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><li>Law, J. W., Ab Mutalib, N. S., Chan, K. G., Lee, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+methods+for+the+detection+of+foodborne+bacterial+pathogens%3A+principles%2C+applications%2C+advantages+and+limitations%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations.</a> Front Microbiol. 5, 770(2014).</li>
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<li>Mahony, J. B., Chong, S., Luinstra, K., Petrich, A., Smieja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+novel+bead-based+multiplex+PCR+assay+for+combined+subtyping+and+oseltamivir+resistance+genotyping+%28H275Y%29+of+seasonal+and+pandemic+H1N1+influenza+A+viruses%5BTitle%5D)+AND+%22J+Clin+Virol%22%5BJournal%5D)">Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses.</a> J Clin Virol. 49 (4), 277-282 (2010).</li>
<li>Alvarez, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+recognition+of+influenza+A%2FH1N1%2F2009+antibodies+in+human+serum%3A+a+simple+virus-free+ELISA+method%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Specific recognition of influenza A/H1N1/2009 antibodies in human serum: a simple virus-free ELISA method.</a> PLoS One. 5 (4), e10176(2010).</li>
<li>Huang, C. J., Dostalek, J., Sessitsch, A., Knoll, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-range+surface+plasmon-enhanced+fluorescence+spectroscopy+biosensor+for+ultrasensitive+detection+of+E.+coli+O157%3AH7%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">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/pubmed?term=((Rapid+visual+detection+of+highly+pathogenic+Streptococcus+suis+serotype+2+isolates+by+use+of+loop-mediated+isothermal+amplification%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Detecting+potentially+virulent+Vibrio+vulnificus+strains+in+raw+oysters+by+quantitative+loop-mediated+isothermal+amplification%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">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>
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</ol>

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, may also be used as targeting molecules. As long as the targeting molecule has a primary amine or alcohol group, or can be functionalized to have one, it may be conjugated to the surface of iron oxide nanoparticles according to the EDC/NHS conjugation chemistry listed in the protocol. Secondly, the amount of MFnS used in each detection solution may be varied. It is important to use enough so that the T2 value for the baseline solution (containing only solvent and nanosensor without target pathogen) is in the range of 100-250 ms. If the baseline value is below 100, then the changes in T2 values will be less sensitive, due to an overabundance of nanosensor. To raise the baseline T2 value, reduce the amount of nanosensor in solution. If, however, the baseline is above 250, it becomes overly sensitive, and false positives may become more likely. For these reasons, it is suggested to aim for a baseline solution T2 value between 100-250 ms. 
Current limitations of this protocol, when considering its use as a point-of-care diagnostic, are the reliance on the current benchtop magnetic relaxometer. While effective and stable, this magnetic relaxation platform could be reduced in size to increase its portability. This would increase the ease with which this platform could be used in different environments, such as at water sources or in grocery markets, for effective on-site bacterial screening. This could also reduce the cost of this machine, making it more feasible for point-of-care pathogen detection.
Concerning disease diagnosis and pathogen detection in low-resource settings, this platform has the potential to be more effective than current pathogen diagnosis techniques used today, including PCR and ELISA. While these traditional techniques have proven to be largely efficient, they are highly complex, costly, and slow. As such, there is a need for an accurate diagnostic platform that could be developed for use in low-resource settings for point-of-care detection of pathogens and disease diagnosis. This platform is relatively simple to use, cost effective and rapid. Aside from initial start-up costs of the required machines (relaxometer and plate reader), the actual MFnS are relatively cheap and very stable. In addition, the collection of T2 values is simple and does not require a professional lab technician. 
In the future, collaboration with chemical and biological engineers will be pursued to design a more portable, hand-held device that could be used for both the magnetic and fluorescent detection of bacterial contamination using MFnS. The development of a smaller device would help to further reduce the costs associated with this detection platform and increase its effectiveness in the field. Furthermore, this platform has the potential to be used to analyze the effectiveness of developing biocide agents. As shown in our data, our MFnS were only able to bind with live-targeted bacteria, and produced little to no signal when incubated with heat-killed bacteria. Based upon this, our platform could be used to test the effectiveness of biocide candidates, and we plan to explore this further in the future. 
Critical steps in this protocol include the initial synthesis of MFnS and collection of T2 data. It is important to properly purify the MFnS to ensure that free-floating antibodies do not interfere with T2 data collection. In addition, the amount of MFnS placed in each test sample must be consistent, as changes in the MFnS concentration will alter T2 values, risking false positives/negatives. 
In conclusion, MFnS are a novel step forward in the fight against bacterial contamination, and will be further developed as the targeting of additional pathogens is achieved and the design more portable machinery is pursued. The low cost and low-complexity associated with the use of MFnS makes them a viable candidate to be used for on-site detection of bacterial contamination. While the synthesis of these nanosensors requires careful preparation, their actual use is relatively simple, and gives them the potential to reduce foodborne and waterborne illnesses. With further development, the implementation of these nanosensors may be seen in the screening of water reservoirs and commercially produced foods at packaging sites, points of sale, and perhaps even homes.

