution after each addition for mixing.</li>
		<li>Immediately begin to add Solution B to Solution D in small increments over a three-minute period. Mix by inverting the solution after each addition.</li>
		<li>Add Solution C to solution in increments of 5 &#181;L, inverting the solution after each addition for mixing.</li>
		<li>Allow reaction to continue for 3 h at room temperature and then continue the incubation at 4 &#176;C overnight.</li>
		<li>Purify the resulting Ab-conjugated IONPs via magnetic column using PBS (pH = 7.4, final [Fe] = 3.5 mmol) to remove any unconjugated antibodies.
		<ol>
			<li>Wash the magnetic column with PBS (1 mL). Attach the column to the magnetic plate, and add IONP solution.</li>
			<li>Wash the magnetic column with PBS (1 mL) again. Remove the magnetic column from magnetic plate. Add PBS to the column and collect the solution. Store at 4 &#176;C</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Encapsulation of fluorescent 1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-Tetramethylindocarbocyanine Perchlorate (DiI) dye into the polyacrylic acid (PAA) coating of the antibody-conjugated IONPs using a solvent diffusion method.
	<ol>
		<li>To 4 mL of the IONPs, add dropwise 2.0 &#181;L of DiI dye (2 mmol) in 100 &#181;L of DMSO with continuous mixing at 1100 rpm.</li>
		<li>Dialyze the resulting solution (MWCO 6-8 KDa, 12 h) against PBS, creating a final MFnS solution with a final iron concentration of [Fe] = 2 mmol.</li>
	</ol>
	</li>
	<li>Characterization of MFnS
	<ol>
		<li>For spectrophotometric analysis, use a plate reader to detect encapsulated DiI dye with fluorescence emission at 595 nM.</li>
		<li>Dynamic Light Scattering
		<ol>
			<li>Place a sample of the MFnS solution into a zetasizer to determine the average size and surface charge of the MFnS. 
			Note: Here, the average size and surface charge of functional MFnS were found to be 77.09 nm and -22.3 mV, respectively.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Bacterial Culturing and Stock Solution Preparation
<ol>
	<li>Bacterial Culturing
	<ol>
		<li>Hydrate a freeze-dried pellet (E. coli O157:H7) in 1 mL of nutrient broth and then add an additional 5 mL of broth.</li>
		<li>Apply 100 &#181;L of this solution to an agar plate and streak using a sterile inoculation loop, followed by incubation at 37 &#176;C for 24 h.</li>
		<li>After 24 h, select an isolated colony from the agar plate and add it to a 15 mL of culture broth. Incubate at 37 &#176;C for 4-6 h, while monitoring the optical density value (absorbance at 600 nm).</li>
		<li>Once an optical density value of 0.1 is obtained, stop culturing, and perform serial dilutions.</li>
	</ol>
	</li>
	<li>Serial dilutions
	<ol>
		<li>Add 900 &#181;L of nutrient broth to eight sterile microcentrifuge tubes.</li>
		<li>Add 100 &#181;L of the bacterial suspension to the first tube (dilution 10-1).</li>
		<li>Take 100 &#181;L from the first tube and add it to the second, to obtain a dilution of 10-2. Continue this pattern for the remaining tubes, ending with a final dilution of 10-8.</li>
		<li>Transfer 100 &#181;L from each tube to separate agar plates, and incubate for 24 h at 37 &#176;C.</li>
		<li>The next day, count the colony forming units (CFUs) on each plate. Select the plate with ~100 CFUs. Use the corresponding dilutions for further experiments (100 &#181;L = ~100 CFUs). 
		Note: Here it was the 10-6 dilution that resulted in ~100 CFUs.</li>
	</ol>
	</li>
</ol>
3. Rapid Detection of E. coli O157:H7 using MFnS
<ol>
	<li>Spike various PBS solutions (1X, pH 7.4, 300 &#181;L) with increasing amounts of the 10-6 bacterial stock, resulting in CFU ranges from 1-100. Add a consistent amount of MFnS (100 &#181;L) to these solutions.</li>
	<li>Create one baseline solution that contains only PBS (1X, pH 7.4, 300 &#181;L) and MFnS (100 &#181;L).</li>
	<li>Incubate solutions for 30 min at 37 &#176;C and then allow them to cool to room temperature.</li>
	<li>Transfer individual solutions to the magnetic relaxometer (0.47 Tesla) and record changes in relaxation times (T2) relative to the CFUs in each solution.
	<ol>
		<li>To record changes in T2 values, begin by measuring the T2 value of the baseline solution that contains only PBS and MFnS.
		<ol>
			<li>Place the solution&#160;in the magnetic relaxometer. Open the respective software, select the &#34;T2 relaxation&#34; setting and press &#34;measure.&#34;</li>
		</ol>
		</li>
		<li>Following the collection of the baseline T2, measure the T2 values of the additional solutions that were spiked with various concentrations of bacteria. 
		Note: The change in T2 is equivalent to baseline T2 subtracted from the spiked T2.</li>
	</ol>
	</li>
	<li>Remove samples from the magnetic relaxometer and centrifuge the tubes at 2880 x g for 10 min.</li>
	<li>Decant the supernatant and resuspend bacterial pellets in 100 &#181;L of PBS (1X, pH 7.4).</li>
	<li>Add 80 &#181;L of each resuspension to a 96-well plate and record fluorescence intensities at 595 nM. 
	Note: Testing can be repeated using different solvents, including lake water, milk and others as described in the results section</li>
</ol>

<ol>
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The demonstrated application of this nanotechnology is not yet approved by the FDA.

