Name: foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7
Uploaded: 2017-09-17T14:00:00
Description: Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

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 ...

<ol>
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J., Dostalek, J., Sessitsch, A., Knoll, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+surface+plasmon-enhanced+fluorescence+spectroscopy+biosensor+for+ultrasensitive+detection+of+E.+coli+O157:H7.">Long-range surface plasmon-enhanced fluorescence spectroscopy biosensor for ultrasensitive detection of E. coli O157:H7.</a> Anal Chem. 83 (3), 674-677 (2011).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+visual+detection+of+highly+pathogenic+Streptococcus+suis+serotype+2+isolates+by+use+of+loop-mediated+isothermal+amplification.">Rapid visual detection of highly pathogenic Streptococcus suis serotype 2 isolates by use of loop-mediated isothermal amplification.</a> J Clin Microbiol. 51 (10), 3250-3256 (2013).</li><li>Han, F., Wang, F., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+potentially+virulent+Vibrio+vulnificus+strains+in+raw+oysters+by+quantitative+loop-mediated+isothermal+amplification.">Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.</a> Appl Environ Microbiol. 77 (8), 2589-2595 (2011).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+pathogenic+bacteria+and+screening+of+phage-derived+peptides+using+microcantilevers.">Rapid detection of pathogenic bacteria and screening of phage-derived peptides using microcantilevers.</a> Anal Chem. 86 (3), 1671-1678 (2014).</li><li>Banerjee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+Magneto-fluorescent+Nanosensors+for+the+Ultrasensitive+Detection+of+Escherichia+coli+O157:H7.">Multiparametric Magneto-fluorescent Nanosensors for the Ultrasensitive Detection of Escherichia coli O157:H7.</a> ACS Infect Dis. 2 (10), 667-673 (2016).</li><li>Shelby, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+magnetic+relaxation+nanosensors:+an+unparalleled+&amp;#34;spin&amp;#34;+on+influenza+diagnosis.">Novel magnetic relaxation nanosensors: an unparalleled &amp;#34;spin&amp;#34; on influenza diagnosis.</a> Nanoscale. 8, 19605-19613 (2016).</li><li>Bui, M. 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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 border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">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 ...