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 bacterial concentration increases, the strength of the fluorescent signal produced by MFnS increases, allowing for the sensitive detection of bacterial contaminants in both low and high concentration ranges.
Following the protocol,16 MFnS were synthesized and first tested in solutions of PBS (1X, pH 7.4). The initial modality used for detection, magnetic relaxation, was used to record changes in T2 values. In accordance with the protocol, the samples were then centrifuged, pellets were resuspended, and fluorescence data were collected. The corresponding &#916;T2 and fluorescence emission values are presented in Figure 2. As shown, the &#916;T2 value was greatest at lower concentrations of bacterial contamination, and reached saturation at roughly 20 CFUs. Fluorescence emission, on the other hand, became more accurate at &#62;20 CFUs, displaying the importance of combining these two modalities. Following these primary assays, similar tests were conducted in complex media, including lake water and milk. This was done to ensure that the nanosensors are still effective in more complex media. The data collected from these assays are presented in Figure 3 and are very similar to the assays conducted in PBS. This verifies that these nanosensors remain effective in complex media.
After confirming that MFnS could detect the presence of bacterial contaminants in both simple and complex media, a number of tests were then conducted to determine the level of specificity maintained by the nanosensors. To this end, MFnS were incubated in solutions containing target bacteria, heat-killed target bacteria, non-target bacteria (non-pathogenic E. coli and S. typhimurium) and a mixture. As this was only a specificity assay, the collection of magnetic relaxation data alone was sufficient, and the corresponding data is presented in Figure 4. As can be seen, the MFnS bound only to live E. coli O157:H7, and not to the other heat-killed or non-target bacteria. This level of specificity is attributed to the conjugated targeting antibodies, and further confirms that MFnS will be effective at detecting specific pathogen contaminants, even when in the presence of non-pathogenic bacterial species.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55821/55821fig1.jpg" /> 
Figure 1: Magneto-Fluorescent Detection Mechanism. Schematic representation of MFnS synthesis and the dual-modal mechanism of bacterial detection. Following a short incubation period (30 min), MFnS are able to sensitively detect the target bacteria via a combination of magnetic resonance and fluorescence emission. Clustering of MFnS around the surface of bacterial contaminants causes changes in magnetic relaxation values, which are detectable by a magnetic relaxometer. In addition, fluorescent signals may be collected from the MFnS directly bound to bacterial contaminants. This provides a dual-modal detection platform capable of detection bacterial contaminants in both low and high concentration ranges.16 This figure has been modified with permission from.16
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55821/55821fig2v3.jpg" /> 
Figure 2: Proof of Concept. A) Magnetic relaxation data (&#916;T2) was first collected from dilutions of E. coli O157:H7 (1-100 CFU) in PBS solvent (1X, pH = 7.4). MR detection of bacteria was highly sensitive at lower CFU counts (inset: ranging from 1-20 CFU). However, the &#916;T2 readings became saturated at higher bacterial concentrations (&#62;20 CFU), indicating that MR is more valuable for the detection and quantification of early-stage bacterial contamination. B) Fluorescence emission data from the same spiked PBS solutions (inset: linearity plot). The results showed that the fluorescence detection method is more sensitive at higher CFU counts, while it is lacking in sensitivity for low CFU samples. Together, these data demonstrate the ability of both magnetic and fluorescent modalities to detect and quantify bacterial contamination in early- and late-stages of bacterial contamination.16 Average values of three measurements are depicted &#177; standard error. This figure has been modified with permission from.16
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55821/55821fig3v3.jpg" /> 
Figure 3: Bacterial Detection in Complex Media. Magnetic relaxation &#916;T2 data collected from more complex media, including A) lake water and B) whole milk. Similar to the data collected from the previous PBS solutions, it was noted that detection of bacterial contaminants was more sensitive in the range of 1-20 CFU (insets). Corresponding fluorescence emission data was collected for the C) lake water and D) milk samples (insets: linearity plots), which showed higher sensitivity with increasing CFU counts. Once again, these data demonstrate the effectiveness with which each modality complements the other, and validate their ability to remain functional in complex media.16 Average values of three measurements are depicted &#177; standard error. This figure has been modified with permission from.16
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55821/55821fig4v3.jpg" /> 
Figure 4: MFnS Specificity. The specificity of MFnS was tested using MR analysis in nutrient broth solutions containing A) a number of bacterial cross-contaminants and a mixture, and B) heat-inactivating E. coli O157:H7. As shown, it is clear that the nanosensors have little to no binding with non-targeted bacteria, and are still capable of detecting the target bacteria in the presence of other contaminants, ensuring that this form of screening would be viable for use in complex media that contain a number of non-pathogenic bacterial species. C) Further specificity testing was conducted by synthesizing MFnS with an isotype antibody (red circles: Anti-E. coli O111), which resulted in little to no binding compared to the O157:H7 antibody-conjugated MFnS (black squares). These data demonstrate that MFnS only react with viable target bacteria, further verifying their effectiveness as a point-of-care diagnostic platform.16 Average values of three measurements are depicted &#177; standard error. This figure has been modified with permission from.16

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 Científica del Sur

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

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biology

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, ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

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 ...

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

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. ...

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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