This protocol has been designed to produce fully functional MFnS as simply as possible. However, there are many key points at which alteration of the protocol may be useful, depending on the user's end goal. For example, the use of different antibodies would allow for targeting of many other pathogens. In addition, this protocol is not limited to the use of antibodies as targeting molecules. Any molecule which has specific binding affinity for target pathogens, such as host cell receptors...

The persistent occurrence of bacterial contamination in both commercially produced food and water sources has created a need for increasingly rapid and specific diagnostic platforms.1,2 Some of the more common bacterial contaminants responsible for food and water contamination are from the Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus, and Escherichia genera.3,4 Bacterial contamination by these pathogens often results in symptoms such as fever, cholera, gastroenteritis, and diarrhea.4 Contamination of water sources often has drastic and adverse effects on communities without access to sufficiently filtered water, and food contamination has led to a great number of illnesses and product recall efforts.5,6
In order to reduce the occurrence of illnesses caused by bacterial contamination, there have been a number of efforts to develop methods by which water and food can be efficiently scanned prior to sale or consumption.3 Techniques such as PCR,1,7,8,9,10 ELISA,11,12 loop-mediated isothermal amplification (LAMP),13,14 among others,15,16,17,18,19,20,21,22,23,24 have recently been used for detection of various pathogens. Compared to traditional bacterial culturing methods, these techniques are far more efficient with regards to specificity and time. However, these techniques still struggle with false positives and negatives, complex procedures, and cost.1,3,25 It is for this very reason that multiparametric magneto-fluorescent nanosensors (MFnS) are proposed as an alternative method for bacterial detection.
These nanosensors uniquely pair together magnetic relaxation and fluorescent modalities, allowing for a dual-detection platform that is both rapid and accurate. Using E. coli O157:H7 as a sample contaminant, the ability of MFnS to detect as little as 1 CFU within minutes is demonstrated. Pathogen-specific antibodies are used to increase specificity, and the combination of both magnetic and fluorescent modalities allows for the detection and quantification of bacterial contaminants in both low- and high-contamination ranges.16 In the case of bacterial contamination, the nanosensors will swarm around the bacteria due to the targeting abilities of the pathogen-specific antibodies. The binding between the magnetic nanosensors and bacteria limits the interaction between the magnetic iron core and the surrounding water protons. This causes an increase in the T2 relaxation times, as recorded by a magnetic relaxometer. As the concentration of bacteria in solution rises, the nanosensors disperse with the increased number of bacteria, resulting in lower T2 values. Conversely, fluorescence emission will increase in proportion with the concentration of bacteria, due to the increased number of nanosensors directly bound to pathogen. Centrifugation of the samples, and isolation of the bacterial pellet, will only conserve the nanoparticles directly attached to the bacteria, removing any free-floating nanosensors, and directly correlating the fluorescence emission with the number of bacteria present in solution. A schematic representation of this mechanism is represented in Figure 1.
This MFnS platform has been designed with point-of-care screening in mind, resulting in low-cost and portable characteristics. MFnS are stable at room temperature, and are only required in very low concentrations for accurate detection of bacterial contaminants. Furthermore, after synthesis, use of the MFnS is simple and does not require the use of trained professionals in the field. Lastly, this diagnostic platform allows for highly customizable targeting, providing a means by which this one platform may be used to detect pathogens of all kinds, in many different settings.

<table><tbody><tr><td>Ferrous Chloride Tetrahydrate</td><td>Fisher Scientific</td><td>I90-500</td><td></td></tr><tr><td>Ferric Chloride Hexahydrate</td><td>Fisher Scientific</td><td>I88-500</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Polyacryllic Acid</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>EDC</td><td>Thermofisher Scientific</td><td>22980</td><td></td></tr><tr><td>NHS</td><td>Fisher Scientific</td><td>AC157270250</td><td></td></tr><tr><td>Anti-E. coli O111 antibody&amp;nbsp;</td><td>sera care</td><td>5310-0352</td><td></td></tr><tr><td>Anti-E. coli O157:H7 antibody [P3C6]&amp;nbsp;</td><td>Abcam</td><td>ab75244</td><td></td></tr><tr><td>DiI Stain</td><td>Fisher Scientific</td><td>D282</td><td></td></tr><tr><td>Nutrient Broth</td><td>Difco</td><td>233000</td><td></td></tr><tr><td>Freeze-dried E. coli O157:H7 pellet</td><td>ATCC</td><td>700728</td><td></td></tr><tr><td>Magnetic Relaxomteter&amp;nbsp;</td><td>Bruker</td><td>mq20</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern</td><td>NANO-ZS90</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>Tecan</td><td>Infinite M200 PRO</td><td></td></tr><tr><td>Magnetic Column&amp;nbsp;</td><td>QuadroMACS</td><td>130-090-976</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 Series</td><td></td></tr><tr><td>Centrifuge (accuSpin Micro 17)</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>Floor Model Shaking Incubator</td><td>SHEL LAB</td><td>SSI5</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>ME104E</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Open-Air Rocking Shaker</td><td>Fisher Scientific</td><td>02-217-765</td><td></td></tr></tbody></table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

The mechanism of MFnS action is represented in Figure 1. The clustering of MFnS around the surface of bacterial contaminants interferes with the interactions between the magnetic cores of the MFnS and the surrounding hydrogen nuclei. As a result of this clustering, magnetic relaxation values increase. As the concentration of bacterial contaminants increases, clustering reduces, and the change in T2 values decreases. Therefore, the addition of a fluorescent modality is crucial. As the bacteri...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.6K Views.  Pittsburg State University. The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Video: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

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 1016 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.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

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

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Bioengineering

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Chemistry

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Biology

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Summary

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Chapters in this video

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

Detection of Bacteria Using Fluorogenic DNAzymes

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Colorimetric Detection of Bacteria Using Litmus Test