The main goal of this protocol is to design and synthesize dual-modal nanosensors capable of detecting bacterial contaminants such as E.coli O157:H7. Magneto-fluorescent nanosensors are synthesized by functionalizing iron oxide nanoparticles via a two-step procedure. First, targeting antibodies are conjugated to the surface of the nanoparticle.Then fluorescent dye is loaded into its coding. The paring of these modalities allows for rapid and sensitive detection of bacterial contaminants in both low and highly concentrated solutions. In the presence of a small amount of bacteria, the nanosensors will swarm around the bacteria due to the targeting of the conjugated antibodies.This swarming alters the interactions of the nanosensors magnetic core with the surrounding water protons, allowing for sensitive detection of changes in magnetic relaxation values. As the concentration of bacterial contaminants in solutions increases, swarming decreases and the magnetic modality's ability to quantify contamination is reduced. However, as the bacterial concentration increases, so does the fluorescent submission of the bio nanosensors.It is for this reason that the pairing of magnetic and fluorescent modalities is important. The combination has allowed for the detection of as little as one colony forming unit of E.coli O157:H7 within minutes. The first step in the systhesis of magneto-fluorescent nanosensors is the preparation of a iron salt solution consisting of both ferous and ferric chloride.The iron will provide the nanosensors with a magnetic core which allows for its adaptability to magnetic relaxation platforms. Additional solutions needed are polyacrylic acid and ammonium hydroxide. Hydrochloric acid is introduced to the iron salt solution, which is then added to the ammonium hydroxide solution while vortexing.Finally the polyacrylic acid solution is added, and the resulting mixture is vortexed for an additional hour while the reaction continues. The resulting solution is centrifuged in order to precipitate the larger molecules and isolate nanoparticles that are smaller than 100 nanometers in size. The solution is then dialyzed overnight for purification.The next step is the conjugation of targeting antibodies to the polyacrylic acid coding of the newly synthesized iron acid nanoparticles. Materials needed include MES buffer, EDC, NHS and the selected antibodies. EDC is first added to the nanoparticle solution, followed by NHS and finally the antibody.The mixture is then placed on a tabletop mixer for three hours while the reaction continues. The solution is purified using a magnetic column. The magnetized column captures the nanoparticles, allowing only free floating antibodies to exit.Afterwards the column is washed with PBS buffer, and the conjugated nanosensors are collected. The nanoparticles are then ready for the addition of fluorescent dye, providing the second key modality used for detection. One of the most key features of our nanosensor is the combination of the magnetic and fluorescent modalities, which is important for a number of reasons.Firstly, the combination of the modalities allows each one to sort of cross check the other one, which greatly reduces the risk of false positives and false negatives, which is one of the greatest hurdles faced by different diagnostic platforms used today. And secondly, the combination of the modalities greatly widens the range within which we can not only detect but also quantify bacterial contamination. The final step in the preparation of the nanosensors is the encapsulation of fluorescent dye in the coding of the nanoparticle.This is simply achieved by adding dye to the solution while vortexing, and then allowing further time for the dye to diffuse into the coding of the nanosensor. The fully functionalized nanosensors are then dialyzed one final time for purification. In order to characterize the nanosensors, the size and fluorescent submission are recorded using a zetasizer and a fluorescence plate reader.A small sample of the nanosensor is added to a cuvette water solution and placed in the zetasizer for the recording of size. For the fluorescence analysis a sample of the nanosensor is placed on a 96 well plate and inserted into the fluorescence plate reader. Ideally the nanosensors will be around 70 nanometers in diameter, and have a fluorescent submission of 575 nanometers.In addition to the synthesis of the nanosensors, the culturing of bacteria is necessary to provide the bacterial targets in the lab. A glycerol stock of bacteria is used to prepare a culture in nutrient broth. This solution is then allowed to incubate.Following the initial incubatory period, serial dilutions are performed in order to achieve a wide range of bacterial concentrations. One hundred microliters of each concentration are plated to an nutrient auger plate and then incubated for 24 hours. Following this incubation the colonies on each plate are counted in order to determine the colony forming unit, or CFU count per mil of each diluted stock.Now that the bacterial stock solutions have been prepared, they are able to be used in conjunction with the prepared nanosensors. And one important aspect of this particular serotype of the bacteria that distinguishes it from other serotypes is like suppose if you get infected with the other serotypes, you need to have many colony forming units to get an infection. But in this particular E.coli 10 to 100 CFUs or colony forming units are good enough to give you an infection.Our technology is established in such a way that you don't miss even a single colony bacterial contamination is there. In order to prepare solutions for reading in the magnetic relaxometer, 300 microliters of PBS is first pipetted into an Eppendorf tube. Then a sample of bacterial stock is added, followed by the addition of nanosensors.The solution is then transferred to a glass tube, and a piece of parafilm is placed on top in order to prevent evaporation. The glass tube is then placed inside a larger NMR tube and inserted into the magnetic relaxometer. A baseline solution containing no bacteria and only nanosensor and PBS is used to get a baseline T2 reading as shown.Then solutions containing various concentrations of bacteria are inserted into the magnetic relaxometer for analysis, and the changes in T2 values are caused by the binding between the nanosensors and the bacteria. As shown, the presence of as few as one bacterial CFU can be detected within minutes using this modality. However, as the bacterial concentration increases the MR reading are less quantifiable, which is why the use of fluorescent subsmission data is also tantamount to an accurate bacterial quantification.Before fluorescence data can be collected the sample must first be centrifuged. The solution is transferred from the glass tube to an Eppendorf tube and then centrifuged. This separates the bacteria and the nanosensors bound to it from free floating nanosensors in solution.The supernatent is discarded, and the bacterial pellet is resuspended in PBS. Finally the sample may be analyzed via fluorescent submission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore also the amount of bacterial present.As can be seen, fluorescent submission strengthens as the bacterial concentration increases, and it becomes more sensitive as well. This is why the pairing of both magnetic and fluorescent modalities allows for the detection and quantification of bacterial contamination in both early and late developmental stages. In addition to the detection of bacteria in simple solutions such as PBS, these nanosensors also possess the capability to function in more complex media, such as lake water or milk.Furthermore, these nanosensors have been tested for the specificity in the presence of non-target bacteria and heat inactivated target bacteria. As shown magneto-fluorescent nanosensors have little to no reaction with non-targeted or non-living bacteria due to the specificity of the conjugated antibodies. This demonstrates their effectiveness for the detection of specific bacteria while in the presence of other species.Finally, it is also important to note that these nanosensors may be easily customized for the detection of other pathogens as well. You can synthesize or you can formulate these nanosensors that we did in our lab within one month now. You can do it within one month, and that one month of product can give you like 10 years of application.

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

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Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

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Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

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Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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

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 ...